Download Internal affairs: investigating the Brucella intracellular lifestyle

REVIEW ARTICLE
Internal affairs: investigating the Brucella intracellular lifestyle
Kristine von Bargen1,2,3, Jean-Pierre Gorvel1,2,3 & Suzana P. Salcedo1,2,3
1
Faculté de Sciences de Luminy, Centre d’Immunologie de Marseille-Luminy, Aix Marseille Université, UM 2, Marseille Cedex, France; 2INSERM,
U 1104, Marseille, France; and 3CNRS, UMR 7280, Marseille, France
Correspondence: Suzana P. Salcedo, Centre
d’Immunologie de Marseille-Luminy, Aix
Marseille Université, Faculté de Sciences de
Luminy, Case 906, Marseille, 13288 Cedex 9,
France. Tel.: +33 491 269 116 and
+33 491 269 115 ; fax: +33 491 269 430;
e-mail: [email protected]
Received 4 July 2011; revised 10 January
2012; accepted 16 February 2012.
Final version published online 22 March
2012.
DOI: 10.1111/j.1574-6976.2012.00334.x
MICROBIOLOGY REVIEWS
Editor: Christoph Dehio
Keywords
Brucella-containing vacuole; trafficking; type
IV secretion system; pathogenicity; bacteria;
brucellosis.
Abstract
Bacteria of the genus Brucella are Gram-negative pathogens of several animal
species that cause a zoonotic disease in humans known as brucellosis or Malta
fever. Within their hosts, brucellae reside within different cell types where they
establish a replicative niche and remain protected from the immune response.
The aim of this article is to discuss recent advances in the field in the specific
context of the Brucella intracellular ‘lifestyle’. We initially discuss the different
host cell targets and their relevance during infection. As it represents the key to
intracellular replication, the focus is then set on the maturation of the Brucella
phagosome, with particular emphasis on the Brucella factors that are directly
implicated in intracellular trafficking and modulation of host cell signalling
pathways. Recent data on the role of the type IV secretion system are discussed, novel effector molecules identified and how some of them impact on
trafficking events. Current knowledge on Brucella gene regulation and control
of host cell death are summarized, as they directly affect intracellular persistence. Understanding how Brucella molecules interplay with their host cell targets to modulate cellular functions and establish the intracellular niche will
help unravel how this pathogen causes disease.
Introduction
Brucellosis is the most common bacterial zoonotic disease
worldwide, with over half a million infected people annually (Pappas, 2010). It remains endemic in many parts of
the world, including the Middle East, Africa, Latin America, central Asia and several regions of the Mediterranean
basin. Human brucellosis is often misdiagnosed and
underreported. It is an important travel-associated disease
(Memish & Balkhy, 2004), for which no vaccine is available. The treatment requires a combination of different
antibiotics for a prolonged period of time (Ariza et al.,
2007). Many countries have succeeded in control of the
disease in the recent years by nearly eradicating it from
livestock, the predominant reservoir for human infections. However, there is growing concern for re-emerging
foci of brucellosis in numerous countries, particularly in
central Asia, where it is causing significant morbidity
(World Health Organization, 2005).
Brucella spp. are Gram-negative bacteria that were first
isolated in 1887 in Malta by Sir David Bruce from the
FEMS Microbiol Rev 36 (2012) 533–562
spleens of soldiers with fatal cases of brucellosis, also
known as undulant fever or Malta fever. Brucella spp.
belong to the a-2 subdivision of Proteobacteria which
includes bacteria that co-evolved with animal or plant
hosts, either in a beneficial symbiotic manner such as
Sinorhizobium meliloti (a plant symbiont) or as pathogens
such as Agrobacterium tumefaciens, Rickettsia spp. and
Bartonella spp.
At present, ten species of the genus Brucella have been
recognized with the nomenclature based on their respective preferential host (Moreno et al., 2002; Audic et al.,
2009). The species most pathogenic for humans and that
are most relevant for domestic animals are Brucella melitensis that infects goats, sheep and camels; Brucella abortus
that causes bovine brucellosis and Brucella suis that is
associated with brucellosis in swine. Three other species,
Brucella canis (dogs), Brucella ovis (sheep and rams) and
Brucella neotomae (desert wood rats) are of low pathogenicity for humans. Since the 1990s, Brucella strains infecting marine mammals have been described in the
literature, illustrating the broad mammal host range of
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
534
this bacterial pathogen. Two distinct strains were identified and referred to as Brucella pinnipediae and Brucella
cetaceae (Cloeckaert et al., 2001). However, they had not
been validly published until 2007 when the species’ names
were slightly altered into Brucella ceti and Brucella pinnipedialis (Foster et al., 2007). Brucella ceti is found in dolphins, porpoises and whales, and B. pinnipedialis affects
mainly seals. An increasing number of reports associate
marine mammal strains with human brucellosis, suggesting that these Brucella species may be pathogenic for
humans (Sohn et al., 2003; Whatmore et al., 2008). A
more recently described species, Brucella microti, has been
isolated from the common vole and from foxes and has
been found as a soil contaminant but so far it has not
been reported as a cause for human brucellosis (Scholz
et al., 2008a, b). Additional strains have been isolated
from Australian rodents but have not yet been confirmed
as new Brucella species (Tiller et al., 2010a). Finally, so
far unknown strains have recently been isolated from brucellosis patients. Brucella inopinata has been isolated from
an infected breast implant (Scholz et al., 2010). The second strain is phenotypically and molecularly similar to
B. inopinata and was isolated from a patient with chronic
lung infection (Tiller et al., 2010b). These newly described
strains highlight the complexity of the growing genus
Brucella.
Humans are not natural hosts for brucellae, and the
source of the infection is usually found in domestic or
wild animal reservoirs. Occupational exposure and ingestion of contaminated food products are the main routes
of infection for humans. However, its high infectivity via
aerosols places Brucella on the category B list for agents
of biological warfare and accounts for it being the most
common laboratory-acquired bacterial infection. Containment of human brucellosis is dependent on successful
vaccination of livestock and imposing strict farming
hygiene, surveillance and infection control measures.
Although Brucella epidemiological control has seen considerable progress in the last years, several recent reports
raise concerns. In some European countries, wild boars
and hares are widely infected with B. suis biovar 2
(Bergagna et al., 2009; Cvetnic et al., 2009; Galindo et al.,
2010). Approximately 50% of the bison population in the
Greater Yellowstone Area in the USA have been found
infected with B. abortus (Olsen, 2010; Scurlock &
Edwards, 2010). In addition, there are concerns of new
habitats being colonized by virulent strains, as illustrated
by a report of B. melitensis isolated from fish in the Nile
river (El-Tras et al., 2010). Marine Brucella species have
been isolated from human brucellosis patients (Sohn
et al., 2003; McDonald et al., 2006), demonstrating possible transmission from a yet unconsidered reservoir. The
growing incidence of brucellosis in wildlife as a potential
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
K. von Bargen et al.
source for human infection poses a new challenge to
eradication of the disease worldwide.
The disease in different hosts
In animal primary hosts, Brucella have a particular tropism for the reproductive system, often leading to abortion in pregnant female animals following extensive
bacterial colonization of the placenta and sterility in male
animals. Transmission between animals is facilitated by
the presence of high numbers of bacteria in the aborted
foetus, reproductive tract discharges and milk. Animals
can present severe complications but may also become
carriers and continuously shed bacteria for many years
(Moreno & Moriyon, 2006). The chronic nature of brucellosis is a key aspect of the disease that is observed in
both the natural host, experimental animal models and
during infection in humans. In marine mammals, Brucella
can affect the reproductive tract and cause abortion but
the disease may also present life-threatening complications such as meningoencephalitis, splenic and liver
necrosis (Gonzalez et al., 2002; Nymo et al., 2011).
In humans, the onset of brucellosis is most commonly
manifested as a flu-like illness, with recurrent fevers associated with muscle and joint pain. As bacteria disseminate
systemically, patients present enlarged liver and spleen
and swollen lymph nodes. If patients are not treated,
Brucella have the propensity to form granulomas and can
colonize multiple sites in the body. The most common
complication is arthritis, which is often seriously debilitating and difficult to treat. More severe complications
include liver abscess formation, neurobrucellosis and
endocarditis. Antibiotic treatment is normally effective
when administered promptly and mortality is rare. Nonetheless, because of the chronic nature of brucellosis, its
tendency to relapse and its propensity to affect joints,
patients can suffer long-term disability (Franco et al.,
2007). In addition, allergic hypersensitivity to Brucella
antigens is a frequent consequence of brucellosis following re-infection or repeated contact with Brucella antigens
(Moreno & Moriyon, 2006).
The mouse model of brucellosis is the most widely used
laboratory experimental model to study Brucella virulence
in vivo (extensively reviewed by Silva et al., 2011). In this
model, Brucella can be found in multiple tissues including
the spleen and liver, where microgranulomas are formed
during the chronic stages of the infection. Interestingly, a
recent study using bioluminescent Brucella strains (Rajashekara et al., 2005) also found them to target the salivary glands, which could be of significance in relation to
human infection, where inoculation occurs through ingestion of contaminated food (Rajashekara et al., 2005). In
addition, mice presented chronic infection of tail joints
FEMS Microbiol Rev 36 (2012) 533–562
Brucella intracellular lifestyle
with Brucella resembling osteoarticular brucellosis in
humans (Rajashekara et al., 2005). The mouse model has
been particularly useful to characterize Brucella factors
that mediate intra-host survival and to understand the
immune response elicited during brucellosis that ultimately leads to development of chronic disease (reviewed
by Martirosyan et al., 2011).
An intracellular lifestyle
The most common portals of entry for Brucella in animals and humans are mucous membranes of the respiratory and digestive tracts, and in the natural host, also the
conjunctiva and membranes covering the sexual organs.
The cellular sites of entry remain poorly characterized.
Bacteria are eventually taken up by phagocytic cells and
reach the regional lymph nodes, leading to subsequent
systemic dissemination. Brucella can efficiently colonize
cells of the monocyte/macrophage lineage and replicate to
high numbers in the liver and spleen. In animals, Brucella
also multiply in mammary glands and reproductive
organs. In humans, any organ can become infected. Histopathology of the tissues from infected animals and
patients clearly shows that Brucella extensively replicate
within host cells. Much work remains to fully characterize
the cellular targets of Brucella during different stages of
the infection but it is clear that intracellular replication is
535
directly linked to Brucella pathogenicity. The progression
of brucellosis into a chronic disease in either animals or
humans is related to the ability of Brucella to persist for
prolonged periods within host cells and to resist the host
immune response (Martirosyan et al., 2011). Study of
Brucella interaction with host cells has mostly relied on in
vitro analysis using cultured murine, bovine or human
cells (Fig. 1) and more recently, the mouse experimental
model of brucellosis.
Macrophages
Macrophages have been shown to constitute an important
site for Brucella intracellular replication within tissues.
Despite the fact that over 90% of Brucella internalized by
macrophages are killed soon after phagocytosis, a few bacteria escape and establish an intracellular niche permissive
for replication without affecting the survival of these
phagocytic cells. Activated macrophages are more efficient
at killing intracellular Brucella (Jiang & Baldwin, 1993; Eze
et al., 2000; Sathiyaseelan et al., 2000). However, wild-type
virulent strains are still able to replicate, albeit at later
time points after infection (Baldwin & Goenka, 2006).
Comparison of different Brucella species shows efficient
survival and replication within human and murine macrophages for B. abortus, B. suis and B. melitensis. In the case
of the marine species B. ceti and B. pinnipedialis, some
Fig. 1. The various cell types of different hosts where Brucella establishes intracellular niches. Depiction of the main differences between the
intracellular replication niches set up by Brucella (Brucella abortus, Brucella melitensis or Brucella suis) within specific cell types originating from
different hosts: murine (top), human (middle) and natural hosts, mostly caprine and bovine (bottom). White ellipses containing question marks
refer to cells or compartment types that have not yet been described in the context of Brucella infection. ER-derived replicative niches are
established within murine macrophages infected with either opsonized or nonopsonized Brucella, murine bone marrow-derived DCs infected with
nonopsonized Brucella, trophoblast giant cells from infected pregnant mice, human epithelial cells and trophoblasts from infected goats and
cows. Although Brucella has been shown to replicate within bovine macrophage cell lines, human monocyte/macrophage cell lines and human
DCs, the respective intracellular niches remain uncharacterized in the case of infection with nonopsonized bacteria. In the human monocyte/
macrophage cell line THP1, opsonized Brucella have been shown to replicate in a non-ER vacuole that retains LAMP1. The VirB T4SS has been
shown to be required for intracellular replication within murine macrophages, murine DCs, human monocyte/macrophage cell lines, human DCs,
human epithelial cells and bovine macrophages. Lys, lysosomes; AP, autophagosome.
FEMS Microbiol Rev 36 (2012) 533–562
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
K. von Bargen et al.
536
strains were also able to replicate within human macrophages to the same level as B. melitensis, including a clinical isolate obtained from a patient with osteomyelitis,
whereas other strains were cleared within 48 h or were
not phagocytosed at all (Maquart et al., 2009). Interestingly, B. microti replicate more efficiently than the virulent
B. suis in human and murine macrophages and showed
enhanced lethality in mice (Jimenez de Bagues et al.,
2010). This emphasizes the high pathogenic potential of
these newly isolated Brucella species for humans.
Infection of macrophages is not only associated with
intracellular bacterial multiplication but also results in
interference with macrophage functions. In human
monocytes/macrophages, B. abortus inhibits the IFN-cinduced expression of the FccRI receptor and FccRI-mediated phagocytosis (Barrionuevo et al., 2011); in murine
macrophages, B. abortus lipopolysaccharide (LPS) reduces
their capacity for antigen presentation and subsequent
T cell activation (Forestier et al., 2000). Moreover, yet
unidentified Brucella factor(s) specifically act on human,
but not on murine, macrophage-like cells to inhibit TNFa expression (Caron et al., 1996).
Dendritic cells
Another important cellular target recently identified is the
dendritic cell (DC). Brucella was found to efficiently proliferate within human and murine DCs (Billard et al., 2005;
Salcedo et al., 2008). In vitro, Brucella intracellular replication within DCs results in reduction of DC susceptibility to
activating signals which may compromise their ability to
induce an appropriate immune response (Billard et al.,
2007; Salcedo et al., 2008) and might contribute to the
development of a chronic infection. However, Brucella
infection in these cells is not silent as there is induction of
low levels of pro-inflammatory cytokines and increased
expression of co-stimulatory molecules as well as MHC class
II on the surface of murine DCs (Salcedo et al., 2008). This
was also observed in human DCs (Billard et al., 2007; Zwerdling et al., 2008). However, Zwerdling et al. (2008) noted
high expression of surface co-stimulatory molecules following Brucella infection comparable to that of Escherichiacoliinfected DCs (but not pro-inflammatory cytokine secretion) at 24 h postinfection and therefore suggested that
Brucella induces significant activation of DCs. It is possible
that the induction of MHC class II and co-stimulatory molecules achieves maximum intensity at different time points
after infection with Brucella compared with E. coli. Consistent with this hypothesis, Billard et al. (2007) found lower
levels of DC activation at 48 h postinfection with Brucella
in contrast to E. coli. Alternatively, the differences between
these two studies could be due to the method of preparation of human DCs or the different cell densities used.
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
Because of the migratory properties of these cells, DCs
may contribute to systemic dissemination of Brucella during infection. In mice, infected DCs were observed in the
lung-draining mediastinal lymph nodes following intranasal inoculation with B. abortus (Archambaud et al., 2010).
When the number of alveolar macrophages was artificially
reduced prior to Brucella inoculation, slightly higher
numbers of pulmonary DCs were infected, resulting in
significant increase in replication within the lungs as well
as dissemination to the liver and spleen. These results
show that DCs are indeed an attractive niche for intracellular replication and that alveolar macrophages have an
important role not only as a replicative niche but also in
initial containment of the bacterial load in the lung. In
the absence of alveolar macrophages, both CD11b- and
CD103-positive lung DCs become activated following
infection, and there is significant recruitment of iNOSand TNFa-producing monocyte-derived inflammatory
DCs. Alveolar macrophages are then contributing to the
containment of the inflammatory response and reduction
of tissue damage in the lungs during Brucella pulmonary
infection. Further work is necessary to understand how
the interplay between Brucella and these two phagocytic
cells, macrophages and DCs, determines disease outcome
in vivo.
Using a murine intestinal loop infection model, DCs in
the subepithelial dome of ileal Peyer’s patches were also
found to efficiently internalize Brucella and could therefore constitute a portal of entry in the gut. These are
likely to be the recently identified LysoDCs, which secrete
high levels of lysozyme and have been shown to very efficiently capture Salmonella and dead cells (Lelouard et al.,
2010, 2011).
Trophoblasts
In animals, abortion is associated with a rapid proliferation of Brucella within the placenta. The presence of high
bacterial loads within placental trophoblasts ultimately
results in disruption of the placenta and infection of the
foetus (Anderson & Cheville, 1986; Meador & Deyoe,
1989). Trophoblasts are therefore a primary cellular target
for Brucella in the natural host. However, little is known
about the infectious process in these cells. In ruminants,
placental trophoblasts produce substantial amounts of
erythritol in the third trimester, which is a favoured carbon source of Brucella. Erythritol may therefore contribute to exacerbated replication of Brucella and subsequent
abortion or stillbirth of the infected foetus, predominantly occurring during the third trimester. Brucellainduced abortion in humans is not a frequent outcome of
brucellosis but is of medical concern in endemic regions
(Khan et al., 2001), suggesting that if Brucella can reach
FEMS Microbiol Rev 36 (2012) 533–562
Brucella intracellular lifestyle
the placenta in pregnant women, trophoblasts may also
constitute an important cellular niche. In vitro studies are
lacking, particularly regarding intracellular trafficking.
One study has demonstrated significant differences in the
ability of B. abortus to replicate within bovine trophoblastic cell lines (Samartino et al., 1994). Replication was
observed in cells corresponding to middle and late gestation but not early gestational stages, consistent with abortion being predominant in the third trimester. It would
be interesting to perform a comparative study with
human trophoblasts as abortion in humans, in contrast
to the situation in cattle, is mainly reported in the first
trimester of pregnancy.
Only a few studies have been carried out in the mouse
model using murine placenta or placental-derived cells.
In mice, transmission to foetus or high colonization of
the placenta is mainly observed following high inocula
doses. Brucella induces placental damage (Tobias et al.,
1993) and targets mouse giant trophoblasts (Kim et al.,
2005b). However, as there are important anatomical differences in placentation between ruminants and mice,
caution should be taken in comparing to abortion in the
natural host or humans.
Other cellular niches
Although poorly investigated to date, additional cell types
may be relevant in brucellosis. A rich amount of literature
describes the ability of Brucella to survive and replicate in
nonphagocytic cells such as fibroblasts and epithelial cells.
Human epithelial cell lines are often used for in vitro
studies of Brucella interaction with host cells. For example, human alveolar and bronchial epithelial cell lines
have been used in the context of understanding pulmonary brucellosis (Ferrero et al., 2009). In vivo, brucellae
can be found in uterine epithelial cells of experimentally
infected pregnant goats (Meador et al., 1988). Invasion of
epithelial cells is likely to be a relevant step in crossing
the mucosal barrier during initial infection but this
remains to be demonstrated.
