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Molecular survival strategies of Borrelia burgdorferi
Review
Molecular survival strategies of the Lyme
disease spirochete Borrelia burgdorferi
Sunit Kumar Singh and Hermann Josef Girschick
Lyme disease is a tick-transmitted disease caused by the
spirochete Borrelia burgdorferi. The bacterium adopts
different strategies for its survival inside the immunocompetent host from the time of infection until dissemination
in different parts of body tissues. The success of this
spirochete depends on its ability to colonise the host tissues
and counteract the host’s defence mechanisms. During
this process borrelia seems to maintain its vitality to ensure
long-term survival in the host. Borrelia’s proteins are
encoded by plasmid and chromosomal genes. These genes
are differentially regulated and expressed by different
environmental factors in ticks as well as in the mammalian
host during infection. In addition, antigenic diversity enables
the spirochete to escape host defence mechanisms and
maintain infection. In this review we focus on the differential
expression of proteins and genes, and further molecular
mechanisms used by borrelia to maintain its survival in the
host. In light of these pathogenetic mechanisms, further
studies on spirochete host interaction are needed to
understand the complex interplay that finally lead to host
autoimmunity.
Lancet Infect Dis 2004; 4: 575-83
Lyme disease is caused by a group of related tick-borne
spirochetes classified as Borrelia burgdorferi sensu lato
(including B burgdorferi sensu stricto, Borellia afzelii, and
Borellia garinii). They are transmitted by ticks of the Ixodes
species including Ixodes ricinus in Europe and Asia, Ixodes
persulcatus in eastern Europe and Asia, and Ixodes scapularis
and Ixodes pacificus in North America. After attachment,
ticks induce local inflammatory and immunological
responses against the components present in saliva of the
tick.1 To overcome the host immune responses, tick saliva
also contains substances that suppress or divert these
responses.1,2 In addition to these vector/host interactions,
B burgdorferi undergoes dynamic changes within the vector
during both acquisition and transmission, and likewise
within vertebrate hosts after transmission. To survive in
nature, B burgdorferi must sense its environment and
synthesise appropriate proteins for interactions with the
different tick and mammalian tissues. Although much
progress has been made in characterisation of the host
immune reaction, spirochetal factors responsible for
infectivity, immune evasion, and disease pathogenesis
remain largely obscure. Several studies have demonstrated
that different borrelia isolates are highly heterogeneous in
their phenotypic and genotypic properties. A high degree of
Infectious Diseases Vol 4 September 2004
variability exists in plasmid and protein profiles of different
B burgdorferi isolates.3 In this review, the role of major
proteins, their differential expression in different
environments, and protective molecular strategies adopted
by B burgdorferi to cope with the host’s immunological
attack are reviewed and discussed.
Biology and genomic organisation
B burgdorferi is a microaerophilic, wavelike shaped
bacterium. It is generally 20–30 m in length and 0·2–0·5
m in width. In addition to a typical bacterial plasma
membrane, it has an outer lipid membrane termed an outer
membrane sheath (figure 1). The space between the
protoplasmic cell cylinder and the outer membrane sheath is
called the periplasm, which contains the flagella.4,5 The
protoplasmic cylinder contains the cytoplasm. The genome
size is relatively small, about 1·5 mb.6 Borrelia has a linear
chromosome of 950 kb in length, and at least 21 extra
chromosomal DNA elements or plasmids.7 Since genes for
biosynthetic reactions are absent, growth of B burgdorferi in
vitro needs tissue culture medium supplemented with
mammalian serum.8 In addition to the linear chromosome,
B burgdorferi isolate B31 contains 12 linear plasmids (lp)
(lp5, lp17, lp21, lp25, lp28-1, lp28-2, lp28-3, lp28-4, lp36,
lp38, lp54, and lp56), and nine circular plasmids (cp) (cp9,
cp26, cp32-1, cp32-3, cp32-4, cp32-6, cp32-7, cp32-8, and
cp32-9).8 B burgdorferi has the largest number of plasmids
known for any bacterium. Genes related with pathogenicity
including outer surface protein genes (osp) are primarily
located on the plasmids.
