Download Bacillus cereus immune escape: a journey

MINIREVIEW
Bacillus cereus immune escape: a journey within macrophages
Seav-Ly Tran & Nalini Ramarao
INRA, Unit
e MICALIS, AgroParisTech, UMR-1319, La Miniere, Guyancourt, France
Correspondence: Nalini Ramarao, INRA,
Unit
e MICALIS, UMR-1319, equipe GME,
La Mini
ere, 78280 Guyancourt, France.
Tel.: +33 1 30 83 36 36;
fax: +33 1 30 43 80 97;
e-mail: [email protected]
Present address: Seav-Ly Tran, Institute of
Food Research, Norwich Research Park,
Norwich, NR4 7UA-7TJ, UK
Norwich Medical School, University of East
Anglia, Norwich NR4 7UA-7TJ, UK
Received 26 June 2013; accepted 28 June
2013. Final version published online 13
August 2013.
Abstract
During bacterial infection, professional phagocytes are attracted to the site of
infection, where they constitute a first line of host cell defense. Their function
is to engulf and destroy the pathogens. Thus, bacteria must withstand the bactericidal activity of professional phagocytes, including macrophages to counteract the host immune system. Bacillus cereus infections are characterized by
bacteremia despite the accumulation of inflammatory cells at the site of infection. This implies that the bacteria have developed means of resisting the host
immune system. Bacillus cereus spores survive, germinate, and multiply in
contact with macrophages, eventually producing toxins that kill these cells.
However, the exact mechanism by which B. cereus evades immune attack
remains unclear. This review addresses the interaction between B. cereus and
macrophages, highlighting, in particular, the ways in which the bacteria escape
the microbicidal activities of professional phagocytes.
DOI: 10.1111/1574-6968.12209
MICROBIOLOGY LETTERS
Editor: Andre Klier
Keywords
Bacillus cereus; macrophage; immune escape.
Introduction
During bacterial infection, professional phagocytes, such
as monocytes, macrophages, dendritic cells, and polymorphonuclear leukocytes, are attracted to the site of infection, where they constitute a first line of host cell defense.
Their function is to engulf the infectious agents, internalizing, and destroying them (Underhill & Goodridge,
2012; Fig. 1). The uptake of infectious agents by phagocytic cells involves the binding of the pathogen to receptors
on the cell surface. Phagocytes can recognize pathogens
directly, through specific motifs called pathogenassociated molecular patterns (PAMPs) or after opsonization, a process in which the pathogen is coated with antibodies or complement factors (Kumar et al., 2011).
Interaction of the pathogen with the receptor triggers an
intracellular cascade, leading to the reorganization of the
actin machinery into a pseudopod that engulfs the pathogen. The internalized pathogens are then held within a
vacuole, the phagosome. The phagosome undergoes a ser-
FEMS Microbiol Lett 347 (2013) 1–6
ies of transformations by sequential fusion with the endosome and lysosome, culminating in the formation of a
mature degradative phagolysosome (Fairn & Grinstein,
2012). This organelle has diverse microbicidal weapons,
including an acidic environment, oxidative and nitrative
responses, and the production of digestive enzymes and
several anti- microbial peptides. These elements eventually
eliminate the pathogen.