Recent studies have looked at Brucella infection of
more peculiar cells such as human osteoblastic cell lines
(Delpino et al., 2009; Scian et al., 2011), which may be
relevant cellular targets during joint and bone complications. Infection of primary cell cultures of mouse astrocytes and microglia has also been carried out (Garcia
Samartino et al., 2010). These cells of the central nervous
system may be relevant in the context of neurobrucellosis
(McLean et al., 1992; Sohn et al., 2003). Moreover,
Brucella has been shown to survive within bovine and
human polymorphonuclear cells (PMNs) but data are still
limited. Brucella inhibits PMN degranulation preventing
release of antimicrobial hydrogen peroxide derivatives
FEMS Microbiol Rev 36 (2012) 533–562
537
(Riley & Robertson, 1984; Orduna et al., 1991; Iyankan &
Singh, 2002). Consistently, Brucella was shown to efficiently resist killing by rat and human PMNs as it fails to
induce a strong respiratory burst and degranulation in
these cells (Barquero-Calvo et al., 2007). Depletion of
PMNs has no significant effect on Brucella proliferation
in the mouse model of brucellosis (Barquero-Calvo et al.,
2007).
Brucella are generally regarded as intracellular pathogens. However, extracellular growth may also be of relevance during disease. Brucellae are well equipped to
withstand attacks of the mammalian arsenal of humoral
immunity including complement (Barquero-Calvo et al.,
2007). At 21 days postinfection of C57BL/6 mice, approximately one third of B. abortus is present not inside but
outside of spleen cells. In mice lacking functional B cells
(Igh6 / ), the ratio is inverse and just one-third of brucellae are intracellular, yet the bacterial loads in the spleen
in both mice strains are very similar (Rolan et al., 2009).
These high percentages of extracellular bacteria suggest
that bacterial growth outside of their cellular niches may
be important during certain stages of infection, at least in
the mouse model of brucellosis. However, the fact that
Brucella mutants that fail to replicate inside mammalian
cells or whose vacuoles become phagolysosomal are generally attenuated in animal models and the fact that
extensive analysis of infected animal tissues finds them to
be inside cells indicate the intracellular lifestyle to be a
key to successful establishment and/or maintenance of
infection.
Brucella intracellular trafficking
The early stages
The mechanism for Brucella entry into host cells remains
poorly characterized and some data are contentious.
However, the early interactions between Brucella and host
cells are decisive for intracellular survival. Both antibodyand complement-opsonized Brucella as well as nonopsonized Brucella survive and replicate within macrophages
(Baldwin & Goenka, 2006). In contrast, opsonized
Brucella fail to replicate in murine bone marrow-derived
DCs (S.P. Salcedo and J.P. Gorvel, unpublished results)
suggesting that in these cells, different uptake mechanisms
result in a different intracellular compartmentalization
and survival. Nonetheless, it is clear that Brucella can
enter host cells in the absence of opsonin receptors as
they can infect nonprofessional phagocytic cells such as
trophoblasts, fibroblasts and epithelial cells.
In the case of nonopsonized Brucella, entry into both
murine macrophages and human monocytes is mediated
by lipid rafts (Naroeni & Porte, 2002; Watarai et al.,
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
538
2002; Fig. 2). This process is dependent on PI3-kinase
and TLR4 (Guzman-Verri et al., 2001; Pei et al., 2008a)
and does not result in a significant activation of these
cells. Uptake of Brucella by human DCs was also shown
to be partially dependent on lipid rafts (Billard et al.,
2005). This mode of entry seems to contribute to intracellular survival within phagocytic cells. Engineered rough
mutants (lacking the O-polysaccharide of the LPS, see
below) that enter independently of lipid rafts fail to
reduce macrophage activation and are subsequently killed
(Porte et al., 2003; Rittig et al., 2003; Pei & Ficht, 2004).
These results suggest that the Brucella O-polysaccharide
(also known as O-antigen) mediates interaction with specific receptors at the cell surface and thereby dictates early
events in the maturation of Brucella-containing vacuoles
(BCVs). Alternatively, the O-polysaccharide could be
K. von Bargen et al.
modifying the fusogenic properties of the early BCV.
However, mutations in LPS and outer membrane proteins
are highly pleiotropic and affect multiple surface components making it difficult to interpret the specificity of the
phenotypes observed with these mutants. For example,
loss of the O-antigen may unmask other surface molecules that would act as PAMPs, which would trigger cell
signalling and induce cell death irrespective of the LPS
structure. Furthermore, uptake of the naturally rough
mutants B. ovis and B. canis is dependent on lipid rafts
(Martin-Martin et al., 2010) arguing against a role for the
O-polysaccharide in mediating entry in macrophages.
Brucella entry into host cells is also dependent on the
two-component regulator BvrR/BvrS, which controls the
expression of numerous genes including those affecting
the acylation status of the LPS lipid A as well as several
Fig. 2. Brucellae entry and intracellular trafficking in host cells. Eucaryotic molecules recruited to the BCV or necessary for its maturation are
summarized. Green zooms indicate bacterial factors implicated at each step. Brucellae transiently interact with different compartments of the
endocytic pathway. Avirulent mutants (e.g. lacking a functional VirB T4SS) are degraded in phagolysosomes, whereas wild-type Brucella strains
are capable of controlling fusion with late endosomes and lysosomes and instead interact with the ER exit sites and efficiently replicate in an ERderived phagosome.
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
FEMS Microbiol Rev 36 (2012) 533–562
Brucella intracellular lifestyle
outer membrane proteins, such as Omp3a and Omp3b
(Guzman-Verri et al., 2002; Manterola et al., 2005;
Lamontagne et al., 2007).
Two receptors have been proposed to mediate lipid
raft-dependent internalization of Brucella by macrophages: (1) the class A scavenger receptor (Kim et al., 2004b)
and (2) the cellular prion protein PrPc (Watarai et al.,
2003). The putative receptor PrPc was shown to bind the
Brucella heat-shock protein Hsp60. However, the specific
role of this receptor in Brucella infection remains controversial as a separate study failed to demonstrate its
involvement in entry of B. suis and B. abortus in human
monocytes (THP1), murine macrophages (J774 and
BMDM) or in vivo (Fontes et al., 2005). In both the case
of macrophages and epithelial cells, Brucella adhesion
seems to be mediated by eucaryotic receptors containing
sialic acid residues that interact with the Brucella surface
protein 41 (SP41) encoded by the ugpB locus (CastanedaRoldan et al., 2006). This locus encodes for a glycerol-3phosphate binding ABC transporter present in the outer
membrane of pathogenic and nonpathogenic bacteria.
Interestingly, Brucella was shown to bind the extracellular
matrix proteins fibronectin and vitronectin, which may
contribute to tissue colonization (Castaneda-Roldan et al.,
2004). Additional recent studies have implicated other
Brucella proteins in adhesion/internalization into nonprofessional phagocytes. A null mutant of the BMEI0216
gene (annotation in B. melitensis) has a severe entry deficiency in HeLa cells (Hernandez-Castro et al., 2008). The
B. abortus efp gene was also shown to have a defect in
internalization but no attenuation in macrophages or
in vivo (Iannino et al., 2012). In addition, the pathogenicity island Bab1_2009–2012 encoding a Brucella adhesin
has been identified. Deletion of the entire island leads to
a decrease in internalization in both HeLa cells and J774
macrophages and reduced adhesion to HeLa cells. More
interestingly, the mutant was not attenuated in mice
inoculated via the intraperitoneal route but displayed
reduced spleen colonization in mice inoculated by oral
gavage at 7 and 21 days postinfection (Czibener & Ugalde, 2012). This is the first adhesin identified for Brucella
that also displays a role in virulence in vivo.
In both phagocytic and nonphagocytic cells, Brucella
entry relies on the actin cytoskeleton and partially on the
microtubule network. The small GTPases Cdc42, Rac and
Rho are necessary for invasion and Cdc42 is directly
recruited and activated at the site of entry (Guzman-Verri
et al., 2001). In murine trophoblast giant cells, bacterial
entry was also dependent on ezrin (Watanabe et al., 2009),
a member of the ezrin-radixin-moesin family of proteins
that tether actin filaments to the plasma membrane.
Once inside cells, Brucella resides in a vacuole, designated the BCV, which interacts with components of the
FEMS Microbiol Rev 36 (2012) 533–562
539
endocytic and secretory pathways to ensure bacterial survival. The nascent BCVs undergo interactions with early
endosomes (Fig. 2), acquiring markers such as the tethering protein early endosomal antigen EEA1 and the GTPbinding protein Rab5 (Pizarro-Cerda et al., 1998b;
Chaves-Olarte et al., 2002; Celli et al., 2003). These interactions occur immediately after internalization and are
very transient. Early BCVs are enriched in cholesterol and
flotilin-1, a protein involved in lipid raft signalling associated with phagosome maturation and interaction with the
endocytic pathway (Arellano-Reynoso et al., 2005). The
Brucella cyclic ß-1,2-glucan has been proposed to modify
cholesterol-rich lipid rafts present on the BCV membrane
(Fig. 2) and control BCV maturation in both epithelial
cells and macrophages (Arellano-Reynoso et al., 2005).
Interestingly, it does not seem to play a role in virulence
in DCs suggesting the composition of the vacuolar membrane in these cells may be different (Salcedo et al., 2008).
Analysis of early stage trafficking, particularly in macrophages, is hampered by the fact that more than 90% of
bacteria are killed within the first hours of infection.
Therefore, the majority of BCVs will become phagolysosomal and bacteria will be degraded. In consequence, analysis of the remaining 10% that will actually successfully
establish a replicative niche will be difficult and will likely
require live cell imaging.
An intermediate stage
As BCVs lose early endosomal markers, they acquire the
late endosomal/lysosomal membrane protein LAMP1
(Fig. 2). Although multiple studies have shown that BCVs
(unlike latex-beads and heat-killed bacteria) do not
acquire most of the markers of late endosomes and lysosomes, live imaging in infected epithelial cells has recently
challenged this hypothesis (Starr et al., 2008). According
to this study, a significant proportion of BCVs are accessible to fluid phase markers preloaded to lysosomes,
although at a much reduced rate compared with heatkilled BCVs (Starr et al., 2008). Furthermore, BCVs
acquire late endocytic markers such as Rab7 and its effector the Rab-interacting lysosomal protein (RILP) and this
is necessary for further BCV trafficking. These results
demonstrate that maturation of BCVs does involve controlled and limited fusion events with late endosomes and
lysosomes, a step necessary for reaching the replicative
niche. Consistently, numerous studies using fixed samples
had previously shown that luminal lysosomal enzymes
such as cathepsin D cannot be detected on wild-type
BCVs, in contrast to heat-killed BCVs (Pizarro-Cerda
et al., 1998a; Comerci et al., 2001; Celli et al., 2003; Arellano-Reynoso et al., 2005), suggesting they are not extensively fusing with lysosomes.
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
540
An important phase in phagosome maturation is the
acidification of BCVs, an essential step for Brucella survival within host cells (Porte et al., 1999; Boschiroli et al.,
2002) and essential for diversion of intracellular trafficking (Starr et al., 2008). In fact, inhibition of BCV acidification at early stages of the phagosome maturation
(within 1 h) completely abolishes intracellular replication.
This is likely due to the fact that an acidic vacuolar pH
induces the expression of Brucella genes that are required
for virulence. For example, BCV acidification induces the
expression of the virB operon that encodes a type IV
secretion system (T4SS) crucial for the establishment of
the Brucella replication niche (Boschiroli et al., 2002).
The endoplasmic reticulum-derived vacuole:
the Brucella safe haven
Following transient fusion events with the endocytic pathway, BCVs undergo extensive interactions with the secretory pathway before finally fusing with the endoplasmic
reticulum (ER) (Fig. 2).
BCV interaction with endocytic compartments is followed by the acquisition of features of autophagosomes
in epithelial cells but not in macrophages: they become
multimembranous and positive for monodansylcadaverine
(Pizarro-Cerda et al., 1998a, b), although this compound
is likely rather a general marker for late endocytic/lysosomal and acidic compartments (Mizushima, 2004). This
transient passage through an autophagosome-like compartment is probably a consequence of fusion events with
autophagic vacuoles already present in the cell, perhaps
induced upon infection as an innate immune response to
clear the invading bacterial pathogen (Gorvel & de
Chastellier, 2005). Virulent Brucella strains, however, efficiently escape these autophagosomal compartments to
reach the ER.
Interestingly, an RNAi screen carried out in Drosophila
S2 cells identified 52 host factors that when inhibited
either increased or reduced intracellular replication of
Brucella, one of which has been related to autophagy
(Qin et al., 2008). Drosophila S2 cells cannot be incubated
above 30 °C and may therefore be problematic to study
pathogens requiring 37 °C for maximal expression of
genes involved in virulence. However, they have been
shown to be a powerful system to study Brucella entry
and replication, because the pathways involved are very
similar to those described for mammalian cells, including
dependency on Rho subfamily GTPases and PI3 kinase
for entry and replication occurring within compartments
positive for ER markers. Twenty-nine genes identified in
this ER-directed RNAi screen had never been correlated
to bacterial virulence. Interesting factors include specific
SNAREs, kinases, proteins related to cytoskeleton, chaperª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
K. von Bargen et al.
ones and biosynthetic or metabolic enzymes. Inhibition
of the inositol-requiring enzyme 1 (IRE1a), an important
kinase that regulates the host cell unfolded protein
response (UPR) and also controls autophagosome biogenesis, resulted in a strong reduction in Brucella survival in
both insect and mammalian cells. Because other kinases
of the UPR were not involved in control of Brucella replication, it has been proposed that IRE1a activation by
Brucella may induce the biogenesis of autophagosomes
from ribosomal-free regions of the ER, which would
interact with BCVs, modulate fusion events with lysosomes, and contribute to complete association with the
ER (Qin et al., 2008). Further work is now required to
test this hypothesis and determine the role of IRE1a during Brucella survival within phagocytic cells, for which no
autophagosome-derived BCV has yet been described.
Brucella replication in ER-derived vacuoles has been
described for all cell types analysed with the exception of
human monocytes, in which opsonized Brucella was
shown to replicate in ER-negative LAMP1-positive large
vacuoles containing multiple bacteria (Bellaire et al.,
2005). In murine immortalized macrophages, murine
bone marrow-derived macrophages and DCs as well as
human epithelial cell lines, BCVs acquire numerous proteins of the ER such as the lectin chaperones calnexin and
calreticulin as well as the ER protein translocator Sec61
(Pizarro-Cerda et al., 1998b; Celli et al., 2003). The vacuolar membrane presents numerous ribosomes visible by
electron microscopy, and the ER lumen enzyme, glucose
6-phosphatase can be found within BCVs. At this stage,
Brucella has reached a safe niche for intracellular persistence. Therefore, the ER is not only an important source
of membrane for replicating bacteria but also creates an
environment suited for Brucella replication. These late
BCVs behave as ‘extensions’ of the ER illustrated by the
fact that induction of ER vacuolation by treating cells
with the toxin aerolysin also results in vacuolation of
BCVs (Pizarro-Cerda et al., 1998b; Celli et al., 2003).
Importantly, Brucella can be found in ER-associated compartments in placental trophoblasts from tissue obtained
from B. abortus-infected cattle and goats (Anderson &
Cheville, 1986), highlighting the importance of the
ER-derived BCVs as an intracellular niche for infection
in vivo.
The molecular mechanisms that enable Brucella to initiate fusion with the ER still remain to be deciphered.
The initial contact with the ER is established at ER exit
sites, where BCVs interact with the Sar1/COPII complex.
Inhibition of the Sar1 activity, which results in disruption
of ER exit sites, blocks intracellular replication by preventing BCVs from fusing with the ER (Celli et al., 2005).
The Brucella proteins that mediate these interactions are
still unknown but there is clear evidence that the T4SS
FEMS Microbiol Rev 36 (2012) 533–562
Brucella intracellular lifestyle
encoded by the virB operon is necessary for sustained
interactions with the ER (Comerci et al., 2001; Celli et al.,
2003) (see below).
A recent proteomic approach to characterize the composition of the BCV membrane has revealed the role of
the small GTPase Rab2 in Brucella intracellular replication
(Fugier et al., 2009). Rab2 was recruited to BCVs, and
inhibition of Rab2 prevented the fusion of BCVs with
ER-derived vesicles and instead BCVs retained LAMP1.
Rab2 is known to interact with the glyceraldehyde-3phosphate dehydrogenase (GAPDH), the coat COPI
complex and the protein kinase C (PKC ι/k) to control
vesicular trafficking from the Golgi to the ER, via vesicular tubular clusters (Tisdale et al., 2004). All the members
of the GAPDH/COPI/Rab2/PKC ι/k complex are required
for Brucella intracellular replication, suggesting that
maturing BCVs are interacting with vesicular tubular
clusters and may intercept retrograde vesicular trafficking
(Fig. 2). It is therefore likely that the final maturation of
BCVs as well as their interaction with the ER is mediated
by specific Brucella effectors translocated across the BCV
membrane into host cells. Consistent with this hypothesis,
the Brucella-translocated RicA was recently identified in a
high-throughput yeast two-hybrid screen and shown to
interact with Rab2 (de Barsy et al., 2011). Further work is
now crucial to understand at the molecular level how this
Brucella protein in conjunction with other uncharacterized effectors directs BCV interactions with the secretory
pathway to sustain successful interactions with the ER
and ensure intracellular replication.
The knowledge of the trafficking events described
above represents a summary of numerous studies
using multiple cell lines infected with either B. abortus,
B. melitensis or B. suis strains. No analysis of the
intracellular compartment of any of the newly described
Brucella strains has yet been carried out. Brucella microti,
which is highly virulent in mice, has been shown to efficiently replicate within human and murine macrophages
(Jimenez de Bagues et al., 2010). Three lines of evidence
are indicative that the intracellular trafficking of B. microti phagosomes is equivalent to that of other described
Brucella strains (Hanna et al., 2011): (1) intracellular replication is dependent on the VirB T4SS, (2) the VirB
operon is induced in acidic media and (3) neutralization
of acidification during early steps of infection blocks
intracellular replication. However, the nature of B. microti
BCVs remains uncharacterized. It would be interesting to
investigate further the intracellular trafficking of these
strains and other more clinically relevant Brucella strains
such as B. ceti, B. pinnipedialis and B. inopinata.
Once Brucella reach their replicative niche within host
cells, extensive replication occurs without much disruption of host cell integrity. This is seen both in cell culture
FEMS Microbiol Rev 36 (2012) 533–562
541
and in infected animals. It remains completely unknown
what happens after reaching high bacterial numbers
within host cells; how bacteria exit host cells and how
cell-to-cell spread may occur. One possible hypothesis for
cell-to-cell spread may be the induction of cell death by
spontaneously occurring rough mutants (see below) but
this remains to be verified. It is also not clear whether
low replication observed in some cells may actually constitute an important reservoir for Brucella relevant during
chronic stages of infection. These are all important new
avenues of research that must be undertaken.
The problem of defining virulence in
Brucella and identifying factors
required for intracellular trafficking and
disease
The mechanistic basis for Brucella trafficking diversion is
so far poorly understood. Most studies screen for attenuated mutants based on intra-host replication/survival and
few control for actual defects in intracellular trafficking.