Outer surface proteins
The borrelia strains possess several outer surface
lipoproteins (Osps). The first major Osps isolated were
termed alphabetically—namely OspA,9 OspB, OspC,10
OspD,11 OspE, and OspF.11–16 Afterwards these proteins were
termed according to their function or by their molecular
mass—eg, decorin binding protein, or lp6·6 for a 6·6 kDa
protein. Osps seem to interact with cellular and interstitial
components of the tick and the mammalian tissue. During
feeding of the tick in addition to migration and transmission
SKS is at the Department of Paediatric Rheumatology, Children’s
Hospital, University of Würzburg, Germany.
Correspondence: Dr Hermann Girschick, Paediatric Rheumatology,
Children’s Hospital, University of Würzburg, Josef-Schneider-Str 2,
97080 Würzburg, Germany. Tel +49 931 201 27728;
fax +49 931 201 27242;
email [email protected]
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Molecular survival strategies of Borrelia burgdorferi
Outer membrane
Multiple
wavelike-shaped
periplasmic
flagella
Transmission electron
micrographs of
Borrelia burgdorferi
Protoplasmic
cylinder
Embedded
proteins
Outer
membrane
Periplasmic
flagella
Protoplasmic
cylinder
FlaB
FlaA
Anchor of flagella
in cell membrane
Figure 1. Ultrastructural morphology of B burgdorferi electron micrographs and schematic line drawings illustrate the structural morphology of
B burgdorferi. The spirochetes consist of a protoplasmic cylinder covered by the cell membrane. A second outer membrane covers the periplasm
containing the flagella. Flagella consist of two major proteins, flagellin A and flagellin B (FlaA and FlaB).
of B burgdorferi, many borrelial lipoproteins (OspC, OspE,
and OspF) have been shown to be upregulated17 or
downregulated by B burgdorferi (OspA, OspB, and lp6·6).18
Many differentially expressed proteins are plasmid
encoded.18,19 B burgdorferi lacks lipopolysaccharide,20
suggesting that Osps, which possess a potent stimulatory
capacity on mammalian cells,21 are in part responsible for the
inflammation associated with an infection. Among these are
the OspE, OspF, and the outer surface E/F-like leader
peptide (Elp) paralogues,18,22 which together have been
termed OspE/F-related proteins (Erps).7,8,18
The most studied B burgdorferi membrane lipoprotein is
OspA expressed on borrelia in resting (unfed) nymphal and
adult ticks and in-vitro culture.23 OspA serves as an anchor
for the spirochete in the midgut of the unfed tick. Digestion
of blood is done intracellularly by tick gut cells using
endocytosis as the mode of uptake. OspA-mediated
adherence of spirochetes to the surface of gut cells might
prevent internalisation of spirochetes during initial blood
meal digestion (figure 2).6 Host antibody responses to OspA
might therefore interfere with the midgut colonisation.24
Some reports show that spirochetes in the midgut of feeding
ticks start to downregulate OspA.17,25 Therefore, suppression
of OspA expression during the tick’s blood meal could help
576
B burgdorferi’s detachment from the gut. Transversion into
the haemolymphatic fluid, subsequent migration to the
salivary glands, and ultimately transmission to the vertebrate
host could be helped.26 The role of OspA in transmission of
the pathogen is further illustrated by a report demonstrating,
that mice immunised with OspA were not protected from
infection when they were challenged with skin implants of
infected mice containing B burgdorferi. This study indirectly
suggested that host adapted spirochetes no longer produced
OspA.27 This finding was confirmed by in-vivo culture
experiments on B burgdorferi with dialysis membrane
chambers.28 During the blood meal, the population of the
spirochetes becomes more heterogeneous, because Osp
expression is quite variable in one single strain. After
transmission, the role of OspA expressed on B burgdorferi in
the mammalian host is not known. A correlation between
the presence of OspA specific antibodies and T lymphocytes
with primary stages of Lyme borreliosis as well as with
chronic antibiotic-resistant Lyme arthritis have been shown.