Thus, if pathogens are to survive, to replicate, and to
disseminate within the host, they must adapt to the
highly hostile environment created by the immune
response. Interactions between host and pathogen should
not be seen as a static phenomenon, but as an ‘arms
race’, in which each opponent tries to respond as effectively as possible (Sarantis & Grinstein, 2012). This has
led to pathogens evolving highly sophisticated strategies
for overcoming the host immune response. As such,
phagocytosis may be seen as an opportunity or an obstacle for microbial pathogens, depending on the lifestyle of
the pathogens concerned. Intracellular bacteria have
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Published by John Wiley & Sons Ltd. All rights reserved
2
S.-L. Tran & N. Ramarao
Pathogen-associated molecular pattern
Bacterium
Pattern recognition receptor
Lysosome
Phagosome
Phagolysosome
Macrophage
DNA
RNA
Nucleus
Pro-inflammatory cytokines
Fig. 1. Macrophages are specialized leukocytes that respond to
invading pathogens by initiating phagocytosis and the synthesis and
release of pro-inflammatory cytokines. Microorganisms like bacteria
have PAMPs that are small molecular motifs conserved within a class
of microorganisms. Bacterial lipopolysaccharide, an endotoxin found
on the bacterial cell membrane of Gram-negative bacteria, is
considered to be the prototypical PAMP. Other PAMPs include
bacterial flagellin, lipoteichoic acid from Gram-positive bacteria,
peptidoglycan, and nucleic acid variants. PAMPs are recognized by
cells of the innate immune system and identified by pattern
recognition receptors on macrophages. The left side of the figure
illustrates the process of phagocytosis, which involves engulfment of
the bacterium into an intracellular vesicle called a phagosome,
phagosome–lysosome fusion to form a phagolysosome, degradation
of the bacterium by enzymes, and cellular release of the degraded
material by exocytosis. The right side of the figure illustrates that
bacterial binding to surface receptors of the macrophage also signals
the transcription of pro-inflammatory cytokines in the cell’s nucleus.
Cytokines are then produced in the cytoplasm, and these proinflammatory proteins are secreted from the cell to affect behavior of
nearby cells.
developed many sophisticated strategies for entering
phagocytic cells and surviving within them, whereas other
bacterial pathogens have evolved mechanisms for
preventing phagocytosis or escaping this process.
In this review, we consider an example of the bacterial
mechanisms used to counteract the host immune system.
We address the interaction between Bacillus cereus and
macrophages, focusing particularly on the ways in which
the bacteria escape the microbicidal activities of these
professional phagocytes.
B. cereus uptake and release by host
macrophages
Bacillus cereus belongs to the Bacillus cereus group, which
contains seven species of diverse sporulating Gram-positive bacteria (i.e. Bacillus anthracis, Bacillus thuringiensis,
and Bacillus cereus). These highly related bacilli are ubiquitous pathogenic bacteria, able to colonize hosts as
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Published by John Wiley & Sons Ltd. All rights reserved
diverse as insects and mammals. They differ in terms of
their plasmid-encoded factors: a capsule and toxins causing anthrax for B. anthracis, and insecticidal crystal proteins for B. thuringiensis (Schnepf et al., 1998; Mock &
Fouet, 2001). Apart from the specific genes borne by plasmids, the genomes of the three species, B. anthracis,
B. thuringiensis, and B. cereus, are very similar and the
genetic determinants required for nonspecies-specific
aspects of infection may be common to all the bacteria of
the B. cereus group (Ivanova et al., 2003).
Bacillus cereus is an emerging human pathogen initially
characterized as a causal agent of gastroenteritis. It is the
third most important cause of collective food poisoning
infections, after Salmonella and Staphylococcus aureus
(Anonymus, 2009). Bacillus cereus food poisoning is generally mild, but bloody diarrhea and emetic poisoning, which
may be fatal in some cases, have been reported (Mahler
et al., 1997; Lund et al., 2000; Dierick et al., 2005; Decousser et al., 2013). The number of cases of B. cereus foodborne infections is probably largely underreported, as the
reporting of such cases is not mandatory. Bacillus cereus is
also an opportunistic pathogen causing severe local and
systemic infections in humans (Auger et al., 2009; Bottone,
2010; Cadot et al., 2010; Ramarao, 2012). The increasing
number of opportunistic infections described highlights
the importance of studying this emerging pathogen. The
most frequently described conditions are endophthalmitis
(Callegan et al., 2006), necrotizing infections (Darbar
et al., 2005), endocarditis (Abusin et al., 2008), bacteremia
(Hernaiz et al., 2003), osteomyelitis (Schricker et al.,
1994), septicemia (Matsumoto et al., 2000), liver abscesses
(Latsios et al., 2003), pneumonia, and meningitis, particularly in neonates, leading to death of the infant within
days (Miller et al., 1997; Gray et al., 1999; Evreux et al.,
2007). These infections are characterized by bacteremia
despite the accumulation of inflammatory cells at the site
of infection (Hernandez et al., 1998). This implies that
the bacteria have developed by means of resisting the host
(Bouillaut et al., 2005; Ramarao & Lereclus, 2006; Gilois
et al., 2007; Tran et al., 2010; Kamar et al., 2013) and, in
particular, the action of the inflammatory cells, enabling
them to escape from the host immune system. Macrophages, through their strategic position throughout the
body, are key actors in immune surveillance. They play
an essential role in the sensing and elimination of invasive microorganisms, but also orchestrate the adaptive
immunity. Thus, macrophages form an essential barrier
that pathogens must overcome to be successful.