A major drawback in the search for factors involved in
the establishment of the intracellular niche is the fact that
killed brucellae are delivered to phagolysosomes. This
renders the distinction between the actual effects of mutations rather difficult. The reason that the vacuole of a
Brucella mutant is retained in lysosomes may be its deficiency in reaching the ER-derived intracellular niche.
However, particularly because the conditions in the early
BCV are acidic and proteolytic (Starr et al., 2008), it
might just be more sensitive to environmental stresses
such as reactive metabolites or, at later stages, auxotroph
for an essential nutrient lacking in the bacterial vacuole.
In such a scenario, the bacteria would be perfectly
equipped to establish a replicative niche in their host
cells, and the difference in trafficking to wild-type
Brucella would reflect merely bacterial death because of
nontrafficking-related events. For instance, the Brucella
membrane components phosphatidylcholine and phosphatidylethanolamine have been described to be involved
in intracellular survival (Conde-Alvarez et al., 2006; Bukata et al., 2008). BCVs of mutants of the phosphatidylcholine pathway show a higher acquisition of LAMP at
early time points in bone marrow-derived macrophages
which then progressively moves to wild-type levels
(Conde-Alvarez et al., 2006). Similarly, vacuoles of a
phosphatidylethanolamine-deficient mutant early acquire
and maintain LAMP-1 (Bukata et al., 2008). In both
cases, LAMP acquisition coincides with a more pronounced initial bacterial killing, which is followed by late
stage replication. However, some mutants of the phosphatidylcholine pathway are more sensitive to killing by
nonimmune serum and poly-L-ornithine (Conde-Alvarez
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
542
et al., 2006), and the B. abortus mutant of phosphatidylethanolamine is more sensitive to anionic detergents and
polycationic peptides (Bukata et al., 2008), both of which
seems to be related to general changes in membrane
properties. This increased sensitivity to macrophage antimicrobial actions complicates a differentiation. The mutation could result in an actual trafficking defect leading to
killing of the bacteria. Alternatively, their increased sensitivity to macrophage bactericidal mechanisms may lead to
enhanced phagolysosome formation of vacuoles containing dead Brucella.
Likewise, although Brucella strains found in phagolysosomes usually do not replicate, a lack of replication is not
indicative for their incapability to avoid lysosomal degradation. For instance, a mutant for nicotinamidase/pyrazinamidase of B. abortus does not replicate in HeLa cells
and macrophages. However, the mutant’s vacuole does
not fuse with lysosomes and bacterial replication can be
restored upon external addition of nicotinic acid to the
medium (Kim et al., 2004a). A spoT mutant of B. abortus
that is more sensitive to various stress conditions as compared with wild-type bacteria has a lower intracellular replication within macrophages but does not co-localize with
either late endosomes or lysosomes (Kim et al., 2005a).
Similar questions remain to be solved with respect to
the correlation between intracellular trafficking and disease. Brucella mutants whose vacuoles become phagolysosomal are generally attenuated in a mouse model (see
below) and factors involved in trafficking diversion crucial
for bacterial pathogenicity therefore usually defined as
virulence factors. However, although being widely used,
this classical concept of ‘virulence factors’ (Falkow, 1988;
Casadevall & Pirofski, 1999; Wassenaar & Gaastra, 2001)
is problematic in the analysis of bacterial pathogens such
as Brucella whose overall morphology and metabolism
have seen a long-term evolutionary adaptation to their
respective hosts (Seleem et al., 2008). Even though they
can be grown in culture medium, brucellae are usually not
found in a noninfectious context (Gorvel & Moreno,
2002), rendering it difficult to draw the line between genes
required for (intra-host) survival in general and actual
virulence genes. Unbiased screens for virulence determinants based on classical (Delrue et al., 2001; Kohler et al.,
2002) or signature-tagged transposon mutagenesis (STM)
(Lestrate et al., 2003) in the last decade have identified a
multitude of genes involved in metabolism, nutrition and
cell wall or inner and outer membrane constituents rather
than those homologous to known virulence factors (in
their various definitions including to cause host damage,
not to be essential for bacterial fitness or to be present
only in pathogenic species). This has led to the idea of
Brucella being a pathogen ‘without classic virulence genes’,
such as, for example, haemolytic toxins (Seleem et al.,
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
K. von Bargen et al.
2008). Although many open reading frames identified to
be crucial for intra-host survival have no homology to any
known bacterial protein and may code for factors
that match the concept of classical virulence factors
(Kohler et al., 2002; Lestrate et al., 2003; Wu et al., 2006),
adaptation processes might have led to overall optimization of morphology, metabolism and effector proteins
for survival in the host. Particularities of the cell envelope
or amino acid metabolism – although present in nonpathogenic species and vital for bacterial fitness – may
likewise serve to promote host invasion. An evolutionary
adaptation to its host ultimately leads to a bacterium
whose morphology, physiology and potential to cause disease form an inseparable entity. Accordingly, rather than
terming them virulence factors, for clarity, we will refer to
bacterial factors in regard to their particular functions and
effects such as on trafficking diversion or intracellular
multiplication.
The mutant as a mirror of intracellular
requirements
Although they may not serve to understand the mechanisms of how Brucella establish their intracellular niche,
the multitude of transport and metabolism-associated
genes (reviewed in Roop et al., 2009 and Barbier et al.,
2011) that have been found to mediate Brucella intracellular/intra-host survival and replication collectively draft
an indirect yet detailed picture of the environment
encountered by the bacteria during infection. For
instance, the high number of Brucella attenuated mutants
that are affected in carbohydrate metabolism or transport
suggests that these may serve as energy and/or carbon
source for intracellular brucellae (Foulongne et al., 2000;
Hong et al., 2000; Lestrate et al., 2000; Kohler et al.,
2002). Kinetic studies of regulation of carbon metabolism
genes suggest a constant process of adaptation to the
environmental changes in the BCV: enzymes of sugar
metabolism and uptake systems are downregulated early
in infection, whereas an increase in amino acid catabolism might open up an alternative carbon source for anabolic processes; the sugar-based catabolism might then be
reactivated once the replicative ER-derived niche is established (McKinney et al., 2000; Kohler et al., 2002; Barbier
et al., 2011). The requirement for several genes involved
in metabolism of different amino acids and nucleotide
synthesis sketches the picture of a nutrient-poor environment allowing for few to no auxotroph defects (Hong
et al., 2000; Foulongne et al., 2001; Kohler et al., 2002;
Kim et al., 2003; Lestrate et al., 2003). Successful infection additionally requires the presence of several ion
transporters such as the manganese transporter MntH
(Anderson et al., 2009).
FEMS Microbiol Rev 36 (2012) 533–562
Brucella intracellular lifestyle
The BCV seems to display a low oxygen tension.
Brucella mutants that are deficient in cytochromes that
are particularly efficient at low O2 concentrations (the
cbb3-type cytochrome c oxidase or cytochrome bd ubiquinol oxidase) are attenuated in cellular and animal infection models (Endley et al., 2001; Kohler et al., 2002), just
like Brucella lacking components of the denitrification
pathway (Kohler et al., 2002; Haine et al., 2006; LoiselMeyer et al., 2006) which may allow for the use of terminal electron acceptors different from O2. Alternatively (or
additionally), these proteins could be involved in scavenging and detoxification of host-derived reactive oxygen or
nitrogen species, respectively. Several attenuated mutants
suggest that Brucella is exposed to these bactericidal compounds in the course of infection such as a strain defective in a Cu–Zn superoxide dismutase (Tatum et al.,
1992; Gee et al., 2005) or with a double deletion in catalase (KatE) and peroxiredoxin (AhpC) (Steele et al.,
2010). Exposure to reactive intermediates may also
account for the requirement of DNA repair systems such
as RecA for survival in mice (Tatum et al., 1993).
These mutant-based indications of the Brucella intrahost environment have been complemented in recent
years by the determination of Brucella intracellular gene
expression profiles. The quorum sensor regulator BvrR/
BvrS controls intracellular expression of several genes
such as genes involved in carbon metabolism and denitrification and seems to be critically involved in the switch
from an extracellular to an intracellular lifestyle (Viadas
et al., 2010). Particularly, the analysis of early time points
of infection and in vivo has recently been improved by an
optimized methodology to obtain pure RNA from limited
amounts of intracellular Brucella (Rossetti et al., 2010).
Brucella factors involved in
establishment of the intracellular niche
The type IV secretion system VirB
Perhaps one of the most studied factors required for
Brucella trafficking diversion is the T4SS encoded by the
VirB operon (de Jong & Tsolis, 2012). The VirB genes are
homologous to those of type IV DNA transfer systems
known from other Gram-negative bacteria such as
A. tumefaciens or Legionella pneumophila (Alvarez-Martinez & Christie, 2009). T4SS are classified in two subgroups, IVA and IVB. The latter is closely related to
conjugation systems and includes those of pathogens such
as L. pneumophila and Coxiella burnetii. The Brucella VirB
T4SS apparatus, which belongs to the subgroup IVA, was
first identified in B. suis and is encoded by 12 open reading frames (virB1 to virB12) on chromosome II (O’Callaghan et al., 1999). It is essential to the virulence of all
FEMS Microbiol Rev 36 (2012) 533–562
543
Brucella strains investigated in their respective models
including the more recently described species B. microti
(Hanna et al., 2011). As mentioned earlier, the VirB
apparatus is crucial for the intracellular trafficking of
Brucella within professional phagocytes and nonprofessional phagocytes including macrophages, DCs and epithelial cells (Fig.1, O’Callaghan et al., 1999; Foulongne
et al., 2000; Sieira et al., 2000; Comerci et al., 2001; Delrue et al., 2001; Celli et al., 2003; Kim et al., 2003; Billard
et al., 2005; Rajashekara et al., 2006; den Hartigh et al.,
2008; Salcedo et al., 2008). BCVs of VirB deficient strains
are unable to sustain interaction with the ER. The vacuoles of most of these mutants eventually fuse with lysosomes where they are degraded (Sieira et al., 2000;
Comerci et al., 2001; Delrue et al., 2001; Celli et al., 2003;
Salcedo et al., 2008; Starr et al., 2008). Interestingly, a
polar mutant of virB10 that lacks transcription of downstream genes is degraded in phagolysosomes, whereas a
nonpolar mutant of virB10 is recycled to the cell surface
(Comerci et al., 2001), suggesting that the escape from
the degradative pathway and establishment of the
ER-derived niche for intracellular replication are two distinct events mediated by different mechanisms (Comerci
et al., 2001).
The inability of virB mutants to establish an intracellular replicative niche reflects their attenuation in the
mouse infection model (Hong et al., 2000; Lestrate et al.,
2000; Sieira et al., 2000; Kahl-McDonagh & Ficht, 2006;
Rajashekara et al., 2006) and in goats (Kahl-McDonagh
et al., 2006; Zygmunt et al., 2006). After infection by
gavage, virB-deficient B. melitensis present lower bacterial
numbers in liver, spleen and intestinal tissues (Paixao
et al., 2009). Bypassing the digestive tract using intraperitoneal injection, virB-deficient strains are able to disseminate to lymph nodes, liver and spleen (Rajashekara et al.,
2005; Rolan & Tsolis, 2007; Paixao et al., 2009). However,
after the initial phase, Brucella virB mutant strains are
cleared much faster than wild-type bacteria, suggesting
defects in persistence to maintain chronic infection. The
difference in survival of virB mutants within infected cells
in vitro (eliminated in 24 h) and in vivo (persistence
equivalent to wild type for the first few days) could reflect
different bacterial localization during mouse infections
(e.g. intra- vs. extracellular) or that cells that are able to
kill in vitro cannot do so in vivo because, for example,
they are regulated by other cells or a particular cytokine
environment.
In contrast to wild-type brucellae, virB mutants with a
deficient T4SS induce essentially no transcriptional
changes in spleen cells. They do not induce inflammation-related genes and the infection remains quiescent,
suggesting that T4SS function triggers innate immune
responses (Roux et al., 2007). However, the level of
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
K. von Bargen et al.
544
response is much lower than that induced by pathogens
such as Salmonella (Barquero-Calvo et al., 2007), underlining the immune evasive strategies of Brucella.
The regulation of the virB operon expression correlates
to its virulence functions. The B. suis virB operon is
expressed maximally in minimal medium (rather than
rich medium) at early exponential phase, at temperature
of 37 °C and inside of the host cell (Boschiroli et al.,
2002). In all Brucella species investigated, an induction of
VirB protein expression is observed in response to an
acidic environment, and this environmental stimulus
seems to account for the major part of induction
observed intracellularly (Boschiroli et al., 2002; Rouot
et al., 2003). The initial acidification of phagosomes
which is essential for B. suis intramacrophage replication
(Porte et al., 1999) is required to induce VirB expression.
Interestingly, there seem to be some differences in the
regulation of VirB expression among Brucella strains:
whereas B. abortus, B. melitensis and B. ovis express VirB
at neutral pH in a rich medium, B. suis, B. canis and the
vaccine strains S19, RB51 (both derived from B. abortus)
and Rev1 (derived from B. melitensis) show little to no
VirB protein expression in these conditions (Rouot et al.,
2003).
Transcriptional regulators of VirB synthesis are the
quorum-sensing regulators VjbR (Delrue et al., 2005) and
BlxR (Rambow-Larsen et al., 2008), the histidine utilization regulator HutC (Sieira et al., 2010), the transcription
factors DeoR, AraC8, AraC2, GntR4 and NolR (Haine
et al., 2005), the two-component system (TCS) BvrR/BvrS
(Martinez-Nunez et al., 2010), the stringent response regulator encoded by Rsh (Dozot et al., 2006) (see below)
and the integration host factor that specifically interacts
with the virB promoter during intracellular and vegetative
growth (Sieira et al., 2004).
Recent findings suggest that apart from being subject
to regulation, mutation of VirB may itself affect expression of other genes. Relative transcription levels of several
genes including the quorum-sensing regulator VjbR are
downregulated in a B. melitensis VirB mutant during
growth in culture and inside macrophages (Wang et al.,
2009). Differential expression at transcriptional and posttranscriptional levels can also be observed with several
outer membrane proteins, which may explain higher sensitivity of virB mutants to polymyxin B and several environmental stresses (Wang et al., 2010).
Substrates of the T4SS
Bacterial proteins (‘effectors’) translocated into the host
cell are important elements of T4SS in the intracellular
survival of pathogens. Although the VirB Brucella T4SS
was first described more than 10 years ago, few potential
effector proteins have been identified and only one for
which a function has been ascribed (Table 1). Two proteins that are translocated into the macrophage cytoplasm
in a T4SS-dependent manner are VceA and VceC (de
Jong et al., 2008). Translocation of VceA and VceC fused
with the TEM1 b-lactamase (N-terminus) into mouse
macrophage-like J774 cells can be detected from 7 h
onwards and is dependent on the last C-terminal 20
amino acids, which contain a motif reminiscent of the
secretion signal identified in Agrobacterium T4SS substrates. The open reading frames of VceA and VceC are
conserved in all Brucella species sequenced, including
B. suis, canis, ovis, abortus and melitensis (de Jong et al.,
Table 1. Brucella proteins translocated into host cells during infection
Name
ORF*
VceA
BAB1_1652/BMEI0390
VceC
BAB1_1058/BMEI0948
RicA
BAB1_1279/BMEI0736
BPE123
BAB2_0123/BMEII1111
BPE043
BPE005
BPE275
BPE865
BPE159
BAB1_1043/BMEI0961
BAB1_2005/BMEI0067
BAB1_1275/BMEI0739
BAB1_1865/BMEI0198
BAB2_0159/BMEII1079
Signals required for
secretion/translocation
T4SS
translocation
Tag used
Function
Reference
C terminus required for
translocation
N-terminus Sec signal
present
C terminus required for
translocation
nd
Yes
TEM1
Unknown
de Jong et al. (2008)
Yes
TEM1
Unknown
Yes
TEM1
Yes
CyA and 3FLAG
Interacts
with Rab2
Unknown
Yes
Yes
Yes
No
No
CyA
CyA
CyA
CyA
CyA
Unknown
Unknown
Unknown
Unknown
Unknown
N-terminus required for
translocation (Sec signal)
nd
nd
nd
nd
nd
de Barsy et al. (2011)
Marchesini et al. (2011)
*ORF nomenclature is given for the genomes of Brucella abortus 2308 (BAB) and B. melitensis 16M (BME).
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
FEMS Microbiol Rev 36 (2012) 533–562
Brucella intracellular lifestyle
2008). VceA and VceC are co-regulated with the T4SS as
they belong to the VjbR regulon (de Jong et al., 2008),
which controls expression of several virulence-associated
factors including VirB. The cellular targets and the functions of VceA and VceC remain unknown.
The third Brucella protein found to be translocated into
the cytoplasm of RAW264.7 macrophages is RicA (de
Barsy et al., 2011). It has been identified because of its
interaction with the guanosine diphosphate (GDP)-bound
form of the eucaryotic protein Rab2, a small GTPase that
is crucial for Brucella intracellular replication (Fugier
et al., 2009). A B. abortus ricA deletion mutant replicates
faster in HeLa cells and shows an accelerated loss of
LAMP1 from its BCVs than wild-type bacteria. The ricA
mutant resides in BCVs, which recruit less GTP-locked
Rab2 (de Barsy et al., 2011) suggesting that RicA plays a
role in control of BCV maturation. However, the fact that
inhibition of Rab2 blocks intracellular replication but the
ricA mutant is not attenuated in virulence suggests other
effector proteins are involved in the control of Rab2 function. At this stage, it is not clear what is the activity of
RicA on Rab2. Results indicate that RicA does not act as a
guanine nucleotide exchange factor catalysing the replacement of GDP by GTP on Rab GTPases. Because RicA
interacts with the GDP-bound form of Rab2, it is unlikely
to be a GTPase-activating protein (GAP) that stimulates
GTP hydrolysis by converting the GTPase back to its
GDP-bound form. It is possible that RicA is functioning
as a guanine nucleotide dissociation inhibitor (GDI) that
stabilizes the inactive Rab form by preventing GDP dissociation or GDI displacement factor that removes GDI and
allows membrane insertion of Rab2 by its geranylgeranyl
anchor. Alternatively, RicA could be interacting with Rab2
without having a direct control on its activity.
Translocation of TEM1-RicA is observed in the wildtype strain but not in the virB mutant and only 24 h after
infection of RAW264.7 macrophages. At this time point,
there is strong attenuation of virB mutants, which are in
very different compartments than the virulent strain, as
they are degraded in lysosomes. This could account for
the lack of translocation of TEM1-RicA in the virB
mutant. Interestingly, there is no obvious C-terminal
motif like for VceA and VceC, and secretion of RicA into
the culture media was independent of the T4SS. It is possible that the in vitro secretion assay used in this study
induces an alternative pathway for effector proteins to
cross the bacterial membranes. Another explanation could
be that the secretion across the bacterial membranes is
independent of VirB and uncoupled from the translocation across the vacuolar membrane. This has recently been
shown to occur in the case of T3SS (Akopyan et al.,
2011). Alternatively, RicA may not be a T4SS effector.
Additional work is now necessary to test these hypotheses.