These studies indirectly suggested that OspA might again be
present at some stages inside the mammalian host.29
Another Osp seems to have a role in spirochete/host
interaction. OspC synthesis was shown to correlate with
borrelia’s migration from the tick midgut to the salivary
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Molecular survival strategies of Borrelia burgdorferi
Mammalian
host
OspA
OspC
Tick
OspC
Midgut
pC
Os
OspA
OspA
Erp
Salivary
glands
Erp and CRASPs
bind complement
Factor H FHL-1 regulators Factor H
Skin
and FHL-1
Factor H FHL-1
Complement C3
C3
C3
C3
components are
C3
recruited to
FactorH/FHL-1
C3
and subsequently
C3
inactivated
C3
Spirochetal
complement
resistance
CRASPs
Figure 2. Regulation of gene expression in B burgdorferi. Borrelia residing
in the midgut of ticks show upregulated outer surface protein, Osp A and
downregulated OspC expression on their surface. OspA is of major
relevance for the colonisation of the tick midgut. During tick feeding OspA
is downregulated, while OspC is upregulated when the spirochetes move
from the midgut into the salivary glands. Inside the host OspA is
downregulated, whereas OspC is upregulated, but shows a complex time
and location dependent expression pattern shown by three arrows
indicating variable upregulation Erp genes generally are upregulated in
the mammalian host. Erp proteins, in addition to complement acquiring
surface proteins (CRASPs), are of importance to inhibit complementmediated killing by the host’s innate immune system. Using Erp and
CRASP proteins, spirochetes are able to bind complement regulatory
proteins, Factor H and FHL-1 present in the host’s serum to their own
surface. Subsequently, complement components C3b, c, and d are
bound to Factor H and FHL-1 and are inactivated. Impaired activation of
the complement cascade in the alternative pathway represents an
important mechanism in spirochete transmission, and propagation of the
infection.
gland, suggesting a role in transmission.30 OspC is not
expressed by most spirochetes in unfed ticks.31 The synthesis
of OspC is upregulated by B burgdorferi in the midgut during
tick feeding.17,32,33 Once the spirochetes reach the tick’s
salivary gland, they begin to downregulate OspC production
again (figure 2). Antibodies against OspC block the
movement of spirochetes from the gut to the tick’s salivary
glands.30 Even though most bacteria evading the salivary
glands and entering the host did not produce OspC any
more, small numbers of bacteria still could have OspC on
their surface. This possibility might be sufficient to stimulate
the early OspC antibody response that is regularly reported
in early stages of Lyme disease.34
Each B burgdorferi spirochete contains a single ospC gene
copy.35 By contrast, individual spirochetes carry many Erp
operons on the cp32 plasmid.36,37 As many as ten different
cp32 family members have been identified in clonal
populations of B burgdorferi.38 A single bacterium can
express its entire Erp repertoire simultaneously.39 A variety of
names were used to describe erp genes including ospE, ospF,
elp, p21, bbk2·10, bbk2·11, pG, and upstream homology box
(UHB)-flanked genes.40,41 The last term was introduced
Infectious Diseases Vol 4 September 2004
Review
because these families share a common feature. They are
flanked at the 5 end by a highly conserved sequence called
UHB.42 The erp genes seen within an individual bacterium
often exhibit a considerable range of sequence variation.7
Hefty and colleagues39 classified the Erp polypeptides of
B burgdorferi strain 297 into three separate groups: OspErelated orthologues, OspF-related orthologues, and Elps,
which contain OspE/F-like leader peptides. Erp proteins are
located on the bacterial outer surface and have an important
role in complement resistance of B burgdorferi .43–45
Strategies of antigenic variation and immune
escape
Immunodominant antigens are commonly used to
distinguish strains of a pathogen. These antigens can vary
from one strain to another resulting in strain-specific
immune responses of vertebrate hosts.46 Different strains of
B burgdorferi express different OspC proteins in rodents.46
Even though each B burgdorferi spirochete contains one
single ospC gene copy, sequences are diverse and immune
responses against OspC seem to influence selection of the
pathogen.46 This diversity of an immunodominant antigen
between strains is called antigenic variation. The adaptive
immune system of infected vertebrates mounts an immune
response against the original infecting serotype, but this
specific response might be ineffective against new emerging
variants.46
Genetic recombination at the vls gene locus
Another mechanism that could contribute to B burgdorferi
survival is the recombination at the variable major protein
like sequence (vls) gene locus.47,48 The vlsE (vls expression
site) encodes a surface protein of 34 kDa with three defined
domains: two constant regions at the amino and carboxyl