In the case of systemic disease or after crossing the
intestinal barrier upon ingestion, it is likely as spore that
B. cereus first interacts with the immune cells. Previous
studies have shown that B. cereus spores can survive
within macrophages, subsequently escaping from this hosFEMS Microbiol Lett 347 (2013) 1–6
Bacillus cereus and macrophages
tile environment (Ramarao & Lereclus, 2005). Spores are
composed of structured layers. The outermost layer of
B. cereus spores is called the exosporium; it forms a loose
balloon-like structure around the spore that may contribute to bacterial resistance to its host (Henriques &
Moran, 2007). The metalloprotease InhA1 is secreted into
the extracellular medium and is also a major proteinaceous component of the spore exosporium (Charlton
et al., 1999). We have previously demonstrated that
InhA1 is involved in the escape of B. cereus from macrophages, as B. cereus strains deleted in inhA1 remain transiently blocked within the cell (Ramarao & Lereclus,
2005). Moreover, heterologous production of the protein
in Bacillus subtilis is sufficient to promote escape from
macrophages. Interestingly, the B. cereus inhA1 mutant
can germinate even when trapped within the macrophage,
providing support for the notion that escape and germination are independent events. It seems likely that germination begins after uptake, as the bacteria are sensitive to
heat even in the presence of an intact external structure.
However, complete germination does not seem to be
required for escape, as germinating bacteria are found
extracellularly and vegetative bacteria are also able to
escape from macrophages after engulfment (Ramarao &
Lereclus, 2005).
B. cereus-induced cell toxicity
It remains unclear whether B. cereus escapes macrophage
by ‘hijacking’ an active cellular process or by causing the
lysis of the cells, either through the action of a cytotoxic
factor or mechanically, due to the accumulation of growing
numbers of intracellular bacteria. The lifestyle of B. cereus
suggests it is unlikely to remain intracellular. Once the vegetative bacteria are released from the cell, they must avoid
reuptake by the in situ phagocytes to escape the immune
response. It is tempting to speculate that InhA1 is involved
in the lysis of macrophages, as its production in B. subtilis
is sufficient to induce toxicity in macrophages although by
an unknown mechanism (Ramarao & Lereclus, 2005).