FEMS Microbiol Rev 36 (2012) 533–562
545
An in silico screen has recently identified four additional substrates of the VirB T4SS (Marchesini et al.,
2011). Furthermore, it identified two Brucella proteins
that are translocated into macrophages in a VirB-independent manner suggesting that Brucella has another
secretion system yet to be identified. Because of the structural similarities of bacterial export machineries and flagella, it has been speculated that in Brucella (normally
considered nonmotile), the flagella genes may serve as a
secretion apparatus rather than as an organelle mediating
bacterial movement (Lestrate et al., 2003). Moreover, the
co-regulation of flagellum components with the VirB
secretion system and the attenuation of a flagellum
mutant during persistence of B. melitensis in the mouse
model of infection suggest a role for this organelle in
Brucella pathogenicity (Delrue et al., 2005; Fretin et al.,
2005). However, it remains to be demonstrated if these
proteins are translocated via the flagella system.
Translocation of all proteins identified in the screen
was assayed 5 h after infection of murine macrophages,
using C-terminal fusions with the adenylate cyclase reporter CyA from Bordetella pertussis. In the case of the T4SS
substrate BPE123-CyA, a protein with no predicted function, the concentration of cAMP peaked between 2 and
5 h postinfection consistent with maximal activation of
VirB (Sieira et al., 2004). In murine bone marrow-derived
macrophages, a 3xFLAG-tagged BPE123 was found in the
proximity of BCVs at 4 h after infection. In contrast with
VceA and VceC, translocation of BPE123 was dependent
on the N-terminal 25 amino acids, which contain a Sec
secretion signal. Although no obvious C-terminal motif
was identified, BPE123 contains several positively charged
amino acids that could constitute a signal for the T4SS. It
is curious that for some substrates (e.g. BPE123), translocation by the VirB T4SS would be coupled to Sec-dependent secretion but not for all effectors (e.g. VceA and
VceC). T4SS substrates of other bacteria have been shown
to depend on a two-step translocation, including the pertussis toxin which contains a sec-dependent signal to
cross the inner membrane (Gauthier et al., 2003) and
some substrates in Agrobacterium that lack a signal peptide but form a soluble complex with VirJ in the periplasm, which then enables further interaction with T4SS
components and translocation across the bacterial outer
membrane and host cell membrane (Pantoja et al., 2002).
Other potential translocated effectors
Although Brucella induces a minimal inflammatory
response in the host, it has acquired proteins that help
modulate host innate and adaptive immune response
mechanisms and which may therefore be important in the
development of a chronic infection. One such interesting
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
K. von Bargen et al.
546
protein contains a Toll/interleukin-1 receptor domain
(TIR), which is essential in TLR signalling. It has been
designated Btp1 for B. abortus (Salcedo et al., 2008) and
TcpB for B. melitensis (Cirl et al., 2008) but for clarity we
will refer to it as Btp1 (Brucella TIR-containing protein 1).
This nomenclature is in our opinion more appropriate
because of the presence of a second TIR-containing protein in the genome of Brucella (Btp2; Salcedo and Gorvel,
unpublished results). Btp1 does not have a role in trafficking but is seems to contribute to infection by manipulating intracellular host pathways. Btp1 has been shown to
interfere with TLR4- and TLR2-mediated NF-jB activation as well as cytokine secretion (Cirl et al., 2008; Salcedo
et al., 2008; Radhakrishnan et al., 2009). Although one
study suggested that Btp1 interacted with Myd88 (Cirl
et al., 2008), there is also good evidence that it targets the
adaptor protein TIRAP/MAL required for both TLR2 and
TLR4 signalling. A more recent study describes a stronger
interaction between Btp1 and Myd88 when compared
with TIRAP and that Btp1 specifically targets the death
domain of Myd88 (Chaudhary et al., 2011). Differences
observed by various research groups regarding the eucaryotic target of Btp1 may simply reflect the different methodology used, and it is now essential to determine what is
the ‘real’ target of Btp1 during infection. Btp1 binds phosphoinositides similarly to TIRAP, so it may be targeted to
the plasma membrane and interfere with the Myd88-TIRAP complex (Radhakrishnan et al., 2009) where it can
induce ubiquitination and degradation of TIRAP (Sengupta et al., 2010). More recently, Btp1 was shown to modulate microtubule dynamics. Btp1 stabilizes polymerized
microtubules and it enhances their rate of nucleation and
polymerization (Radhakrishnan et al., 2011). However,
most of the work regarding Btp1 has been performed
in vitro, by ectopically expressing the protein in host cells.
It is now important to determine the cellular localization
of translocated Btp1 and its molecular interactions during
infection to confirm these hypotheses. During Brucella
infection, Btp1 has been shown to contribute to the modulation of TLR2-dependent activation of murine bone
marrow-derived DCs (Salcedo et al., 2008) and to
decrease TIRAP degradation in macrophage-like J774 cells
(Sengupta et al., 2010). Although the btp1 mutant is attenuated neither in any of the cell systems tested so far (cultured macrophages, epithelial cells and DCs) nor in
immune competent intraperitoneally inoculated mice, it
plays a role in control of the immune response during
infection. In intranasally infected mice, specific subsets of
lung DCs showed higher maturation levels when infected
with the btp1 mutant compared with the wild-type strain
(C. Archambaud, S.P. Salcedo and J.P. Gorvel, unpublished results). It will be interesting to determine whether
Btp1 is a substrate for the VirB T4SS and whether it has a
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
role in control of microtubules or microtubule-dependent
vesicular trafficking during infection.
One poorly characterized candidate effector is BvfA, a
small protein of 11 kDa which is unique to the genus
Brucella (Lavigne et al., 2005). It has been identified in a
random screen using the Yersinia YopP as a reporter system, which induces apoptosis when the fusion protein is
translocated into the host cytosol. Although the function
of BvfA is unknown, it is necessary for intracellular survival within both human and murine macrophages, and
bvfA mutants are highly attenuated in mice. Interestingly,
the promoter of BvfA is induced intracellularly upon
acidification but it is unclear whether this small protein
predicted to be periplasmic is translocated into host cells.
It would be worth re-analysing the translocation of BvfA
into host cells using the TEM1 or CyA reporter system as
it may be another substrate for the VirB T4SS. Its specific
role during intracellular trafficking needs to be investigated.
Brucella LPS
Being a Gram-negative bacterium, Brucella is enclosed by
an outer membrane containing LPS. LPS molecules consist of three major subcomponents (Fig. 3): (1) a lipid A,
which serves to anchor the molecule in the outer membrane, (2) a sugar-based outer and inner core and (3) a
chain of several sugar molecules, the O-polysaccharide
that reaches into the extracellular space (Haag et al.,
2010). Brucella strains naturally occur as strains with
Smooth LPS that contains a terminal O-polysaccharide or
Rough strains with LPS that is lacking the O-polysaccharide (Haag et al., 2010). Brucella strains found in human
infections are generally characterized by a Smooth LPS
phenotype.
The outer membrane is the first site of contact between
eucaryotic cell and bacterium. In the constant co-evolution of host and pathogen, LPS compounds have become
a major pathogen-associated pattern (PAMP) recognized
by innate immune cells to mount an efficient antibacterial
response. The LPS of Brucella, however, has evolved to
avoid these mechanisms. Brucella LPS reduces binding of
complement to the bacterial surface, it does not induce
pro-inflammatory responses in mice as indicated by no
change in blood leucocyte numbers, absence of recruitment of polymorphonuclear neutrophils to the site of
infection, low levels of pro-inflammatory cytokines and
low cytotoxic activity (Moreno et al., 1981; BarqueroCalvo et al., 2007) which might represent a specific adaption to the intracellular lifestyle of this pathogen.
The structure of Brucella LPS differs from the canonical
pattern (Moreno & Moriyon, 2006). Its nonclassic lipid A
possesses a diaminoglucose backbone and very long chain
FEMS Microbiol Rev 36 (2012) 533–562
Brucella intracellular lifestyle
547
Fig. 3. Brucella LPS. The O-polysaccharide of Brucella abortus is composed of homopolymers of a-1,2-linked 4,6-dideoxy-4-formamido-a-Dmannopyranosyl (formyl-perosamine) subunits with an average chain length of 96 to100 subunits. The oligosaccharide core is still poorly
characterized. It contains glucose, mannose, quinovosamine, glucosamine, 3-deoxy-D-manno-2-octulosonic acid (Kdo) and several unidentified
sugars. Unlike other members of a-Proteobacteria, it lacks heptose, phosphates, galucturonic and glucuronic acid. The lipid A of Brucella is
considered nonclassical because of the following particular features: (1) backbone is composed of 2,3-diamino-2,3-dideoxy-D-glucose
(diaminoglucose) instead of glucosamine present in classical lipid A; (2) all acyl substitutions are in amide linkages; (3) sugar backbone is modified
with unusually very long chain hydroxylated fatty acids. These remarkable features are highlighted in red boxes in the figure.
fatty acid groups (Iriarte et al., 2004; Fig. 3). These particular features seem to be crucial for Brucella-specific
infection responses. In contrast to the lipid A of other
Gram-negative bacteria, that of Brucella is not efficiently
recognized by TLR4 (Lapaque et al., 2006). Infection with
a Brucella bacA mutant that is deficient in the very long
chain fatty acid content of lipid A results in higher
inflammation and the mutant is attenuated in mice (Ferguson et al., 2004; Parent et al., 2007). Likewise, the
increased amounts of underacylated lipid A species in a
Brucella bvrRS mutant (Manterola et al., 2005) may partially account for their attenuation in the mouse model of
infection (Sola-Landa et al., 1998). Brucella LPS interferes
with the MHCII-dependent antigen processing machinery
of macrophages by clustering with MHCII molecules,
likely resulting in a downregulated T cell activation
(Forestier et al., 1999, 2000).
The LPS O-polysaccharide crucially impacts outcome
of infection with B. abortus, B. melitensis and B. suis.
Strains bearing mutations in several genes involved in
FEMS Microbiol Rev 36 (2012) 533–562
various stages of the LPS biosynthesis pathway such as
the mannosyltransferase wbdA (Kohler et al., 2002) or the
phosphomannomutase manB (Allen et al., 1998; Foulongne et al., 2000; Kohler et al., 2002) and whose mutation
generally result in a ‘Rough’ phenotype are avirulent in
cellular or animal infection models (Roop et al., 1991;
Cheville et al., 1992; Winter et al., 1996; Allen et al.,
1998; Elzer et al., 1998; Godfroid et al., 1998; McQuiston
et al., 1999; Foulongne et al., 2000; Hong et al., 2000;
Ugalde et al., 2000; Kohler et al., 2002; Lestrate et al.,
2003). The degree of this attenuation depends on the particular gene and pathway affected. For instance, mutants
with defective O-polysaccharide are able to replicate to
varying levels, whereas those defective in synthesis of the
LPS core are more sensitive to macrophage killing (Gonzalez et al., 2008). However, it has been suggested that the
decrease in intra-macrophage survival of Rough mutants
is not because of general defects in survival and persistence abilities but because of their cytotoxic potential
which results in destruction of their replicative niche. The
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
K. von Bargen et al.
548
attenuation in vivo may therefore result from Rough
strain cytotoxicity as well as several changes in strain
infection biology. Rough Brucella strains induce more
cytokine and chemokine production than their Smooth
counterparts (Rittig et al., 2003). They induce maturation
of human DCs (Billard et al., 2007), and their BCVs fuse
more frequently with lysosomes (Porte et al., 2003) but
also with neighbour phagosomes to form large communal
vacuoles which cannot be observed with Smooth strain
brucellae (Rittig et al., 2003). These differences in trafficking may result from a different mode of attachment and
subsequent uptake. Brucella strains with Rough LPS show
an increased attachment to and uptake into monocytes
and macrophages (Rittig et al., 2003; Pei & Ficht, 2004),
indicating that they may enter cells upon interaction with
receptors different from those utilized during uptake of
Smooth strains. A more efficient uptake into cells can
also be observed in vivo: Smooth Brucella remain sensitive
to Gentamicin treatment up to 24 h postintraperitoneal
injection, whereas Rough strains are insensitive to the
antibiotic and therefore completely internalized into cells
as early as 1 h postinfection (Turse et al., 2011). Brucella
strains with a Smooth LPS are thought to be internalized
upon interaction with lipid rafts which essentially determines their ability for short-term intracellular survival,
whereas short-term survival of Rough B. suis is not
dependent on interaction with lipid rafts (Naroeni et al.,
2001; Porte et al., 2003).
The spontaneous appearance of Rough mutants
derived from Smooth Brucella strains has been known
for a long time. This conversion from Smooth to Rough
LPS occurs in culture but also during animal infection,
likely due to integration of the unstable genetic element
genomic island 2 (Mancilla et al., 2010). A spontaneous
mutation resulting in a Rough phenotype can be
observed 100–1000 times more frequently than spontaneous Brucella pyrimidine auxotrophs (Turse et al., 2011).
The fact that Rough strains are eliminated by the host
quickly and efficiently raises the question as to why, over
time and under these selective pressures, Brucella maintains an intrinsic instability of one of its most essential
means of intra-host survival. One explanation would be
that LPS conversion actually serves a function during
infection that, in the long run, favours bacterial shortterm survival and/or persistence (e.g. as described below:
Brucella and host cell death). However, because so far
there exist no experimental means to suppress LPS conversion, its actual role during animal infection remains
to be investigated.
Although a Smooth LPS seems to be a major determinant of intra-host survival for most Brucella strains, the
two species B. canis and B. ovis naturally lack the O-polysaccharide yet cause infection in their respective hosts
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
(dogs or sheep and rams). As with in vitro generated
Rough mutants, these naturally Rough strains are internalized more efficiently into host cells than Smooth brucellae without the requirement of opsonization (Detilleux
et al., 1990; Fernandez-Prada et al., 2003; Rittig et al.,
2003; Chen & He, 2009; Ferrero et al., 2009). Their intracellular multiplication is reduced compared with that of
Smooth Brucella strains (Detilleux et al., 1990; Rittig
et al., 2003; Ferrero et al., 2009; Martin-Martin et al.,
2009) and in the infection models used; phagosomes containing B. canis and B. ovis fuse more frequently with
lysosomes than those of Smooth Brucella strains (Porte
et al., 2003; Rittig et al., 2003). In contrast to Rough
mutants of B. abortus or melitensis, the naturally Rough
strains B. ovis or canis have no cytopathic effects (Pei &
Ficht, 2004).
Cyclic glucan
Like their relatives Agrobacterium and Rhizobium, Brucella
strains produce periplasmic cyclic ß-1,2-glucans (CßG)
(Ugalde, 1999). In plant pathogens, CßG has been
described as a factor essential for endosymbiontic invasion of nodules by Rhizobium meliloti or tumour induction by A. tumefaciens, respectively (Breedveld et al.,
1994). A central enzyme in Brucella CßG synthesis is cyclic glucan synthetase (Cgs) (Inon de Iannino et al., 1998).
Brucella strains defective in CßG synthesis have a
decreased survival in mice and do not replicate in HeLa
cells and mouse peritoneal macrophages (Briones et al.,
2001). Interestingly, cyclic glucan is not required for
Brucella replication in DCs (Salcedo et al., 2008).
The functions of cyclic glucan in bacterial physiology
and during infection are not yet fully understood. Cyclic
b-1,2-glucan mutants of Agrobacterium have multiple
altered cell surface properties including loss of motility as
a result of defective flagellum assembly (Breedveld et al.,
1994). Likewise, Brucella cgs mutants seem to have general
defects in their membranes, as suggested by their higher
sensitivity to surface-active molecules (Briones et al.,
2001). However, although a cgs mutant of the B. abortus
wild-type strain or of the attenuated vaccination strain
B. abortus S19 shows the same sensitivity against surfaceactive reagents, respectively, the former is much less
attenuated than the latter, suggesting that membrane
alterations are not the main cause for the decreased persistence (Briones et al., 2001).
During infections, cyclic glucan might be released from
periplasmic space via outer membrane vesicles (Briones
et al., 2001), allowing its interaction with host cell components. It has been proposed that it targets cholesterolrich lipid rafts found on the BCV membrane to control
the vacuole’s interactions with the endocytic pathway
FEMS Microbiol Rev 36 (2012) 533–562
Brucella intracellular lifestyle
549
(Arellano-Reynoso et al., 2005). Consistent with this
hypothesis, purified Brucella CßG was shown to extract
cholesterol from eucaryotic membranes. Furthermore,
CßG enhanced recruitment of flotillin-1 to BCVs, a signalling molecule characteristic of lipid rafts involved in
control of phagosome maturation. Brucella CßG mutants
reside in vacuoles that fuse with lysosomes where the bacteria are degraded (Arellano-Reynoso et al., 2005). This
trafficking defect is restored when the mutant strain is
grown with purified CßG prior to infection (ArellanoReynoso et al., 2005).
Brucella regulation of gene expression
Bacterial genes mediating intra-host survival and intracellular trafficking diversion are usually subject to complex
regulatory networks orchestrating gene expression in the
required amount at the appropriate phase of infection.
Several gene regulatory systems of Brucella belonging to
different protein families have been correlated to infection
success:
•
Two-component regulatory systems (TCS). These systems represent a common bacterial mechanism to
adapt to environmental changes. A transmembrane
sensor kinase is reacting by autophosphorylation to a
particular extracellular signal, for example, a drop in
pH or an increase in temperature TCS; the phosphate
group is then transferred to one or several response
regulators that mediate the change in transcription
(Casino et al., 2010).
TCS mediating Brucella intra-host replication include
FeuQ, NtrY, VsrB, OmpR and BvrR/BvrS. FeuQ
encodes the putative sensor kinase of the FeuP/FeuQ
system (Lestrate et al., 2003) that mediates the regulation of high affinity iron uptake in Rhizobium leguminosarum (Yeoman et al., 1997). However, a B. suis
mutant of the corresponding feuP response regulator
replicates on wild-type levels in mice and macrophages
(Dorrell et al., 1998), suggesting either species-specific
differences (melitensis vs. suis) or that FeuP is not the
only regulator responding to feuQ signalling (Lestrate
et al., 2003). Additional TCS regulating Brucella virulence are the putative nitrogen responsive factor NtrY
(B. suis, Foulongne et al., 2000), VsrB (B. melitensis,
Lestrate et al., 2000) and OmpR (B. melitensis, Wu
et al., 2006).
One of the best studied Brucella TCS is the BvrR/BvrS
system which was also one of the first global gene
expression regulator systems involved in Brucella virulence identified (Sola-Landa et al., 1998). Mutants of
this TCS have a Smooth type LPS yet they do not persist in mouse spleens, they poorly invade and do not
FEMS Microbiol Rev 36 (2012) 533–562
•
replicate in macrophages and HeLa cells, their vacuoles
show enhanced fusion with lysosomes and they have a
decreased resistance to antimicrobial peptide polymyxin B (Sola-Landa et al., 1998) which might result
from changes in the outer membrane (Manterola et al.,
2005). Interestingly, there is a small but significant difference between the residual persistence of the bvrR
and the bvrS mutant in mice (Sola-Landa et al., 1998),
suggesting that the lack of the sensor protein results in
a more severe phenotype. In contrast to wild-type bacteria, bvrS mutants do not recruit the small GTPases
of the Rho subfamily essential for actin polymerization
and they do not activate Cdc42 Brucella that are all
crucial for Brucella uptake into cells (Guzman-Verri
et al., 2001). BvrR/BvrS regulates expression of more
than 100 genes including those involved in cell envelope modulation, carbon and nitrogen metabolism,
and factors that have been shown to be involved in
virulence such as the VirB T4SS and the LuxR-type
regulator VjbR (Lamontagne et al., 2007; MartinezNunez et al., 2010; Viadas et al., 2010).