termini and an internal variable segment.48,49 It has been
postulated that infection induces sequence changes and thus
alters the antigenic properties of the vlsE, and leads to
immune evasion through antigenic variation. The
generation of new antigenic variants is thought to occur
through the exchange of DNA cassettes by the process of
recombination. This recombination could potentially help
spirochetes to escape antibody-mediated defence against the
vlsE protein variants arising during infection.47,48 The vls gene
cluster consists of the single vlsE located near the right
telomere of lp28-1 and 15 silent cassettes upstream.48,50 The
infectivity of B burgdorferi strain B31 diminished after the
loss of vlsE bearing lp25 and lp28-1 plasmids51,52 suggesting
that these plasmids encode virulence factors. Unidirectional
gene conversion events between the silent cassettes and the
vlsE gene generate new vlsE variants.53 Tick feeding
stimulates the recombination process at the vlsE locus.34 The
three-dimensional structure of the vlse outer surface
lipoprotein, VlsE, shows that the most distal outer part of the
protein consists primarily of variable regions. The variable
regions on the outermost surface could thus mask the
invariant regions of the protein. By producing a myriad of
VlsE variants that do not bind effectively to anti-VlsE
antibodies elicited by previous versions of the protein,
B burgdorferi can stay one step ahead of the mammalian
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Molecular survival strategies of Borrelia burgdorferi
immune response.54 VlsE gene expression is downregulated
during tick feeding and then upregulated upon establishment of infection in the mammalian host.55
Lateral gene transfer
Bacteriophages have a role in naturally occurring lateral gene
transfer. They have been reported in association with many
spirochetes,56 including Borrelia species.57 Eggers and coworkers reported on a bacteriophage, which packaged the
32-kb circular plasmids (cp32, table) of the B burgdorferi
genome, which was used to transduce cp32 to other borrelia
strains. Transfer of cp32 DNA via phage particles from one
spirochete to another could be an important mechanism of
pathogenesis. In addition, lateral gene transfer between ospC
genes within the species, and between B burgdorferi sensu
stricto, B garinii, and B afzelii has been reported.58 Genes for
ospC from eight isolates of Borrelia valaisiana were cloned and
compared with the ospC from B burgdorferi sensu lato species.59
Frequent ospC gene transfer was shown between B valaisiana
and B garinii, as well as B valaisiana and B afzelii.60
Simultaneous infection of B valaisiana and B garinii or B afzelii
in ticks and birds61 could explain the high frequency of gene
exchange between different Borrelia species. A high frequency
of gene exchange could enable the spirochetes to react
effectively to environmental selection pressures.
Lipoprotein polymorphism
Molecular changes in genes encoding surface antigens could
provide sufficient antigenic variation to support persistence of
the pathogen.62 OspA shows little heterogeneity within species,
supposedly because it is not under immune selection pressure
by vertebrate hosts.63 OspC as an antigen is, however, much
more polymorphic than OspA.16,63 OspC diversity can be
prompted by several mechanisms, including host-stimulated
immunological selection, gene transfer, intragenomic gene
recombination, and effects of environmental constraints. Of
these factors, selection pressure might be the major force in
maintaining the variation of OspC.64,17 To escape selective
immunological pressure caused by the host, individual
spirochetal strains can change their ospC gene sequences, and
thus cause variation of the ospC gene.
By contrast with ospC genes, erp gene sequences inside
individual strains remain considerably constant. Erp loci
of borrelia re-isolated from laboratory mice after 1 year of
infection were identical to those of the inoculated bacteria.
This finding shows that the erp gene sequences remain
considerably stable during the infection of an individual
mammal.38,41 However, comparative sequence analysis of
ospE gene family members from various borrelia strains
show the existence of hypervariable regions, indicating that
these sequences have been influenced to some extent by
different molecular events like recombinations or mutations
acting in an evolutionary time frame.42,65 Thus, the high
variation in protein sequence translated from erp genes,
compared with ospC, is based on the high number of
different but related genes arranged on up to at least ten
different cp32 plasmids.
There are several different gene families in Lyme disease
spirochetes that enhance the antigenic diversity during
infection in their natural reservoirs or in the human host,
either by genetic variation (eg, ospC), or by recruiting a
variety of closely related genes out of one family (eg, erp).