However, a B. cereus inhA1-deficient mutant although
avirulent (Guillemet et al., 2010) can nevertheless kill
macrophages, indicating that other cytotoxic factors are
involved. Pathogens frequently destroy macrophages by
inducing apoptosis (Navarre & Zychlinsky, 2000). Apoptosis is a programmed multistep cell death pathway that may
be activated in several ways (Elmore, 2007). Physiological
apoptosis plays an essential role in development, differentiation, and tissue homeostasis. Apoptosis can also occur as a
defense mechanism, when cells are damaged by an external
agent. This ability to alter inflammatory responses within
phagocytic cells may confer significant advantages on the
pathogen. The only B. cereus factor known to induce apopFEMS Microbiol Lett 347 (2013) 1–6
3
tosis is hemolysin II (HlyII; Tran et al., 2011a). HlyII is an
oligomeric b-barrel pore-forming toxin. Other toxins of
this group include the a-toxin of S. aureus, b-toxin of
Clostridium perfringens, and B. cereus cytotoxin K (CytK;
Ramarao & Sanchis, 2013). HlyII forms heptameric
transmembrane pores in erythrocytes and artificial membranes (Andreeva et al., 2006, 2007; Andreeva-Kovalevskaya et al., 2008). It induces the apoptosis of host
monocytes and macrophages in vivo, in a death receptordependent pathway (Tran et al., 2011a). Cell death occurs
in two steps: HlyII binds to dendritic cells and/or macrophages and induces the formation of a pore, leading to
transient membrane permeability (Tran et al., 2011b). The
formation of this pore eventually leads to the induction of
apoptosis in the cells. It remains unclear how HlyII interacts with macrophage surfaces to form pores, and it has
been reported that HlyII has no specific receptor in
erythrocytes (Andreeva et al., 2006). However, there is
some evidence for the existence of a specific receptor for
HlyII. First, the susceptibility of cells to HlyII depends
strongly on cell type. HlyII activity may even be specific
within a particular family of cell types, as a previous study
reported an effect on Caco2 cells (Andreeva et al., 2006),
whereas our data suggest that the purified toxin does not
induce permeability or apoptosis in HeLa cells, another
type of epithelial cell (Tran et al., 2011a). Moreover, the
related a-toxin of S. aureus appears to bind to the host cell
receptor phosphocholine (Valeva et al., 2006; Liang & Ji,
2007). This receptor binding may allow the protein to accumulate locally in microdomains enriched in cholesterol and
sphingolipids (lipid rafts). This results in high local concentrations, favoring toxin oligomerization and, thus, stable
membrane-anchored binding to target host cells, suggesting
that certain cell types have high-affinity toxin-binding sites
(Valeva et al., 2006). Thus, HlyII may bind to a specific
receptor possibly present in a lipid-rich microdomain.
The ability of HlyII to kill macrophages may account
for the persistence and dissemination of B. cereus in the
host. The induction of apoptosis by B. cereus may cause
tissue damage and compromise the antimicrobial immune
response, thereby promoting bacterial spread, leading to
the signs and symptoms of disease. The importance of
HlyII for virulence has been demonstrated in various
models (Sineva et al., 2009; Tran et al., 2011a) and by the
presence of a gene encoding this protein in several clinical
isolates of B. cereus (Cadot et al., 2010).
Iron and glucose regulate expression of the hlyII gene,
by activating the regulators Fur and HlyIIR, respectively
(Sineva et al., 2012; Guillemet et al., 2013; Tran et al.,
2013). Both iron and glucose are crucial for bacterial
multiplication and, thus, for the capacity of the bacterium
to colonize its host. HlyII probably induces host cell lysis
to provide the bacteria with access to nutrients. As a
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S.-L. Tran & N. Ramarao
Glucose and iron limiting environment
Glucose consumpƟon
by bacteria
HlyIIR Box
Iron sequestraƟon
by immune cells
Fur Box
Repression of HlyIIR
hlyII
hlyIIR
Repression of Fur
hlyII expression
HlyII
Macrophage apoptosis
Erythrocyte lysis
Iron and nutrient release
Bacterial proliferaƟon
Fig. 2. Model of the role and expression of hlyII during infection. As
long as iron and glucose are abundant in the bacterial environment,
glucose enters the bacteria as glucose 6P (blue rectangles) and binds
HlyIIR. Iron (purple circles) binds Fur. These binding events promote
the repressor activities of HlyIIR and Fur, leading to the HlyIIR- and Furbased transcriptional repression of hlyII gene expression. By contrast,
when glucose and iron become scarce, hlyII expression is activated.
HlyII is then released into the environment and induces macrophage
and erythrocyte lysis. The dead cells release their intracellular content,
providing access to metabolites that are essential for bacterial growth.
From Guillemet et al. (2013) and Tran et al. (2013).
model (Fig. 2), we suggest that when glucose is consumed
by the bacteria and iron is sequestered by phagocytic cells
as a natural host defense (Ratledge & Dover, 2000;
Weinberg, 2009), the HlyIIR and Fur repressors become
inactivated and hlyII expression is triggered. HlyII is then
produced and secreted by the bacteria, triggering the
death of hemocytes and macrophages (Tran et al., 2011a).