Quorum-sensing regulatory systems. Quorum sensing is
based on signalling molecules that can transmit messages between individual bacteria and their corresponding regulators. It is an essential bacterial
mechanism to adequately respond to environmental
changes and also for virulence of many bacterial
pathogens (Antunes et al., 2010) including Brucella
(Rambow-Larsen et al., 2009). Brucella species synthesize a C12-homoserine lactone quorum-sensing signal
(Taminiau et al., 2002), and five putative Brucella
LuxR-like transcriptional regulators have been identified, though only two (VjbR and BlxR) have been
investigated more closely and only one (VjbR) seems
to be crucially involved in virulence (Delrue et al.,
2005; Rambow-Larsen et al., 2008; Weeks et al., 2010).
The LuxR-type quorum-sensing regulator VjbR (Delrue
et al., 2005) controls more than 100 genes including
outer membrane proteins, the VirB operon, the cyclic
β-1,2-glucan synthetase Cgs and genes encoding flagella-related gene products such as FliC (Uzureau
et al., 2007; Weeks et al., 2010). VjbR mutants are
attenuated in macrophages, HeLa cells and the mouse
model of infection (Delrue et al., 2005; ArenasGamboa et al., 2008). The LuxR-type regulator BlxR
(Rambow-Larsen et al., 2008) also affects expression of
the virB operon and flagella-related genes. BlxR and
VjbR are positively autoregulated and they cross-regulate each other (Rambow-Larsen et al., 2008). However, compared to VjbR, BlxR mutation has negligible
effects on B. melitensis 16M virulence (Rambow-Larsen
et al., 2008; Weeks et al., 2010) suggesting an overlap
but not redundancy in regulated genes.
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
K. von Bargen et al.
550
•
•
•
•
The stringent response. Brucella expresses the 3′,5′-bispyrophosphate (ppGpp) synthetase (Rsh for RelA/SpoT
homologue). This enzyme produces ppGpp in response
to starvation to induce global changes of adaptive
responses to cope with low nutrient situations. The
trigger is thought to be an amino acid-free tRNA molecule binding to the ribosome, leading to guanosine
pentaphosphate (pppGpp) production by the RelA
protein. pppGpp is hydrolysed to guanosine tetraphosphate (ppGpp) which is believed to associate with the
RNA polymerase to modify its promoter specificity
(Wells & Long, 2003). In correspondence with the
BCV being a low nutrient environment, Brucella rsh
mutants are less resistant to starvation and are attenuated in macrophages, HeLa cells and mice (Kohler
et al., 2002; Kim et al., 2005a; Dozot et al., 2006).
Starvation also induces virB operon genes (Boschiroli
et al., 2002) which are targets of Rsh regulation (Dozot
et al., 2006).
LysR-type transcription regulators. Members of the family of LysR-type of transcriptional regulators (LTTRs)
are the most abundant type of transcriptional factors
in prokaryotes (Maddocks & Oyston, 2008). Several
LTTRs regulate Brucella intra-host survival although
none of them has been fully investigated (Foulongne
et al., 2000; Wu et al., 2006).
Additional transcriptional regulators. Several other regulators of Brucella belonging to different protein families
have been found to be involved in virulence. A mutant
of a GntR family transcription regulator is attenuated
both in mice and in cellular infection models (Haine
et al., 2005). Several more transcription regulators of
this family are involved in virulence in the mouse,
although interestingly, most of them are not attenuated
in cellular infection models (Lestrate et al., 2003; Haine et al., 2006). Attenuated Brucella mutants of transcription regulators include those disrupted in DeoR
(Kohler et al., 2002; Wu et al., 2006), MerR (Wu et al.,
2006) and MucR (Wu et al., 2006). More recently,
HutC, a transcriptional repressor of histidine utilization genes (Sieira et al., 2010), was also implicated in
virulence as well as a light-responsive histidine kinase
carrying a light, oxygen or voltage (LOV) domain
(Swartz et al., 2007). Nonlight responsive mutants of
this protein are attenuated in mouse macrophages
(Swartz et al., 2007).
Small regulatory RNAs. In addition to regulation by
protein factors, control of bacterial gene expression
can include small regulatory RNAs (sRNAs) which
may bind to mRNA to regulate their translation or
degradation (Zhou & Xie, 2011). Some of these RNAs
require the chaperone hfq for interaction with their
targets. Factors whose expression is controlled by Hfq
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
include the quorum-sensing regulator BlxR and the
VirB T4SS (Caswell et al., 2012). A B. abortus hfq
mutant is more sensitive to several environmental
stresses, including H2O2, acid and starvation (Robertson & Roop, 1999), and is avirulent in murine and
human macrophages as well as in mice (Robertson &
Roop, 1999; Bellaire et al., 2005). A corresponding
mutant of B. melitensis is attenuated in goats (Roop
et al., 2002).
Although the functions of several of these regulatory elements seem to converge in control of expression of the factors such as the T4SS, deletions in these regulators result
in more or less pronounced attenuation. This lack of
redundancy suggests individual roles for these transcriptional factors and supports the notion of a versatile and
complex regulatory network of Brucella gene expression.
Brucella and host cell death
The manipulation of programmed death of cells is one of
the key mechanisms of pathogens to promote their intrahost survival (Lamkanfi & Dixit, 2010). Several bacterial
pathogens such as C. burnetii (Luhrmann et al., 2010) or
Salmonella enterica serovar Typhimurium (Lindgren et al.,
1996) interfere with the cell’s programmed death pathways in promoting or suppressing them which may support infection stage specific bacterial actions such as
dissemination or long-term intracellular persistence.
Brucellae seem to belong to the category of cell death
inhibiting pathogens. Brucella strains with a Smooth LPS
phenotype (see below) inhibit the programmed cell death
(Fig. 4) in murine and human macrophages (Gross et al.,
2000; Tolomeo et al., 2003; He et al., 2006). Infection
with a Smooth B. suis strain suppresses spontaneously
occurring as well as interferon-gamma or Fas-induced
apoptosis in human monocytes (Gross et al., 2000). Likewise, serum-deprived human monocytes are protected
from apoptosis by B. melitensis infection (Fernandez-Prada et al., 2003). A delayed apoptosis has been shown for
lymphocytes and monocytes of naturally Brucella-infected
cattle compared with healthy or vaccinated controls
(Galdiero et al., 2000). An increased resistance to spontaneous or induced apoptosis has also been shown for
monocytes and lymphocytes of brucellosis patients (Tolomeo et al., 2003). Interestingly, this resistance is reversed
more efficiently in acute brucellosis patients compared
with chronic patients following antibiotic therapy (Tolomeo et al., 2003).
These protective effects require bacterial survival (Gross
et al., 2000), they are not mediated by Brucella LPS alone
(Gross et al., 2000) and might be induced by a soluble
factor as suggested by protection from apoptosis of cells
FEMS Microbiol Rev 36 (2012) 533–562
Brucella intracellular lifestyle
551
Fig. 4. Brucella manipulation of macrophage cell death. (a) (1) Macrophages/monocytes infected with virulent smooth Brucella strains are
protected from spontaneous cell death or from death induced by interferon-c treatment, serum deprivation or Fas. This protection might be
mediated by inhibition of cytochrome C and reactive oxygen species release by mitochondria, upregulation of the anti-apoptotic Bcl-2 family
member A1, downregulation of pro-apoptotic Bcl-2 family members and caspase-3 (2) and depends on bacterial viability. Uninfected cells of the
same sample are likewise protected from cell death. (3) However, with overexpression of the VirB secretion system, infection with smooth
brucellae becomes cytotoxic and results in macrophage cell death. (4) Cytopathic effects also occur during infection with high bacterial numbers.
This cytotoxicity can be reduced by (5) deletion of the VirB secretion system. (b) (1) Infection with several Rough mutant derivatives of Brucella
strains induces a caspase-2-dependent cell death that involves secretion of proinflammatory cytokines and NFkB activation. Induction of cell
death requires bacterial protein biosynthesis, (2) the uptake of bacteria into the cell, (3) bacterial viability and (4) the VirB secretion system.
Noninfected neighbour cells are not affected from Rough strain cytotoxicity.
which are not themselves infected (Gross et al., 2000; Tolomeo et al., 2003). The mechanisms could involve
increased expression of the survival-promoting protein of
the bcl-2 family A1 and/or suppression of pro-apoptotic
proteins of the same family as well as several other proapoptotic proteins including caspase-3 (Gross et al., 2000;
Eskra et al., 2003). Another study based on microarray
analysis of B. melitensis infected macrophages has found
that bacteria may inhibit apoptosis by blocking mitochondrial release of cytochrome c as well as the production of reactive oxygen species which may prevent caspase
activation (He et al., 2006).
In contrast to their Smooth counterparts, various
Rough mutants of these strains have been found to be
cytotoxic (Fernandez-Prada et al., 2003; Pei & Ficht, 2004;
Pei et al., 2006; Chen & He, 2009; Chen et al., 2011;
Fig. 4). Because they are spontaneously generated during
animal infection, Rough mutants could provide an exit
strategy for their Smooth parental bacteria in the same
intracellular vacuole to leave their niche for cell-to-cell
spread (Turse et al., 2011). The type of cell death induced
by live, Rough B. abortus and B. melitensis strains includes
necrotic features (Pei & Ficht, 2004; Pei et al., 2006) as
well as those of the mitochondrial apoptosis pathway
(Chen & He, 2009) and seems to be specific for macrophages whereas epithelial cells are not affected (Pei &
Ficht, 2004). A major difference of this particular cell
FEMS Microbiol Rev 36 (2012) 533–562
death from classical apoptosis is the involvement of a
robust pro-inflammatory response including TNF-a and
IL-1b secretion and activation of NF-jB (Chen et al.,
2011). It is also different from what has been described as
‘pyroptotic cell death’ because it is not mediated by caspase-1 but by caspase-2 activity (Chen & He, 2009; Chen
et al., 2011), which occurs at very early time points of
infection with Rough Brucella strains (Chen & He, 2009).
The cell death induced by Rough strains seems to be dominant compared with the protective effects of Smooth Brucellae: preinfection of cells with Smooth Brucella strains or
pretreatment with their LPS does not protect from the cell
death induced by their Rough derivatives (Pei et al.,
2006). Whereas the anti-apoptotic effects of their wildtype parental strains also protect noninfected cells, Rough
strain-induced cytotoxicity is likely not mediated by a soluble factor (such as TNF-a or nitric oxide), but rather
requires the direct contact of host cell and bacterium (Pei
et al., 2006). Furthermore, it requires bacterial uptake,
viability and protein biosynthesis (Pei et al., 2006).
One of the factors that has been related to interference
with host cell death is the VirB secretion system. Although
Smooth brucellae generally inhibit programmed cell death
in murine and human macrophages, infection with B. melitensis becomes increasingly cytotoxic for macrophages
with increasing multiplicities of infection (Zhong et al.,
2009). These Smooth strain cytopathic effects can be
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
552
strongly reduced by deletion of the VirB secretion system
(Zhong et al., 2009). Correspondingly, overexpression of
VirB considerably enhances the cytotoxicity and results in
a decrease in virulence for mice (Zhong et al., 2009).
Likewise, the T4SS seems to be involved in Rough strain
cytotoxicity. In a screen for factors involved in mediating
these cytopathic effects by transposon mutation of a Rough
B. melitensis strain, almost two-third of the open reading
frames whose disruption resulted in a loss of cytotoxicity
induction were part of the VirB operon (Pei et al., 2008b).
At the same time, elimination of T4SS expression in Rough
mutants results in enhanced replication and persistence
(Pei et al., 2008b). All of these findings are consistent with
the hypothesis that the cytotoxic effects of Rough Brucella
mutants may result from enhanced secretion by the VirB
system (Zhong et al., 2009). It has been shown that T3SSmediated secretion of effectors is increased in Shigella with
shorter LPS (West et al., 2005). This enhanced secretion is
thought to result from increased efficiency of the secretion
system because of steric effects. A similar effect could lead
to an uncontrolled effector secretion in Rough Brucella
mutants, resulting in an effector ‘overdose’ and host cell
death. However, different Rough mutants show variable
levels of cytotoxicity; some are not cytotoxic at all (Pei &
Ficht, 2004; Pei et al., 2006), and there are no cytopathic
effects during cell infection with the naturally Rough strain
B. ovis (Martin-Martin et al., 2008), suggesting that the
correlation of LPS phenotype, VirB-mediated secretion and
cytotoxic potential are characterized by strain-specific differences.
The attenuated phenotype of a Rough mutant strain
may not just result from a difference in intracellular trafficking, but also from the cytopathic effects of Rough
brucellae on infected cells which might counteract their
effective colonization of the Brucella intracellular niche
inside of their hosts. The inhibition of Rough straininduced cell death promotes bacterial intracellular survival (Chen & He, 2009). Likewise, the stability in and, at
later time points, decline of Rough strain bacterial numbers in macrophages might not result from bacterial killing by macrophage microbicidal actions, but rather
macrophage death and release of bacteria into the extracellular media, where they are not quantified in gentamicin-protection assays (Pei & Ficht, 2004). A similar effect
might take place inside the host, exposing bacteria from
their protective environment inside of cells and rendering
them vulnerable to humoral antibacterial mechanisms
(Pei & Ficht, 2004).
Future directions
The understanding of the mechanisms that underlie the
pathogenesis of Brucella has greatly advanced in the past
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
K. von Bargen et al.
10 years. The availability of genome sequences has contributed to this success, in particular, in view of the growing number of newly isolated Brucella species, some of
which also have pathogenic potential for humans.
Some recent exciting studies are highlighted in Table 2.
These include use of large-scale methodology such as the
classical STM, allowing the identification of key factors of
Brucella intra-host survival (Lestrate et al., 2003) and
transcriptomic approaches that enable a global view on
Brucella gene expression profiles throughout specific
stages of the infection (Rossetti et al., 2010; Viadas et al.,
2010). Proteomic characterization of the BCV membrane
composition revealed important host factors involved in
Brucella intracellular replication (Fugier et al., 2009). This
study was strengthened by the identification of a Brucella
effector protein involved, via a high-throughput yeast
two-hybrid screen that defined the interactome map
between predicted Brucella proteins and human phagosomal proteins (de Barsy et al., 2011).
Directed RNAi screens are powerful tools to identify
novel eucaryotic molecules relevant during a specific step
of the infection, as demonstrated by the ER-specific RNAi
screen carried out in Drosophila S2 cells (Qin et al.,
2008). This screen has identified a very extensive and
interesting list of eucaryotic proteins, including numerous
proteins involved in vesicle transport along the secretory
pathway, that are likely targeted by Brucella and should
therefore be further characterized.
The recent identification of several Brucella-translocated
effector proteins (de Jong et al., 2008; de Barsy et al., 2011;
Marchesini et al., 2011) will greatly advance the study of
how Brucella manipulates and interacts with host cell pathways at the molecular level. It will enable identification of
the eucaryotic targets, which may include additional Rab
GTPases or microtubule-interacting proteins that might
enable establishment of the ER-derived compartment
where Brucella replicates. For instance, the small GTPases
Rab3d and Rab9 as well as the kinesin motor proteins Kif1
and Kif4 have been shown to be downregulated in Brucellainfected macrophages (Eskra et al., 2003). Perhaps some of
these effectors are dedicated to inhibiting host cell death or
controlling the fusion events between BCVs and late endosomes and lysosomes (Starr et al., 2008). These studies will
be reinforced by use of advanced imaging technology,
including live imaging of Brucella-infected cells (Starr
et al., 2008) as well as the use of bioluminescent strains to
follow the infection in vivo (Rajashekara et al., 2005).
Integration of in vitro studies in the context of in vivo
models and in natura brucellosis taking into account the
complex interplay of cellular and immune factors that
contribute to disease development may help to close the
gap separating results of clinical and fundamental
research. Several target cells have been identified and have
FEMS Microbiol Rev 36 (2012) 533–562
Brucella intracellular lifestyle
553
Table 2. Techniques that provided important advances in understanding Brucella intracellular pathogenesis
Technique
Objective
Advantages
Disadvantages
Reference
(1) Transposon
mutagenesis
and (2)
Signaturetagged
transposon
mutagenesis
Microarray
technology
Identification of
virulence determinants
(1) and (2) Unbiased screen;
(2) reduced number of mice
possible preselection of
mutant pools (e.g. by
growth in minimal media to
exclude auxotrophs)
(1) Kohler et al. (2002);
Delrue et al. (2001) (2)
Foulongne et al. (2000);
Lestrate et al. (2003)
Characterization of host cell
transcriptional responses to
Brucella infection
Thorough analysis of
induction/suppression of
eucaryotic pathways
following Brucella infection
Bioluminescent
Brucella strains
in vivo
Kinetic profiles of bacterial
organ invasion over time
Live imaging
of infected cells
Observation of trafficking
events following individual
bacteria
Kinetic analysis in the same
individual; thorough in vivo
studies of bacterial
dissemination
Avoid artifacts because of
fixation; information on the
dynamics of the events;
kinetic analysis of the fate
of single bacteria
(2) Possible complementation
of attenuated mutants by
nonattenuated strains;
masking of subtle effects by
the high bacterial loads
required to obtain individual
signals for each mutant
May yield results different
from changes on proteomic
level because of
post-transcriptional
regulation
Limited resolution and
sensitivity with respect to
bacterial load observed
RNAi screen in
host cells such as
S2 Drosophila
cells
Genome-wide loss-offunction screen in infected
cells
Proteomics of
BCV membrane
Determination of eucaryotic
and prokaryotic proteins on
vacuole
Amplification of
Brucella mRNA
from infected
samples
Generation of intracellular
Brucella gene expression
profiles
High-throughput
yeast two-hybrid
screen
Identify potential interactions
between subset of
eucaryotic proteins and
Brucella proteins
Identification of effector
molecules
Bioinformatics
Possibility of directed screens
(e.g. ER-directed) relevant
for Brucella; identification of
conserved eucaryotic
proteins involved in Brucella
virulence
Thorough and unbiased
information on the
recruitment of eucaryotic
proteins to the vacuole
Analysis of whole genome or
multiple gene sets
transcripts in same
experiment; global picture
of regulation
Directed screening of whole
bacterial ORFeome against
specific baits
Exhaustive analysis of
potential effectors including
those that would not be
found in mutant screenings
because of mild effects or
functional redundancies
been extensively studied in vitro, including macrophages,
trophoblasts, DCs and epithelial cells but their relevance
and particular role in the course of animal and even more
human infection is poorly understood. Although macrophages seem to be essential to reduce the initial bacterial
load of infection (Archambaud et al., 2010), they seem to
serve as permanent residence for Brucella during persistence of disease. In contrast to other cell types, macro-
FEMS Microbiol Rev 36 (2012) 533–562
Eskra et al. (2003)
Rajashekara et al. (2005)
Over-expression of markers
may affect their location
and/or function;
time-consuming to analyse
statistical significant number
of bacteria and events
Dependent on the efficiency
of the knock-down;
temperature limitation for
S2 cells;
Starr et al. (2008)
Difficult to obtain good
purity and enough quantity
material
Fugier et al. (2009)
Only transcript level (not
protein); only relative
quantification
Rossetti et al. (2010);
Viadas et al. (2010)
Nonspecificity of some
interactions;
de Barsy et al. (2011)
High number of candidate
effectors to test for
translocation in infected
cells
de Jong et al. (2008);
Marchesini et al. (2011)
Qin et al. (2008)
phages are able to initially kill a high percentage of
internalized brucellae which are otherwise perfectly
adapted to intra-host survival, raising the question
whether this killing phase may serve a function during
Brucella infection. Little is known as to where and how
the bacteria cross the epithelial barriers of their hosts,
which cells are targeted first, if/how a switch of host cells
takes place and how these processes relate to the particular
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
554
intracellular trafficking of Brucella. The mouse model of
brucellosis should be further exploited with all the new
tools available to decipher in greater detail the cellular
players and the immune response during infection, maybe
also using the recently described rodent strain B. microti.