Continual genomic change in its gene repertoire, differential
expression of gene families,66,67 and the diversity introduced
by lateral transfer of plasmids15 could provide the Lyme
disease spirochetes with sufficient genetic and antigenic
diversity to be able to react to the host’s immune attack and
thus maintain chronic infection.
Kinetics of expression, location, and function of B burgdorferi genes
Gene/Gene
product
OspA
lp54
Predominant expression Function
in location
Tick
Anchors spirochetes to tick midgut
OspC
cp26
Tick/mammal
Migration of spirochete from tick
midgut to salivary gland
ErpA/I/N, ErpP,
ErpC, and ErpX
DbpA
DbpB
p66
vls
BgP
CRASP
cp32
Mammal
lp54
lp54
Chromosome
lp28-1
Chromosome
Lp54 and cp32
Mammal
Mammal
Mammal
Mammal
Mammal
Mammal
oppA-IV
oppA-V
Bdr D
cp26
lp54
cp32
Mammal
Mammal
Tick/mammal
Bdr E
cp32
Tick/mammal
spoT
Chromosome
Tick/mammal?
Inactivation of the host alternative
complement pathway
Binds decorin
Binds decorin
Binds integrin
Promotes spirochetal immune evasion
Binds glycosaminoglycans
Blocks complement activation
of the host
Environmental adaptation
Environmental adaptation
Sensing and transducing
environmental signals
Sensing and transducing
environmental signals
Important role during fatty
acid starvation ?
578
Gene location
Kinetics/pathogenetic mechanisms
Expressed in unfed tick and
downregulated during tick feeding
Upregulated during migration from tick midgut
to salivary gland but downregulated during
dissemination in mammalian host
Expressed/upregulated in mammalian
host/infection
Responsible for colonisation of the host
Responsible for colonisation of the host
Responsible for colonisation of the host
Recombination leads to antigenic variation
Responsible for colonisation of the host
Upregulated in mammalian host
Upregulated with temperature
Unclear
Expressed in fed and unfed ticks,
induced by serum deprivation
Expressed in fed and unfed ticks,
induced by serum deprivation
Upregulated during fasting/fatty acid
starvation
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Molecular survival strategies of Borrelia burgdorferi
Complement regulator-acquiring surface proteins
(CRASPs)
The complement system of the host’s innate immunity is
important in the first-line defence against invading microbes.
Complement activation destroys bacterial pathogens by
coating them with opsonising molecules (C1q, C3b, and
iC3b) after entry in the human host. This process can be
initiated by antibodies or antibody-independent mechanisms
via direct activation of the classic or alternative pathways.
Assembly of the complement membrane attack complex
usually leads to lysis of Gram-negative bacteria; however,
microbes have evolved effective means to escape this attack.
Microorganisms can evade the destructive effects of this
powerful defence system by binding host complement
regulators of the alternative pathway directly to their surfaces,
leading to inhibition of the complement activation cascade
(figure 2).68,69 B burgdorferi expresses up to five distinct
proteins on its surface (CRASP-1, CRASP-2, CRASP-3,
CRASP-4, and CRASP-5), which display different and unique
binding properties for host immune regulators.70 CRASPs
bind to two major human fluid-phase complement regulators
of the alternative pathway, factor H and factor H like protein1 (FHL-1/reconnectin). This binding can inactivate newly
formed C3b (figure 2).
CRASPs of borrelia have been given names according to
the species of origin, Ba for B afzelii and Bb for B burgdorferi.
Both B afzelii and B burgdorferi contain proteins of identical
binding profiles.69 BaCRASP-1, BbCRASP-1, and BbCRASP-2
bind FHL-1/reconectin strongly but show weak interaction
with factor H.69 By contrast, both of the B afzelii proteins
(BaCRASP-4 and BaCRASP-5) and the three B burgdorferi
proteins (BbCRASP-3, BbCRASP-4, and BbCRASP-5) bind to
factor H, but not FHL-1/reconectin.69 CRASP proteins do
orient amino complementary domains of attached FHL1/reconectin and factor H in such a way that they do not loose
their complement regulatory function.71 This function
prevents the formation of cytolytic complement activation
products on the borrelial surface. Erp proteins are also able
to bind complement inhibitory factor H (figure 2). Like
CRASPs, Erp proteins (OspE, OspF,62,72 Elps,22 p21,73 ErpA,
and ErpP73) can interact with complement regulators.74 Each
Erp protein exhibits different relative affinities for the
complement inhibitors of various potential animal hosts. The
presence of many Erp proteins on the surface can allow a
single B burgdorferi bacterium to resist complement-mediated
killing in any of the potential hosts that it might infect.75
Kurtenbach and co-workers76 reported that B burgdorferi also
binds to other complement control proteins in the tick
midgut and thus is protected against complement-mediated
lysis. Complement resistance might therefore represent one
major pathogenic factor favouring spirochete transmission to
the host. Of note, exposure to the host’s complement cascade
takes place not only in the gut, but also in the subsequent steps
of spirochete transmission into the host.