The contents of the cell are then released into the environment, providing the bacteria with access to nutrients,
allowing them to grow, and promoting a new cycle of
hlyII gene inhibition/expression.
B. cereus response to ROS/NOS
Bacillus cereus encounters oxidants, including superoxide
(O2 ), hydrogen peroxide, and nitric oxide (NO), when
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Published by John Wiley & Sons Ltd. All rights reserved
they germinate and grow in macrophages (MacMicking
et al., 1997; Shatalin et al., 2008). The exposure of B. cereus
to mild and lethal hydrogen peroxide concentrations modifies the expression of numerous genes (Ceragioli et al.,
2010), including those involved in the common response to
general stresses, such as groES, dnaK, and clp proteases.
Genes encoding catalases, thioredoxin reductases, and peroxidases are also induced to remove hydrogen peroxide from
the cells or from the extracellular environment. The induction of perR, iron, and manganese uptake systems suggests
that iron and manganese are involved in the response of
B. cereus to hydrogen peroxide. Tarasenko et al. (2008)
showed that treatment with glycoconjugates increases the
intracellular killing of B. cereus spores by inducing a dosedependent increase in macrophage nitrogen derivatives
(NO) production. Finally, the SOS response, which is activated when DNA is damaged, is induced, together with other
DNA repair and protection mechanisms, by exposure to
hydrogen peroxide, suggesting that exposure to oxygen or
nitrogen derivatives leads to protein and DNA damage
(Mols & Abee, 2011).
We have consistently shown that mutation frequency
decline (Mfd), a bacterial protein known to be involved in
DNA repair mechanisms (Savery, 2007), plays a crucial role
in bacterial resistance to the host nitrogen response
(N. Ramarao, unpublished data). Mfd is essential for
specific resistance to the deleterious effects of the nitrogen
stress imposed by host phagocytes. Moreover, a B. cereus
mfd mutant is avirulent and unable to survive NO stress
in vivo in two animal models (insects and mice). In Escherichia coli, Mfd is known to be required for DNA repair, after
UV irradiation, for example. However, Mfd had never been
implicated in bacterial pathogenicity or NO responses.
As Mfd is widely conserved in the bacterial kingdom,
these data highlight a novel mechanism that may be used
by a large spectrum of bacteria to overcome the host
immune response, including the mutagenic properties of
reactive nitrogen species. This might make it possible to
develop new, potentially universal antimicrobial strategies.
Conclusions
Technological advances in the last decade have facilitated
studies on the mechanisms of interaction between the
pathogen and its host in the context of infection. During
the establishment of infection, bacteria are confronted
and must deal with the immune response of their host.
Macrophages are one of the first actors in immunity. In
addition to their sentinel activities, they degrade pathogens by phagocytosis and activate the appropriate
immune response. The fine balance between the macrophage response and the ability of the pathogen to modulate the cellular response determines the outcome of
FEMS Microbiol Lett 347 (2013) 1–6
Bacillus cereus and macrophages
infection, and the number of known examples of bacteria
duping the immune system of their host is growing.
The interplay between pathogen and host should
certainly not be seen as static. Although similar strategies
are frequently used, the ways in which bacteria combine
them, the timing of their use and their targets differ with
the situation. Bacillus cereus has a genetic background very
similar to that of B. anthracis, another pathogen of the
B. cereus group. Many studies have investigated the way in
which B. anthracis escapes the immune system. However,
although the results of these studies can be used to guide
similar research for B. cereus, it would be dangerous to
assume that the immune evasion strategies of these two
species are the same. On the cell side, it is essential to avoid
the concept of a ‘bad’ or ‘good’ cellular response to the pathogen. The reality is far less black and white and seems to
involve a subtle fine-tuning of a combination of connected
cellular responses. This highlights the importance of using
an appropriate model to study host–pathogen interactions.
Finally, the conversion of some of these advances into
a true understanding of disease should make it possible
to identify weak points in the immune response that
could be corrected and the ‘Achilles’ heel’ of the pathogen, which could then be targeted by treatment. This next
step promises to be a great challenge.
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