How the intracellular lifestyle is contributing to Brucella persistence during the chronic stage of disease is an
important question that merits further investigation.
Intracellular replication to high numbers that has been
observed in vitro and described, for example, in trophoblasts of infected pregnant animals may enable cell-to-cell
spread and disruption of specific tissue or barriers. As
discussed above, control of cell death may also regulate
bacterial escape from cells and dissemination. Perhaps, in
some cells, Brucella is able to survive but restrain its
intracellular growth in a way that would establish a bacterial reservoir. Consistent with this possibility, cells with
only a few bacteria are often observed in cultured cells
even at very late time points after infection. Analysis of
the viability of these bacteria needs to be undertaken to
determine whether they could represent a latent virulent
Brucella. Although Brucella induces a minimal inflammatory response in the host, it is not silent and has therefore
acquired proteins that help modulate host innate and
adaptive immune response mechanisms that may be
important in the development of a chronic infection.
Besides Btp1, another of these factors is PrpA, which
belongs to the proline-racemase family. It is required for
chronic infection in mice. It does not seem specifically
associated with intracellular survival but instead, it is
involved in immune modulation in the host, by eliciting
B-lymphocyte polyclonal activation and IL-10 secretion
(Spera et al., 2006). Additional factors are likely to be
involved in persistence of Brucella within the host.
As summarized in this review, great advances in the
field of Brucella intracellular trafficking have been made
in recent years. To integrate the results from studies at
the cell and organism levels will open new perspectives
and eventually allow for a deeper understanding of the
complex mechanisms of brucellosis.
Acknowledgements
This work was supported by the CNRS (J.P.G.), INSERM
(S.S.) and ANR BruTir (J.P.G., K.V.B. and S.S.). We
thank Steve Garvis for critical reading of the manuscript.
References
Akopyan K, Edgren T, Wang-Edgren H, Rosqvist R, Fahlgren
A, Wolf-Watz H & Fallman M (2011) Translocation of
surface-localized effectors in type III secretion. P Natl Acad
Sci USA 108: 1639–1644.
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
K. von Bargen et al.
Allen CA, Adams LG & Ficht TA (1998) Transposon-derived
Brucella abortus rough mutants are attenuated and exhibit
reduced intracellular survival. Infect Immun 66: 1008–1016.
Alvarez-Martinez CE & Christie PJ (2009) Biological diversity
of prokaryotic type IV secretion systems. Microbiol Mol Biol
Rev 73: 775–808.
Anderson TD & Cheville NF (1986) Ultrastructural
morphometric analysis of Brucella abortus-infected
trophoblasts in experimental placentitis. Bacterial replication
occurs in rough endoplasmic reticulum. Am J Pathol 124:
226–237.
Anderson ES, Paulley JT, Gaines JM, Valderas MW, Martin
DW, Menscher E, Brown TD, Burns CS & Roop RM II
(2009) The manganese transporter MntH is a critical
virulence determinant for Brucella abortus 2308 in
experimentally infected mice. Infect Immun 77:
3466–3474.
Antunes LC, Ferreira RB, Buckner MM & Finlay BB (2010)
Quorum sensing in bacterial virulence. Microbiology 156:
2271–2282.
Archambaud C, Salcedo SP, Lelouard H, Devilard E, de Bovis
B, Van Rooijen N, Gorvel JP & Malissen B (2010)
Contrasting roles of macrophages and dendritic cells in
controlling initial pulmonary Brucella infection. Eur J
Immunol 40: 3458–3471.
Arellano-Reynoso B, Lapaque N, Salcedo S, Briones G,
Ciocchini AE, Ugalde R, Moreno E, Moriyon I & Gorvel
JP (2005) Cyclic beta-1,2-glucan is a Brucella virulence
factor required for intracellular survival. Nat Immunol 6:
618–625.
Arenas-Gamboa AM, Ficht TA, Kahl-McDonagh MM & RiceFicht AC (2008) Immunization with a single dose of a
microencapsulated Brucella melitensis mutant enhances
protection against wild-type challenge. Infect Immun 76:
2448–2455.
Ariza J, Bosilkovski M, Cascio A et al. (2007) Perspectives for
the treatment of brucellosis in the 21st century: the
Ioannina recommendations. PLoS Med 4: e317.
Audic S, Lescot M, Claverie JM & Scholz HC (2009) Brucella
microti: the genome sequence of an emerging pathogen.
BMC Genomics 10: 352.
Baldwin CL & Goenka R (2006) Host immune responses to
the intracellular bacteria Brucella: does the bacteria instruct
the host to facilitate chronic infection? Crit Rev Immunol
26: 407–442.
Barbier T, Nicolas C & Letesson JJ (2011) Brucella adaptation
and survival at the crossroad of metabolism and virulence.
FEBS Lett 585: 2929–2934.
Barquero-Calvo E, Chaves-Olarte E, Weiss DS, Guzman-Verri
C, Chacon-Diaz C, Rucavado A, Moriyon I & Moreno E
(2007) Brucella abortus uses a stealthy strategy to avoid
activation of the innate immune system during the onset of
infection. PLoS ONE 2: e631.
Barrionuevo P, Delpino MV, Velasquez LN, Garcia Samartino
C, Coria LM, Ibanez AE, Rodriguez ME, Cassataro J &
Giambartolomei GH (2011) Brucella abortus inhibits
FEMS Microbiol Rev 36 (2012) 533–562
Brucella intracellular lifestyle
IFN-gamma-induced FcgammaRI expression and
FcgammaRI-restricted phagocytosis via toll-like receptor 2 on
human monocytes/macrophages. Microbes Infect 13: 239–250.
Bellaire BH, Roop RM II & Cardelli JA (2005) Opsonized
virulent Brucella abortus replicates within nonacidic,
endoplasmic reticulum-negative, LAMP-1-positive
phagosomes in human monocytes. Infect Immun 73: 3702–
3713.
Bergagna S, Zoppi S, Ferroglio E, Gobetto M, Dondo A,
Di Giannatale E, Gennero MS & Grattarola C (2009)
Epidemiologic survey for Brucella suis biovar 2 in a wild
boar (Sus scrofa) population in northwest Italy. J Wildl Dis
45: 1178–1181.
Billard E, Cazevieille C, Dornand J & Gross A (2005) High
susceptibility of human dendritic cells to invasion by the
intracellular pathogens Brucella suis, B. abortus, and B.
melitensis. Infect Immun 73: 8418–8424.
Billard E, Dornand J & Gross A (2007) Brucella suis prevents
human dendritic cell maturation and antigen presentation
through regulation of tumor necrosis factor alpha secretion.
Infect Immun 75: 4980–4989.
Boschiroli ML, Ouahrani-Bettache S, Foulongne V, MichauxCharachon S, Bourg G, Allardet-Servent A, Cazevieille C,
Liautard JP, Ramuz M & O’Callaghan D (2002) The
Brucella suis virB operon is induced intracellularly in
macrophages. P Natl Acad Sci USA 99: 1544–1549.
Breedveld MW, Yoo JS, Reinhold VN & Miller KJ (1994)
Synthesis of glycerophosphorylated cyclic beta-(1,2)-glucans
by Rhizobium meliloti ndv mutants. J Bacteriol 176: 1047–
1051.
Briones G, Inon de Iannino N, Roset M, Vigliocco A, Paulo
PS & Ugalde RA (2001) Brucella abortus cyclic beta-1,2glucan mutants have reduced virulence in mice and are
defective in intracellular replication in HeLa cells. Infect
Immun 69: 4528–4535.
Bukata L, Altabe S, de Mendoza D, Ugalde RA & Comerci DJ
(2008) Phosphatidylethanolamine synthesis is required for
optimal virulence of Brucella abortus. J Bacteriol 190: 8197–
8203.
Caron E, Gross A, Liautard JP & Dornand J (1996) Brucella
species release a specific, protease-sensitive, inhibitor of
TNF-alpha expression, active on human macrophage-like
cells. J Immunol 156: 2885–2893.
Casadevall A & Pirofski LA (1999) Host-pathogen interactions:
redefining the basic concepts of virulence and pathogenicity.
Infect Immun 67: 3703–3713.
Casino P, Rubio V & Marina A (2010) The mechanism of
signal transduction by two-component systems. Curr Opin
Struct Biol 20: 763–771.
Castaneda-Roldan EI, Avelino-Flores F, Dall’Agnol M, Freer E,
Cedillo L, Dornand J & Giron JA (2004) Adherence of
Brucella to human epithelial cells and macrophages is
mediated by sialic acid residues. Cell Microbiol 6: 435–445.
Castaneda-Roldan EI, Ouahrani-Bettache S, Saldana Z, Avelino
F, Rendon MA, Dornand J & Giron JA (2006)
Characterization of SP41, a surface protein of Brucella
FEMS Microbiol Rev 36 (2012) 533–562
555
associated with adherence and invasion of host epithelial
cells. Cell Microbiol 8: 1877–1887.
Caswell CC, Gaines JM & Roop RM II (2012) The RNA
chaperone Hfq independently coordinates expression of the
VirB Type IV secretion system and the LuxR-type regulator
BabR in Brucella abortus 2308. J Bacteriol 194: 3–14.
Celli J, de Chastellier C, Franchini DM, Pizarro-Cerda J,
Moreno E & Gorvel JP (2003) Brucella evades macrophage
killing via VirB-dependent sustained interactions with the
endoplasmic reticulum. J Exp Med 198: 545–556.
Celli J, Salcedo SP & Gorvel JP (2005) Brucella coopts the
small GTPase Sar1 for intracellular replication. P Natl Acad
Sci USA 102: 1673–1678.
Chaudhary A, Ganguly K, Cabantous S, Waldo GS, MichevaViteva SN, Nag K, Hlavacek WS & Tung CS (2011) The
Brucella TIR-like protein TcpB interacts with the death
domain of MyD88. Biochem Biophys Res Commun 417: 299–
304.
Chaves-Olarte E, Guzman-Verri C, Meresse S, Desjardins M,
Pizarro-Cerda J, Badilla J, Gorvel JP & Moreno E (2002)
Activation of Rho and Rab GTPases dissociates Brucella
abortus internalization from intracellular trafficking. Cell
Microbiol 4: 663–676.
Chen F & He Y (2009) Caspase-2 mediated apoptotic and
necrotic murine macrophage cell death induced by rough
Brucella abortus. PLoS ONE 4: e6830.
Chen F, Ding X, Ding Y, Xiang Z, Li X, Ghosh D, Schurig
GG, Sriranganathan N, Boyle SM & He Y (2011)
Proinflammatory caspase-2-mediated macrophage cell death
induced by a rough attenuated Brucella suis strain. Infect
Immun 79: 2460–2469.
Cheville NF, Jensen AE, Halling SM, Tatum FM, Morfitt DC,
Hennager SG, Frerichs WM & Schurig G (1992) Bacterial
survival, lymph node changes, and immunologic responses
of cattle vaccinated with standard and mutant strains of
Brucella abortus. Am J Vet Res 53: 1881–1888.
Cirl C, Wieser A, Yadav M et al. (2008) Subversion of Tolllike receptor signaling by a unique family of bacterial Toll/
interleukin-1 receptor domain-containing proteins. Nat Med
14: 399–406.
Cloeckaert A, Verger JM, Grayon M, Paquet JY, Garin-Bastuji
B, Foster G & Godfroid J (2001) Classification of Brucella
spp. isolated from marine mammals by DNA polymorphism
at the omp2 locus. Microbes Infect 3: 729–738.
Comerci DJ, Martinez-Lorenzo MJ, Sieira R, Gorvel JP &
Ugalde RA (2001) Essential role of the VirB machinery in
the maturation of the Brucella abortus-containing vacuole.
Cell Microbiol 3: 159–168.
Conde-Alvarez R, Grillo MJ, Salcedo SP, de Miguel MJ,
Fugier E, Gorvel JP, Moriyon I & Iriarte M (2006)
Synthesis of phosphatidylcholine, a typical eukaryotic
phospholipid, is necessary for full virulence of the
intracellular bacterial parasite Brucella abortus. Cell Microbiol
8: 1322–1335.
Cvetnic Z, Spicic S, Toncic J, Majnaric D, Benic M, Albert D,
Thiebaud M & Garin-Bastuji B (2009) Brucella suis infection
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
556
in domestic pigs and wild boar in Croatia. Rev Sci Tech 28:
1057–1067.
Czibener C & Ugalde JE (2012) Identification of a unique gene
cluster of Brucella spp. that mediates adhesion to host cells.
Microbes Infect 14: 79–85.
de Barsy M, Jamet A, Filopon D et al. (2011) Identification of
a Brucella spp. secreted effector specifically interacting with
human small GTPase Rab2. Cell Microbiol 13: 1044–1058.
de Jong MF & Tsolis R (2012) Brucellosis and type IV
secretion. Future Microbiol 7: 47–58.
de Jong MF, Sun YH, den Hartigh AB, van Dijl JM & Tsolis
RM (2008) Identification of VceA and VceC, two members
of the VjbR regulon that are translocated into macrophages
by the Brucella type IV secretion system. Mol Microbiol 70:
1378–1396.
Delpino MV, Fossati CA & Baldi PC (2009) Proinflammatory
response of human osteoblastic cell lines and osteoblastmonocyte interaction upon infection with Brucella spp.
Infect Immun 77: 984–995.
Delrue RM, Martinez-Lorenzo M, Lestrate P, Danese I, Bielarz
V, Mertens P, De Bolle X, Tibor A, Gorvel JP & Letesson JJ
(2001) Identification of Brucella spp. genes involved in
intracellular trafficking. Cell Microbiol 3: 487–497.
Delrue RM, Deschamps C, Leonard S et al. (2005) A quorumsensing regulator controls expression of both the type IV
secretion system and the flagellar apparatus of Brucella
melitensis. Cell Microbiol 7: 1151–1161.
den Hartigh AB, Rolan HG, de Jong MF & Tsolis RM (2008)
VirB3 to VirB6 and VirB8 to VirB11, but not VirB7, are
essential for mediating persistence of Brucella in the
reticuloendothelial system. J Bacteriol 190: 4427–4436.
Detilleux PG, Deyoe BL & Cheville NF (1990) Entry and
intracellular localization of Brucella spp. in Vero cells:
fluorescence and electron microscopy. Vet Pathol 27: 317–328.
Dorrell N, Spencer S, Foulonge V, Guigue-Talet P,
O’Callaghan D & Wren BW (1998) Identification, cloning
and initial characterisation of FeuPQ in Brucella suis: a new
sub-family of two-component regulatory systems. FEMS
Microbiol Lett 162: 143–150.
Dozot M, Boigegrain RA, Delrue RM, Hallez R, OuahraniBettache S, Danese I, Letesson JJ, De Bolle X & Kohler S
(2006) The stringent response mediator Rsh is required for
Brucella melitensis and Brucella suis virulence, and for
expression of the type IV secretion system virB. Cell
Microbiol 8: 1791–1802.
El-Tras WF, Tayel AA, Eltholth MM & Guitian J (2010)
Brucella infection in fresh water fish: evidence for natural
infection of Nile catfish, Clarias gariepinus, with Brucella
melitensis. Vet Microbiol 141: 321–325.
Elzer PH, Enright FM, McQuiston JR, Boyle SM & Schurig
GG (1998) Evaluation of a rough mutant of Brucella
melitensis in pregnant goats. Res Vet Sci 64: 259–260.
Endley S, McMurray D & Ficht TA (2001) Interruption of the
cydB locus in Brucella abortus attenuates intracellular
survival and virulence in the mouse model of infection.
J Bacteriol 183: 2454–2462.
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
K. von Bargen et al.
Eskra L, Mathison A & Splitter G (2003) Microarray analysis
of mRNA levels from RAW264.7 macrophages infected with
Brucella abortus. Infect Immun 71: 1125–1133.
Eze MO, Yuan L, Crawford RM, Paranavitana CM, Hadfield
TL, Bhattacharjee AK, Warren RL & Hoover DL (2000)
Effects of opsonization and gamma interferon on growth of
Brucella melitensis 16M in mouse peritoneal macrophages in
vitro. Infect Immun 68: 257–263.
Falkow S (1988) Molecular Koch’s postulates applied to
microbial pathogenicity. Rev Infect Dis 10(suppl 2): S274–
S276.
Ferguson GP, Datta A, Baumgartner J, Roop RM II, Carlson
RW & Walker GC (2004) Similarity to peroxisomalmembrane protein family reveals that Sinorhizobium and
Brucella BacA affect lipid-A fatty acids. P Natl Acad Sci USA
101: 5012–5017.
Fernandez-Prada CM, Zelazowska EB, Nikolich M, Hadfield
TL, Roop RM II, Robertson GL & Hoover DL (2003)
Interactions between Brucella melitensis and human
phagocytes: bacterial surface O-Polysaccharide inhibits
phagocytosis, bacterial killing, and subsequent host cell
apoptosis. Infect Immun 71: 2110–2119.
Ferrero MC, Fossati CA & Baldi PC (2009) Smooth Brucella
strains invade and replicate in human lung epithelial
cells without inducing cell death. Microbes Infect 11:
476–483.
Fontes P, Alvarez-Martinez MT, Gross A, Carnaud C,
Kohler S & Liautard JP (2005) Absence of evidence for
the participation of the macrophage cellular prion protein
in infection with Brucella suis. Infect Immun 73: 6229–
6236.
Forestier C, Moreno E, Pizarro-Cerda J & Gorvel JP (1999)
Lysosomal accumulation and recycling of lipopolysaccharide
to the cell surface of murine macrophages, an in vitro and
in vivo study. J Immunol 162: 6784–6791.
Forestier C, Deleuil F, Lapaque N, Moreno E & Gorvel JP
(2000) Brucella abortus lipopolysaccharide in murine
peritoneal macrophages acts as a down-regulator of T cell
activation. J Immunol 165: 5202–5210.
Foster G, Osterman BS, Godfroid J, Jacques I & Cloeckaert
A (2007) Brucella ceti sp. nov. and Brucella pinnipedialis
sp. nov. for Brucella strains with cetaceans and seals as
their preferred hosts. Int J Syst Evol Microbiol 57: 2688–
2693.