Role of tissue localisation of B burgdorferi in survival
and immune escape
Persistence of B burgdorferi in bradytrophic tissue, deep
invaginations of the cell membrane, or even in the cytosol of
Infectious Diseases Vol 4 September 2004
Review
local joint cells, might contribute to the chemotherapeutic
resistance and interference with immune clearance.77
Peripheral blood fibrocytes are a circulating leucocyte
subpopulation.78 B burgdorferi was shown to bind to fibrocytes
in vitro, and to reside within deep invaginations on the cell
surface without being phagocytosed.63,79 This process of hiding
could be a mechanism of protection for B burgdorferi avoiding
the attack of the immune system.63 B burgdorferi can be
recovered a long time after initial infection, even in
ceftriaxone-treated fibroblast cultures, suggesting that it can
resist eradication by host defence mechanisms as well as
antibiotics.80
We have reported that human synovial cells could be a
target for intracellular persistence of B burgdorferi after the
bacteria have passed through the endothelial barriers.81,82 Ma
and colleagues83 reported the intracellular localisation of
B burgdorferi within human endothelial cells. They likewise
reported that internalisation of B burgdorferi into endothelial
cells seemed to be mediated by a constitutively expressed
endothelial receptor, but not by adhesion molecules.83
Montgomery and colleagues84 also reported that nonopsonised B burgdorferi could enter macrophages rapidly,
resulting mainly in degradation but occasionally in apparent
intracellular persistence. Persistence of spirochetes within
macrophages might provide a possible reservoir for chronic or
recurrent Lyme disease in human beings.84 In an experimental
murine Lyme disease model spirochetes have been detected
intracellularly in cardiac myocytes.85 Even though there is
experimental evidence suggesting intracellular persistence
from in vitro and in vivo studies, reports on patients and
human tissues supporting these findings are very rare.86 On
the other hand, it has been reported several times that borrelia
can be detected extracellularly in bradytrophic tissue like
ligaments and collagen tissue of Lyme disease patients, or in
vitro models.85,87–89 Molecular analysis for B burgdorferi DNA in
synovial fluid or tissue samples revealed a higher detection
rate in the tissue samples.90 PCR of synovial tissue biopsies,
however, does not give information on a potential
intracellular or extracellular location. In addition, there are
reports of extracellular matrix localisation of B burgdorferi in
tissues other than joints.91,92 Thus, in conclusion, B burgdorferi
in general seems to reside extracellularly. So far, intracellular
location or persistence seems to be a rare event. However,
because of the scarcity of spirochetes in affected tissues, and
the difficulty in detecting them, further studies are
needed to assess modes of spirochetal long-term persistence.
Adaptive responses of B burgdorferi to variable
environmental factors
During ticks feeding, the spirochetes encounter environmental
changes caused by the influx of mammalian blood into the
midgut. Spirochetes encounter a rise in temperature and a fall
in pH from 7·4 to 6·8.93 At the time of tick feeding the
spirochetes change their surface expression of the outer
surface lipoproteins,17,94 as shown in figure 2. The synthesis of
OspC,17,95 Bbk32, and Bbk50 (B burgdorferi fibronectin binding
protein) proteins94 is induced specifically during the course of
the tick feeding. To survey more broadly B burgdorferi’s
adaptation, various in-vitro model systems have been used to
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Molecular survival strategies of Borrelia burgdorferi
mimic certain environmental conditions. For example, raised
temperature, reduced pH, and an expansion in B burgdorferi
cell density are conditions that ostensibly mimic those during
tick engorgement. They all have been shown to induce
a downregulation of OspA, lipoprotein P22, and an
upregulation of OspC, decorin binding protein A, multicopy
lipoprotein, and the alternative sigma factor RpoS96 (RNA
polymerase sigma S subunit) (table).