Foulongne V, Bourg G, Cazevieille C, Michaux-Charachon S &
O’Callaghan D (2000) Identification of Brucella suis genes
affecting intracellular survival in an in vitro human
macrophage infection model by signature-tagged transposon
mutagenesis. Infect Immun 68: 1297–1303.
Foulongne V, Walravens K, Bourg G, Boschiroli ML, Godfroid
J, Ramuz M & O’Callaghan D (2001) Aromatic compounddependent Brucella suis is attenuated in both cultured cells
and mouse models. Infect Immun 69: 547–550.
Franco MP, Mulder M & Smits HL (2007) Persistence and
relapse in brucellosis and need for improved treatment.
Trans R Soc Trop Med Hyg 101: 854–855.
FEMS Microbiol Rev 36 (2012) 533–562
Brucella intracellular lifestyle
Fretin D, Fauconnier A, Kohler S et al. (2005) The sheathed
flagellum of Brucella melitensis is involved in persistence in
a murine model of infection. Cell Microbiol 7: 687–698.
Fugier E, Salcedo SP, de Chastellier C, Pophillat M, Muller A,
Arce-Gorvel V, Fourquet P & Gorvel JP (2009) The
glyceraldehyde-3-phosphate dehydrogenase and the small
GTPase Rab 2 are crucial for Brucella replication. PLoS
Pathog 5: e1000487.
Galdiero E, Romano Carratelli C, Vitiello M, Nuzzo I, Del
Vecchio E, Bentivoglio C, Perillo G & Galdiero F (2000)
HSP and apoptosis in leukocytes from infected or
vaccinated animals by Brucella abortus. New Microbiol 23:
271.
Galindo RC, Munoz PM, de Miguel MJ, Marin CM, Labairu J,
Revilla M, Blasco JM, Gortazar C & de la Fuente J (2010)
Gene expression changes in spleens of the wildlife reservoir
species, Eurasian wild boar (Sus scrofa), naturally infected
with Brucella suis biovar 2. J Genet Genomics 37: 725–736.
Garcia Samartino C, Delpino MV, Pott Godoy C et al. (2010)
Brucella abortus induces the secretion of proinflammatory
mediators from glial cells leading to astrocyte apoptosis. Am
J Pathol 176: 1323–1338.
Gauthier A, Thomas NA & Finlay BB (2003) Bacterial
injection machines. J Biol Chem 278: 25273–25276.
Gee JM, Valderas MW, Kovach ME, Grippe VK, Robertson
GT, Ng WL, Richardson JM, Winkler ME & Roop RM II
(2005) The Brucella abortus Cu,Zn superoxide dismutase is
required for optimal resistance to oxidative killing by
murine macrophages and wild-type virulence in
experimentally infected mice. Infect Immun 73: 2873–2880.
Godfroid F, Taminiau B, Danese I, Denoel P, Tibor A,
Weynants V, Cloeckaert A, Godfroid J & Letesson JJ (1998)
Identification of the perosamine synthetase gene of Brucella
melitensis 16M and involvement of lipopolysaccharide O
side chain in Brucella survival in mice and in macrophages.
Infect Immun 66: 5485–5493.
Gonzalez L, Patterson IA, Reid RJ et al. (2002) Chronic
meningoencephalitis associated with Brucella sp. infection in
live-stranded striped dolphins (Stenella coeruleoalba).
J Comp Pathol 126: 147–152.
Gonzalez D, Grillo MJ, De Miguel MJ et al. (2008) Brucellosis
vaccines: assessment of Brucella melitensis lipopolysaccharide
rough mutants defective in core and O-polysaccharide
synthesis and export. PLoS ONE 3: e2760.
Gorvel JP & de Chastellier C (2005) Bacteria spurned by selfabsorbed cells. Nat Med 11: 18–19.
Gorvel JP & Moreno E (2002) Brucella intracellular life: from
invasion to intracellular replication. Vet Microbiol 90: 281–
297.
Gross A, Terraza A, Ouahrani-Bettache S, Liautard JP &
Dornand J (2000) In vitro Brucella suis infection prevents
the programmed cell death of human monocytic cells. Infect
Immun 68: 342–351.
Guzman-Verri C, Chaves-Olarte E, von Eichel-Streiber C,
Lopez-Goni I, Thelestam M, Arvidson S, Gorvel JP &
Moreno E (2001) GTPases of the Rho subfamily are
FEMS Microbiol Rev 36 (2012) 533–562
557
required for Brucella abortus internalization in
nonprofessional phagocytes: direct activation of Cdc42.
J Biol Chem 276: 44435–44443.
Guzman-Verri C, Manterola L, Sola-Landa A, Parra A,
Cloeckaert A, Garin J, Gorvel JP, Moriyon I, Moreno E &
Lopez-Goni I (2002) The two-component system BvrR/BvrS
essential for Brucella abortus virulence regulates the
expression of outer membrane proteins with counterparts in
members of the Rhizobiaceae. P Natl Acad Sci USA 99:
12375–12380.
Haag AF, Myka KK, Arnold MF, Caro-Hernandez P &
Ferguson GP (2010) Importance of lipopolysaccharide and
cyclic beta-1,2-glucans in Brucella-mammalian infections.
Int J Microbiol 2010: 124509.
Haine V, Sinon A, Van Steen F, Rousseau S, Dozot M, Lestrate
P, Lambert C, Letesson JJ & De Bolle X (2005) Systematic
targeted mutagenesis of Brucella melitensis 16M reveals a
major role for GntR regulators in the control of virulence.
Infect Immun 73: 5578–5586.
Haine V, Dozot M, Dornand J, Letesson JJ & De Bolle X
(2006) NnrA is required for full virulence and regulates
several Brucella melitensis denitrification genes. J Bacteriol
188: 1615–1619.
Hanna N, de Bagues MP, Ouahrani-Bettache S, El Yakhlifi Z,
Kohler S & Occhialini A (2011) The virB operon is essential
for lethality of Brucella microti in the Balb/c murine model
of infection. J Infect Dis 203: 1129–1135.
He Y, Reichow S, Ramamoorthy S, Ding X, Lathigra R, Craig
JC, Sobral BW, Schurig GG, Sriranganathan N & Boyle SM
(2006) Brucella melitensis triggers time-dependent
modulation of apoptosis and down-regulation of
mitochondrion-associated gene expression in mouse
macrophages. Infect Immun 74: 5035–5046.
Hernandez-Castro R, Verdugo-Rodriguez A, Puente JL &
Suarez-Guemes F (2008) The BMEI0216 gene of Brucella
melitensis is required for internalization in HeLa cells.
Microb Pathog 44: 28–33.
Hong PC, Tsolis RM & Ficht TA (2000) Identification of genes
required for chronic persistence of Brucella abortus in mice.
Infect Immun 68: 4102–4107.
Iannino F, Ugalde JE & Inon de Iannino N (2012) Brucella
abortus efp gene is required for an efficient internalization
in HeLa cells. Microb Pathog 52: 31–40.
Inon de Iannino N, Briones G, Tolmasky M & Ugalde RA
(1998) Molecular cloning and characterization of cgs, the
Brucella abortus cyclic beta(1-2) glucan synthetase gene:
genetic complementation of Rhizobium meliloti ndvB and
Agrobacterium tumefaciens chvB mutants. J Bacteriol 180:
4392–4400.
Iriarte M, González D, Delrue RM, Monreal D, Conde R,
López-Goñi I, Letesson J-J & Moriyón I (2004) Brucella
lipopolysaccharide: structure, biosynthesis and genetics.
Brucella: Molecular and Cellular Biology (López-Goñi I &
Moriyón I, eds.), pp. 152–183. Horizon Bioscience, Norfolk.
Iyankan L & Singh DK (2002) The effect of Brucella abortus
on hydrogen peroxide and nitric oxide production by
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
558
bovine polymorphonuclear cells. Vet Res Commun 26: 93–
102.
Jiang X & Baldwin CL (1993) Effects of cytokines on
intracellular growth of Brucella abortus. Infect Immun 61:
124–134.
Jimenez de Bagues MP, Ouahrani-Bettache S, Quintana JF
et al. (2010) The new species Brucella microti replicates in
macrophages and causes death in murine models of
infection. J Infect Dis 202: 3–10.
Kahl-McDonagh MM & Ficht TA (2006) Evaluation of
protection afforded by Brucella abortus and Brucella
melitensis unmarked deletion mutants exhibiting different
rates of clearance in BALB/c mice. Infect Immun 74: 4048–
4057.
Kahl-McDonagh MM, Elzer PH, Hagius SD et al. (2006)
Evaluation of novel Brucella melitensis unmarked deletion
mutants for safety and efficacy in the goat model of
brucellosis. Vaccine 24: 5169–5177.
Khan MY, Mah MW & Memish ZA (2001) Brucellosis in
pregnant women. Clin Infect Dis 32: 1172–1177.
Kim S, Watarai M, Kondo Y, Erdenebaatar J, Makino S &
Shirahata T (2003) Isolation and characterization of miniTn5Km2 insertion mutants of Brucella abortus deficient in
internalization and intracellular growth in HeLa cells. Infect
Immun 71: 3020–3027.
Kim S, Kurokawa D, Watanabe K, Makino S, Shirahata T &
Watarai M (2004a) Brucella abortus nicotinamidase (PncA)
contributes to its intracellular replication and infectivity in
mice. FEMS Microbiol Lett 234: 289–295.
Kim S, Watarai M, Suzuki H, Makino S, Kodama T &
Shirahata T (2004b) Lipid raft microdomains mediate class
A scavenger receptor-dependent infection of Brucella
abortus. Microb Pathog 37: 11–19.
Kim S, Watanabe K, Suzuki H & Watarai M (2005a) Roles of
Brucella abortus SpoT in morphological differentiation and
intramacrophagic replication. Microbiology 151: 1607–1617.
Kim S, Lee DS, Watanabe K, Furuoka H, Suzuki H & Watarai
M (2005b) Interferon-gamma promotes abortion due to
Brucella infection in pregnant mice. BMC Microbiol 5: 22.
Kohler S, Foulongne V, Ouahrani-Bettache S, Bourg G,
Teyssier J, Ramuz M & Liautard JP (2002) The analysis of
the intramacrophagic virulome of Brucella suis deciphers the
environment encountered by the pathogen inside the
macrophage host cell. P Natl Acad Sci USA 99: 15711–
15716.
Lamkanfi M & Dixit VM (2010) Manipulation of host cell
death pathways during microbial infections. Cell Host
Microbe 8: 44–54.
Lamontagne J, Butler H, Chaves-Olarte E et al. (2007)
Extensive cell envelope modulation is associated with
virulence in Brucella abortus. J Proteome Res 6: 1519–1529.
Lapaque N, Takeuchi O, Corrales F, Akira S, Moriyon I,
Howard JC & Gorvel JP (2006) Differential inductions of
TNF-alpha and IGTP, IIGP by structurally diverse classic
and non-classic lipopolysaccharides. Cell Microbiol 8: 401–
413.
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
K. von Bargen et al.
Lavigne JP, Patey G, Sangari FJ, Bourg G, Ramuz M,
O’Callaghan D & Michaux-Charachon S (2005)
Identification of a new virulence factor, BvfA, in Brucella
suis. Infect Immun 73: 5524–5529.
Lelouard H, Henri S, De Bovis B, Mugnier B, Chollat-Namy
A, Malissen B, Meresse S & Gorvel JP (2010) Pathogenic
bacteria and dead cells are internalized by a unique subset
of Peyer’s patch dendritic cells that express lysozyme.
Gastroenterology 138: 173–184, e171–173.
Lelouard H, Fallet M, de Bovis B, Méresse S & Gorvel JP
(2011) Peyer’s Patch denditric cells sample antigens by
extending dendrites through M cell-specific transcellular
pores. Gastroenterology 142: 592–601.
Lestrate P, Delrue RM, Danese I, Didembourg C, Taminiau B,
Mertens P, De Bolle X, Tibor A, Tang CM & Letesson JJ
(2000) Identification and characterization of in vivo
attenuated mutants of Brucella melitensis. Mol Microbiol 38:
543–551.
Lestrate P, Dricot A, Delrue RM, Lambert C, Martinelli V, De
Bolle X, Letesson JJ & Tibor A (2003) Attenuated signaturetagged mutagenesis mutants of Brucella melitensis identified
during the acute phase of infection in mice. Infect Immun
71: 7053–7060.
Lindgren SW, Stojiljkovic I & Heffron F (1996) Macrophage
killing is an essential virulence mechanism of Salmonella
typhimurium. P Natl Acad Sci USA 93: 4197–4201.
Loisel-Meyer S, Jimenez de Bagues MP, Basseres E, Dornand J,
Kohler S, Liautard JP & Jubier-Maurin V (2006)
Requirement of norD for Brucella suis virulence in a murine
model of in vitro and in vivo infection. Infect Immun 74:
1973–1976.
Luhrmann A, Nogueira CV, Carey KL & Roy CR (2010)
Inhibition of pathogen-induced apoptosis by a Coxiella
burnetii type IV effector protein. P Natl Acad Sci USA 107:
18997–19001.
Maddocks SE & Oyston PC (2008) Structure and function of
the LysR-type transcriptional regulator (LTTR) family
proteins. Microbiology 154: 3609–3623.
Mancilla M, Lopez-Goni I, Moriyon I & Zarraga AM (2010)
Genomic island 2 is an unstable genetic element
contributing to Brucella lipopolysaccharide spontaneous
smooth-to-rough dissociation. J Bacteriol 192: 6346–6351.
Manterola L, Moriyon I, Moreno E, Sola-Landa A, Weiss DS,
Koch MH, Howe J, Brandenburg K & Lopez-Goni I (2005)
The lipopolysaccharide of Brucella abortus BvrS/BvrR
mutants contains lipid A modifications and has higher
affinity for bactericidal cationic peptides. J Bacteriol 187:
5631–5639.
Maquart M, Zygmunt MS & Cloeckaert A (2009) Marine
mammal Brucella isolates with different genomic
characteristics display a differential response when
infecting human macrophages in culture. Microbes Infect
11: 361–366.
Marchesini MI, Herrmann CK, Salcedo SP, Gorvel JP &
Comerci DJ (2011) In search of Brucella abortus type IV
secretion substrates: screening and identification of four
FEMS Microbiol Rev 36 (2012) 533–562
Brucella intracellular lifestyle
proteins translocated into host cells through VirB system.
Cell Microbiol 13: 1261–1274.
Martinez-Nunez C, Altamirano-Silva P, Alvarado-Guillen F,
Moreno E, Guzman-Verri C & Chaves-Olarte E (2010) The
two-component system BvrR/BvrS regulates the expression
of the type IV secretion system VirB in Brucella abortus.
J Bacteriol 192: 5603–5608.
Martin-Martin AI, Caro-Hernandez P, Orduna A, Vizcaino N
& Fernandez-Lago L (2008) Importance of the Omp25/
Omp31 family in the internalization and intracellular
replication of virulent B. ovis in murine macrophages and
HeLa cells. Microbes Infect 10: 706–710.
Martin-Martin AI, Caro-Hernandez P, Sancho P, Tejedor C,
Cloeckaert A, Fernandez-Lago L & Vizcaino N (2009)
Analysis of the occurrence and distribution of the Omp25/
Omp31 family of surface proteins in the six classical
Brucella species. Vet Microbiol 137: 74–82.
Martin-Martin Al, Vizcaino N & Fernandez-Lago L (2010)
Cholesterol, galglioside GM1 and class A scavenger receptor
contribute to infection by Brucella ovis and Brucella canis in
murine macropgaes. Microbes Infect 12: 246–251.
Martirosyan A, Moreno E & Gorvel JP (2011) An evolutionary
strategy for a stealthy intracellular Brucella pathogen.
Immunol Rev 240: 211–234.
McDonald WL, Jamaludin R, Mackereth G et al. (2006)
Characterization of a Brucella sp. strain as a marinemammal type despite isolation from a patient with spinal
osteomyelitis in New Zealand. J Clin Microbiol 44: 4363–
4370.
McKinney JD, Honer zu Bentrup K, Munoz-Elias EJ, Miczak
A, Chen B, Chan WT, Swenson D, Sacchettini JC, Jacobs
WR Jr & Russell DG (2000) Persistence of Mycobacterium
tuberculosis in macrophages and mice requires the glyoxylate
shunt enzyme isocitrate lyase. Nature 406: 735–738.
McLean DR, Russell N & Khan MY (1992) Neurobrucellosis:
clinical and therapeutic features. Clin Infect Dis 15: 582–590.
McQuiston JR, Vemulapalli R, Inzana TJ et al. (1999) Genetic
characterization of a Tn5-disrupted glycosyltransferase gene
homolog in Brucella abortus and its effect on
lipopolysaccharide composition and virulence. Infect Immun
67: 3830–3835.
Meador VP & Deyoe BL (1989) Intracellular localization of
Brucella abortus in bovine placenta. Vet Pathol 26: 513–515.
Meador VP, Hagemoser WA & Deyoe BL (1988)
Histopathologic findings in Brucella abortus-infected,
pregnant goats. Am J Vet Res 49: 274–280.
Memish ZA & Balkhy HH (2004) Brucellosis and international
travel. J Travel Med 11: 49–55.
Mizushima N (2004) Methods for monitoring autophagy. Int J
Biochem Cell Biol 36: 2491–2502.
Moreno E & Moriyon I (2006) The genus Brucella. The
Prokaryotes, Vol. 5 (Dworkin M, Falkow S, Rosenberg E,
Schleifer K & Stackebrandt E, eds), pp. 315–456. Springer,
New York.
Moreno E, Speth SL, Jones LM & Berman DT (1981)
Immunochemical characterization of Brucella
FEMS Microbiol Rev 36 (2012) 533–562
559
lipopolysaccharides and polysaccharides. Infect Immun 31:
214–222.
Moreno E, Cloeckaert A & Moriyon I (2002) Brucella
evolution and taxonomy. Vet Microbiol 90: 209–227.
Naroeni A & Porte F (2002) Role of cholesterol and the
ganglioside GM(1) in entry and short-term survival of
Brucella suis in murine macrophages. Infect Immun 70:
1640–1644.
Naroeni A, Jouy N, Ouahrani-Bettache S, Liautard JP & Porte
F (2001) Brucella suis-impaired specific recognition of
phagosomes by lysosomes due to phagosomal membrane
modifications. Infect Immun 69: 486–493.
Nymo IH, Tryland M & Godfroid J (2011) A review of
Brucella infection in marine mammals, with special
emphasis on Brucella pinnipedialis in the hooded seal
(Cystophora cristata). Vet Res 42: 93.
O’Callaghan D, Cazevieille C, Allardet-Servent A, Boschiroli
ML, Bourg G, Foulongne V, Frutos P, Kulakov Y & Ramuz
M (1999) A homologue of the Agrobacterium tumefaciens
VirB and Bordetella pertussis Ptl type IV secretion systems is
essential for intracellular survival of Brucella suis. Mol
Microbiol 33: 1210–1220.
Olsen SC (2010) Brucellosis in the United States: role and
significance of wildlife reservoirs. Vaccine 28(suppl 5): F73–
F76.
Orduna A, Orduna C, Eiros JM, Bratos MA, Gutierrez P,
Alonso P & Rodriguez Torres A (1991) Inhibition of the
degranulation and myeloperoxidase activity of human
polymorphonuclear neutrophils by Brucella melitensis.