Carroll and colleagues97 reported that a lower pH of the
culture affects the transcription of ospC. This report
confirmed earlier findings showing that OspC was
downregulated in the midgut of unfed ticks.17 This showed
that ospC gene expression could be under the coordinate
regulation of temperature and pH. Kraiczy et al,69 noted an
upregulation of BaCRASP-1, BaCRASP-2, and BaCRASP-5 in
cultures of B afzelii by changing the temperature from 33°C to
37°C. He suggested that the upregulation of CRASPs leads to
complement resistance and, thus, could be relevant for
maintaining bacterial integrity during adaptation to the
human host. Erp proteins, which are regulated by
temperature as well,98 do not show any significant change in
their expression at different pH values, suggesting that not all
proteins under temperature regulation are likewise regulated
by pH.41,97 Other stimulating mammalian factors, that have a
role in regulation of Erp proteins, have been shown by
comparing the transcription of erp genes in spirochetes
cultivated within the mammalian host environment with
solely temperature-shifted spirochetes cultivated in vitro.39
These findings suggested that host-specific signals could
further enhance the positive stimulus provided by raised
temperature.99 Of interest, even inside the erp lipoprotein
family the expression of members in the midgut of the tick
varied during feeding. Some family members were expressed
early, while other members were expressed late during
transmission39 suggesting that there might be factors other
than temperature contributing to gene expression. The
induction of lipoprotein genes at different points in the
borrelial enzootic cycle shows that individual members of a
protein/gene family have evolved differently.39 It is likely that
B burgdorferi grown in BSK-H medium are receiving a set of
regulatory signals. Therefore, studies done in vitro can not
truly mimic the conditions experienced by B burgdorferi
during the infectious cycle.
One striking observation is that B burgdorferi does not
possess genes for the synthesis of aminoacids, fatty acids, and
other essential elements.8,100 Thus, the organism is dependent
on its environment to supply these essential nutrients. Other
bacteria typically possess many peptide transporter systems
with different specificities to help the use of peptides as a
source for aminoacids.100 However, the genome of
B burgdorferi seems to code for a single oligo peptide
transport system only (Opp).100 It is reported that a
chromosomal locus and two plasmid loci (on cp26 and lp54)
in B burgdorferi encode homologues of oligopeptide
permease components.101 During its natural life cycle
B burgdorferi needs to adapt to different hosts, because it
moves between the tick and the mammalian host. Bono and
co-workers101 reported that expression of oligopeptide
permease system A (oppA-ll and oppA-lV) are upregulated
580
during periods of stable nutrient availability. Expression of
oppA-ll seems to be specifically downregulated in spirochetes
grown in ticks compared with in vitro.100 By contrast, oppAlV gene expression is substantially upregulated in spirochetes
located in the mouse.100 Thus, expression of oppA-lV rises
with temperature. Differences in the expression and in
substrate specificity of OppA proteins have evolved to allow
the spirochete to adapt to different environmental niches
and requirements.
Another gene family of B burgdorferi has been identified,
the bdr (borrelia direct repeat-related gene) family. It has
been postulated that bdr proteins have a role in sensing and
transducing environmental signals.102 There are three distinct
subfamilies known as bdrD, bdrE, and bdrF genes.103–105
Production of bdr proteins is influenced to various degrees
by environmental conditions.105 Serum deprivation induced
the production of bdr proteins in B burgdorferi. The loss
of one or more bdr encoding plasmids did not alter
the production level of other bdr genes. In this condition the
loss of a plasmid could be complemented by a different
plasmid.
Morphological changes of B burgdorferi cells in response
to adverse environmental conditions (changes in pH,
depletion of metabolites, ageing, and exposure to
antibiotics) have been reported.106–108 The shape of the
spirochetes changes to blebs or cysts. Blebs have been shown
to contain DNA108,109 and might be implicated in the
exchange of genetic information.108 Unlike cysts, blebs are
not viable and not capable of transforming back into motile
spirochetes.108 In addition, B burgdorferi responded to serum
starvation by inducing the synthesis of two starvationinduced proteins (SSP),108 SSP o, which is a homologue of
OspA, and SSP s, a homologue of vlsE.108 An upregulation of
ospA was also reported during starvation.