Microbiologia 7: 113–119.
Paixao TA, Roux CM, den Hartigh AB, Sankaran-Walters S,
Dandekar S, Santos RL & Tsolis RM (2009) Establishment
of systemic Brucella melitensis infection through the
digestive tract requires urease, the type IV secretion system,
and lipopolysaccharide O antigen. Infect Immun 77: 4197–
4208.
Pantoja M, Chen L, Chen Y & Nester EW (2002)
Agrobacterium type IV secretion is a two-step process in
which export substrates associate with the virulence
protein VirJ in the periplasm. Mol Microbiol 45: 1325–
1335.
Pappas G (2010) The changing Brucella ecology: novel
reservoirs, new threats. Int J Antimicrob Agents 36(suppl 1):
S8–S11.
Parent MA, Goenka R, Murphy E, Levier K, Carreiro N,
Golding B, Ferguson G, Roop RM II, Walker GC & Baldwin
CL (2007) Brucella abortus bacA mutant induces greater
pro-inflammatory cytokines than the wild-type parent
strain. Microbes Infect 9: 55–62.
Pei J & Ficht TA (2004) Brucella abortus rough mutants are
cytopathic for macrophages in culture. Infect Immun 72:
440–450.
Pei J, Turse JE, Wu Q & Ficht TA (2006) Brucella abortus
rough mutants induce macrophage oncosis that requires
bacterial protein synthesis and direct interaction with the
macrophage. Infect Immun 74: 2667–2675.
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
560
Pei J, Turse JE & Ficht TA (2008a) Evidence of Brucella abortus
OPS dictating uptake and restricting NF-kappaB activation
in murine macrophages. Microbes Infect 10: 582–590.
Pei J, Wu Q, Kahl-McDonagh M & Ficht TA (2008b)
Cytotoxicity in macrophages infected with rough Brucella
mutants is type IV secretion system dependent. Infect
Immun 76: 30–37.
Pizarro-Cerda J, Moreno E, Sanguedolce V, Mege JL & Gorvel
JP (1998a) Virulent Brucella abortus prevents lysosome
fusion and is distributed within autophagosome-like
compartments. Infect Immun 66: 2387–2392.
Pizarro-Cerda J, Meresse S, Parton RG, van der Goot G, SolaLanda A, Lopez-Goni I, Moreno E & Gorvel JP (1998b)
Brucella abortus transits through the autophagic pathway
and replicates in the endoplasmic reticulum of
nonprofessional phagocytes. Infect Immun 66: 5711–5724.
Porte F, Liautard JP & Kohler S (1999) Early acidification of
phagosomes containing Brucella suis is essential for
intracellular survival in murine macrophages. Infect Immun
67: 4041–4047.
Porte F, Naroeni A, Ouahrani-Bettache S & Liautard JP (2003)
Role of the Brucella suis lipopolysaccharide O antigen in
phagosomal genesis and in inhibition of phagosomelysosome fusion in murine macrophages. Infect Immun 71:
1481–1490.
Qin QM, Pei J, Ancona V, Shaw BD, Ficht TA & de
Figueiredo P (2008) RNAi screen of endoplasmic reticulumassociated host factors reveals a role for IRE1alpha in
supporting Brucella replication. PLoS Pathog 4: e1000110.
Radhakrishnan GK, Yu Q, Harms JS & Splitter GA (2009)
Brucella TIR domain-containing protein mimics properties
of the Toll-like receptor adaptor protein TIRAP. J Biol
Chem 284: 9892–9898.
Radhakrishnan GK, Harms JS & Splitter GA (2011)
Modulation of microtubule dynamics by a TIR domain
protein from the intracellular pathogen Brucella melitensis.
Biochem J 439: 79–83.
Rajashekara G, Glover DA, Krepps M & Splitter GA (2005)
Temporal analysis of pathogenic events in virulent and
avirulent Brucella melitensis infections. Cell Microbiol 7:
1459–1473.
Rajashekara G, Glover DA, Banai M, O’Callaghan D & Splitter
GA (2006) Attenuated bioluminescent Brucella melitensis
mutants GR019 (virB4), GR024 (galE), and GR026
(BMEI1090-BMEI1091) confer protection in mice. Infect
Immun 74: 2925–2936.
Rambow-Larsen AA, Rajashekara G, Petersen E & Splitter G
(2008) Putative quorum-sensing regulator BlxR of Brucella
melitensis regulates virulence factors including the type IV
secretion system and flagella. J Bacteriol 190: 3274–3282.
Rambow-Larsen AA, Petersen EM, Gourley CR & Splitter GA
(2009) Brucella regulators: self-control in a hostile
environment. Trends Microbiol 17: 371–377.
Riley LK & Robertson DC (1984) Ingestion and intracellular
survival of Brucella abortus in human and bovine
polymorphonuclear leukocytes. Infect Immun 46: 224–230.
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
K. von Bargen et al.
Rittig MG, Kaufmann A, Robins A, Shaw B, Sprenger H,
Gemsa D, Foulongne V, Rouot B & Dornand J (2003)
Smooth and rough lipopolysaccharide phenotypes of
Brucella induce different intracellular trafficking and
cytokine/chemokine release in human monocytes. J Leukoc
Biol 74: 1045–1055.
Robertson GT & Roop RM Jr (1999) The Brucella abortus host
factor I (HF-I) protein contributes to stress resistance
during stationary phase and is a major determinant of
virulence in mice. Mol Microbiol 34: 690–700.
Rolan HG & Tsolis RM (2007) Mice lacking components of
adaptive immunity show increased Brucella abortus virB
mutant colonization. Infect Immun 75: 2965–2973.
Rolan HG, Xavier MN, Santos RL & Tsolis RM (2009) Natural
antibody contributes to host defense against an attenuated
Brucella abortus virB mutant. Infect Immun 77: 3004–3013.
Roop RM II, Jeffers G, Bagchi T, Walker J, Enright FM &
Schurig GG (1991) Experimental infection of goat fetuses
in utero with a stable, rough mutant of Brucella abortus. Res
Vet Sci 51: 123–127.
Roop RM II, Robertson GT, Ferguson GP, Milford LE,
Winkler ME & Walker GC (2002) Seeking a niche: putative
contributions of the hfq and bacA gene products to the
successful adaptation of the brucellae to their intracellular
home. Vet Microbiol 90: 349–363.
Roop RM II, Gaines JM, Anderson ES, Caswell CC & Martin
DW (2009) Survival of the fittest: how Brucella strains adapt
to their intracellular niche in the host. Med Microbiol
Immunol 198: 221–238.
Rossetti CA, Galindo CL, Garner HR & Adams LG (2010)
Selective amplification of Brucella melitensis mRNA from a
mixed host-pathogen total RNA. BMC Res Notes 3: 244.
Rouot B, Alvarez-Martinez MT, Marius C, Menanteau P,
Guilloteau L, Boigegrain RA, Zumbihl R, O’Callaghan D,
Domke N & Baron C (2003) Production of the type IV
secretion system differs among Brucella species as revealed
with VirB5- and VirB8-specific antisera. Infect Immun 71:
1075–1082.
Roux CM, Rolan HG, Santos RL, Beremand PD, Thomas TL,
Adams LG & Tsolis RM (2007) Brucella requires a
functional Type IV secretion system to elicit innate immune
responses in mice. Cell Microbiol 9: 1851–1869.
Salcedo SP, Marchesini MI, Lelouard H et al. (2008) Brucella
control of dendritic cell maturation is dependent on the
TIR-containing protein Btp1. PLoS Pathog 4: e21.
Samartino LE, Traux RE & Enright FM (1994) Invasion and
replication of Brucella abortus in three different trophoblastic
cell lines. Zentralbl Veterinarmed B 41: 229–236.
Sathiyaseelan J, Jiang X & Baldwin CL (2000) Growth of
Brucella abortus in macrophages from resistant and
susceptible mouse strains. Clin Exp Immunol 121: 289–294.
Scholz HC, Hubalek Z, Nesvadbova J et al. (2008a) Isolation of
Brucella microti from soil. Emerg Infect Dis 14: 1316–1317.
Scholz HC, Hubalek Z, Sedlacek I et al. (2008b) Brucella
microti sp. nov., isolated from the common vole Microtus
arvalis. Int J Syst Evol Microbiol 58: 375–382.
FEMS Microbiol Rev 36 (2012) 533–562
Brucella intracellular lifestyle
Scholz HC, Nockler K, Gollner C et al. (2010) Brucella
inopinata sp. nov., isolated from a breast implant infection.
Int J Syst Evol Microbiol 60: 801–808.
Scian R, Barrionuevo P, Giambartolomei GH, Fossati CA,
Baldi PC & Delpino MV (2011) Granulocyte-macrophage
colony-stimulating factor- and tumor necrosis factor alphamediated matrix metalloproteinase production by human
osteoblasts and monocytes after infection with Brucella
abortus. Infect Immun 79: 192–202.
Scurlock BM & Edwards WH (2010) Status of brucellosis in
free-ranging elk and bison in Wyoming. J Wildl Dis 46:
442–449.
Seleem MN, Boyle SM & Sriranganathan N (2008) Brucella: a
pathogen without classic virulence genes. Vet Microbiol 129:
1–14.
Sengupta D, Koblansky A, Gaines J, Brown T, West AP, Zhang
D, Nishikawa T, Park SG, Roop RM II & Ghosh S (2010)
Subversion of innate immune responses by Brucella through
the targeted degradation of the TLR signaling adapter, MAL.
J Immunol 184: 956–964.
Sieira R, Comerci DJ, Sanchez DO & Ugalde RA (2000) A
homologue of an operon required for DNA transfer in
Agrobacterium is required in Brucella abortus for virulence
and intracellular multiplication. J Bacteriol 182: 4849–4855.
Sieira R, Comerci DJ, Pietrasanta LI & Ugalde RA (2004)
Integration host factor is involved in transcriptional
regulation of the Brucella abortus virB operon. Mol
Microbiol 54: 808–822.
Sieira R, Arocena GM, Bukata L, Comerci DJ & Ugalde RA
(2010) Metabolic control of virulence genes in Brucella
abortus: HutC coordinates virB expression and the histidine
utilization pathway by direct binding to both promoters.
J Bacteriol 192: 217–224.
Silva TM, Costa EA, Paixao TA, Tsolis RM & Santos RL
(2011) Laboratory animal models for brucellosis research.
J Biomed Biotechnol 2011: 518323.
Sohn AH, Probert WS, Glaser CA, Gupta N, Bollen AW,
Wong JD, Grace EM & McDonald WC (2003) Human
neurobrucellosis with intracerebral granuloma caused by a
marine mammal Brucella spp. Emerg Infect Dis 9: 485–488.
Sola-Landa A, Pizarro-Cerda J, Grillo MJ, Moreno E, Moriyon
I, Blasco JM, Gorvel JP & Lopez-Goni I (1998) A twocomponent regulatory system playing a critical role in plant
pathogens and endosymbionts is present in Brucella abortus
and controls cell invasion and virulence. Mol Microbiol 29:
125–138.
Spera JM, Ugalde JE, Mucci J, Comerci DJ & Ugalde RA
(2006) A B lymphocyte mitogen is a Brucella abortus
virulence factor required for persistent infection. P Natl
Acad Sci USA 103: 16514–16519.
Starr T, Ng TW, Wehrly TD, Knodler LA & Celli J (2008)
Brucella intracellular replication requires trafficking through
the late endosomal/lysosomal compartment. Traffic 9: 678–
694.
Steele KH, Baumgartner JE, Valderas MW & Roop RM II
(2010) Comparative study of the roles of AhpC and KatE as
FEMS Microbiol Rev 36 (2012) 533–562
561
respiratory antioxidants in Brucella abortus 2308. J Bacteriol
192: 4912–4922.
Swartz TE, Tseng TS, Frederickson MA et al. (2007) Bluelight-activated histidine kinases: two-component sensors in
bacteria. Science 317: 1090–1093.
Taminiau B, Daykin M, Swift S, Boschiroli ML, Tibor A,
Lestrate P, De Bolle X, O’Callaghan D, Williams P &
Letesson JJ (2002) Identification of a quorum-sensing signal
molecule in the facultative intracellular pathogen Brucella
melitensis. Infect Immun 70: 3004–3011.
Tatum FM, Detilleux PG, Sacks JM & Halling SM (1992)
Construction of Cu-Zn superoxide dismutase deletion
mutants of Brucella abortus: analysis of survival in vitro in
epithelial and phagocytic cells and in vivo in mice. Infect
Immun 60: 2863–2869.
Tatum FM, Morfitt DC & Halling SM (1993) Construction of
a Brucella abortus RecA mutant and its survival in mice.
Microb Pathog 14: 177–185.
Tiller RV, Gee JE, Frace MA, Taylor TK, Setubal JC,
Hoffmaster AR & De BK (2010a) Characterization of novel
Brucella strains originating from wild native rodent species
in North Queensland, Australia. Appl Environ Microbiol 76:
5837–5845.
Tiller RV, Gee JE, Lonsway DR, Gribble S, Bell SC, Jennison
AV, Bates J, Coulter C, Hoffmaster AR & De BK (2010b)
Identification of an unusual Brucella strain (BO2) from a
lung biopsy in a 52 year-old patient with chronic
destructive pneumonia. BMC Microbiol 10: 23.
Tisdale EJ, Kelly C & Artalejo CR (2004) Glyceraldehyde-3phosphate dehydrogenase interacts with Rab2 and plays an
essential role in endoplasmic reticulum to Golgi transport
exclusive of its glycolytic activity. J Biol Chem 279: 54046–
54052.
Tobias L, Cordes DO & Schurig GG (1993) Placental
pathology of the pregnant mouse inoculated with Brucella
abortus strain 2308. Vet Pathol 30: 119–129.
Tolomeo M, Di Carlo P, Abbadessa V, Titone L, Miceli S,
Barbusca E, Cannizzo G, Mancuso S, Arista S & Scarlata F
(2003) Monocyte and lymphocyte apoptosis resistance in
acute and chronic brucellosis and its possible implications
in clinical management. Clin Infect Dis 36: 1533–1538.
Turse JE, Pei J & Ficht TA (2011) Lipopolysaccharide-deficient
Brucella variants arise spontaneously during infection. Front
Microbiol 2: 54.
Ugalde RA (1999) Intracellular lifestyle of Brucella spp.
Common genes with other animal pathogens, plant
pathogens, and endosymbionts. Microbes Infect 1: 1211–
1219.
Ugalde JE, Czibener C, Feldman MF & Ugalde RA (2000)
Identification and characterization of the Brucella abortus
phosphoglucomutase gene: role of lipopolysaccharide in
virulence and intracellular multiplication. Infect Immun 68:
5716–5723.
Uzureau S, Godefroid M, Deschamps C, Lemaire J, De Bolle X
& Letesson JJ (2007) Mutations of the quorum sensingdependent regulator VjbR lead to drastic surface
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
562
modifications in Brucella melitensis. J Bacteriol 189: 6035–
6047.
Viadas C, Rodriguez MC, Sangari FJ, Gorvel JP, Garcia-Lobo
JM & Lopez-Goni I (2010) Transcriptome analysis of the
Brucella abortus BvrR/BvrS two-component regulatory
system. PLoS ONE 5: e10216.
Wang Y, Chen Z, Qiao F et al. (2009) Comparative
proteomics analyses reveal the virB of B. melitensis affects
expression of intracellular survival related proteins. PLoS
ONE 4: e5368.
Wang Y, Chen Z, Qiao F et al. (2010) The type IV secretion
system affects the expression of Omp25/Omp31 and the
outer membrane properties of Brucella melitensis. FEMS
Microbiol Lett 303: 92–100.
Wassenaar TM & Gaastra W (2001) Bacterial virulence: can we
draw the line? FEMS Microbiol Lett 201: 1–7.
Watanabe K, Tachibana M, Kim S & Watarai M (2009)
Participation of ezrin in bacterial uptake by trophoblast
giant cells. Reprod Biol Endocrinol 7: 95.
Watarai M, Makino S, Fujii Y, Okamoto K & Shirahata T
(2002) Modulation of Brucella-induced macropinocytosis by
lipid rafts mediates intracellular replication. Cell Microbiol 4:
341–355.
Watarai M, Kim S, Erdenebaatar J, Makino S, Horiuchi M,
Shirahata T, Sakaguchi S & Katamine S (2003) Cellular
prion protein promotes Brucella infection into macrophages.
J Exp Med 198: 5–17.
Weeks JN, Galindo CL, Drake KL, Adams GL, Garner HR &
Ficht TA (2010) Brucella melitensis VjbR and C12-HSL
regulons: contributions of the N-dodecanoyl homoserine
lactone signaling molecule and LuxR homologue VjbR to
gene expression. BMC Microbiol 10: 167.
Wells DH & Long SR (2003) Mutations in rpoBC suppress the
defects of a Sinorhizobium meliloti relA mutant. J Bacteriol
185: 5602–5610.
West NP, Sansonetti P, Mounier J et al. (2005) Optimization
of virulence functions through glucosylation of Shigella LPS.
Science 307: 1313–1317.
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
K. von Bargen et al.
Whatmore AM, Dawson CE, Groussaud P, Koylass MS, King
AC, Shankster SJ, Sohn AH, Probert WS & McDonald WL
(2008) Marine mammal Brucella genotype associated with
zoonotic infection. Emerg Infect Dis 14: 517–518.
Winter AJ, Schurig GG, Boyle SM, Sriranganathan N, Bevins
JS, Enright FM, Elzer PH & Kopec JD (1996) Protection of
BALB/c mice against homologous and heterologous species
of Brucella by rough strain vaccines derived from Brucella
melitensis and Brucella suis biovar 4. Am J Vet Res 57: 677–
683.
World Health Organization (2005) The Control of Neglected
Zoonotic Diseases. World Health Organization, Geneva.
Wu Q, Pei J, Turse C & Ficht TA (2006) Mariner mutagenesis
of Brucella melitensis reveals genes with previously
uncharacterized roles in virulence and survival. BMC
Microbiol 6: 102.
Yeoman KH, Delgado MJ, Wexler M, Downie JA & Johnston
AW (1997) High affinity iron acquisition in Rhizobium
leguminosarum requires the cycHJKL operon and the feuPQ
gene products, which belong to the family of twocomponent transcriptional regulators. Microbiology 143(Pt
1): 127–134.
Zhong Z, Wang Y, Qiao F et al. (2009) Cytotoxicity of
Brucella smooth strains for macrophages is mediated by
increased secretion of the type IV secretion system.
Microbiology 155: 3392–3402.
Zhou Y & Xie J (2011) The roles of pathogen small RNAs.
J Cell Physiol 226: 968–973.
Zwerdling A, Delpino MV, Barrionuevo P, Cassataro J,
Pasquevich KA, Garcia Samartino C, Fossati CA &
Giambartolomei GH (2008) Brucella lipoproteins mimic
dendritic cell maturation induced by Brucella abortus.
Microbes Infect 10: 1346–1354.
Zygmunt MS, Hagius SD, Walker JV & Elzer PH (2006)
Identification of Brucella melitensis 16M genes required for
bacterial survival in the caprine host. Microbes Infect 8:
2849–2854.
FEMS Microbiol Rev 36 (2012) 533–562