Upregulation of the spoT gene (guanosine 3,5bispyrophosphate [ppGpp] synthase/pyrophosphohydrolase
gene) was shown during starvation. It expresses a protein that
acts as a bifunctional enzyme associated in both the
degradation and synthesis of ppGpp. PpGpp is an effector
molecule implicated in the physiological response of some
bacteria to nutritional stress.108 By contrast, serum feeding
suppressed spoT expression in B burgdorferi.110
B burgdorferi has to adapt tremendously to survive in its
life cycle. The ability to adapt to different environmental
conditions like temperature, pH, and cell density enables the
spirochetes to effectively cope with environmental changes
encountered in the transition from the cold-blooded tick to
the warm-blooded mammals. Since B burgdorferi mainly
seems to be located in bradytrophic mammalian tissue,
genes and their products implicated in nutritional transport
have to be used by the spirochete to survive in nonfavourable conditions and locations. On the other hand,
these locations might shelter the spirochetes to some extent
from the attack of the host’s immune system.
Proposed regulatory networks of gene
expression in B burgdorferi
To date, there is restricted information available on the
mechanisms of gene regulation in B burgdorferi.
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Review
Molecular survival strategies of Borrelia burgdorferi
RpoN-RpoS regulatory pathway
RpoS and RpoN (RNA polymerase sigma S/N subunit) are
stress-induced sigma factors of eubacteria and are implicated
in modulating the gene expression of bacterial virulence in
different environmental conditions.11,112 Therefore, the RpoS
and RpoN pathway represent a key regulatory network where
the RpoN protein controls the transcription of rpoS, which
ultimately governs the expression of OspC and decorin
binding protein A.93,112 Yang and co-workers113 reported the
involvement of the RpoN-RpoS pathway in the expression of
multicopy lipoproteins. Environmental signals appearing
during the tick’s blood meal could activate the RpoN activator
protein, which likely binds to an enhancer region upstream of
the rpoN gene leading to the synthesis of rpoS mRNA112 and
the further expression of important outer surface proteins.
Quorum sensing in B burgdorferi
To differentially synthesise proteins during its infection
cycle, B burgdorferi must possess regulatory networks to
sense its environment and regulate the expression of
required genes.114 Quorum sensing is a mechanism by which
bacteria monitor the presence of other bacteria in their
surrounding by producing and responding to signalling
molecules known as autoinducers.115 The sensory reception
of autoinducers can coordinate the production of virulence
factors during host infection.114,116 Two general types of
autoinducers have been reported: those that are specific for
the species producing it (eg, homoserine lactones or certain
polypeptides) and autoinducer type 2 (AI-2). The latter
molecules are conserved across the species. In B burgdorferi a
gene (BB0377) that shows homology to a quorum sensor has
likewise been identified and called luxS. The functional LuxS
enzyme enables B burgdorferi to synthesise an AI-2.114 AI-2mediated quorum sensing can function in both the
vertebrate host and the arthropod vector. Therefore it could
regulate the expression of a different set of proteins in
different environments.114 Miller and colleagues117 suggested
that during feeding of nymphal ticks, B burgdorferi produce
AI-2 to coordinate expression of Erp lipoproteins. The
complex life cycle and mechanism of pathogenesis means
that B burgdorferi precisely senses its environment and
regulates protein expression. An individual bacterium must
interact with many different tissue types during its cycle,
derive nutrition from warm-blooded hosts as well as from
the vector tick, and avoid clearance by the host’s and the
vector’s immune systems. Through quorum sensing, a whole
population of Lyme disease spirochetes can synchronise
production of proteins needed for infection and survival.118
Role of DNA supercoiling in gene regulation
DNA supercoiling can affect gene expression in bacteria. It
can serve as a signal transducer between sensing the
environmental conditions and altering the gene expression.
It has been reported that the temperature-mediated changes
References
1
2
Singh SK, Girschick HJ. Tick-host interactions
and their immunological implications in
tick-borne diseases. Current Science 2003; 85:
101–115.
3
Search strategy and selection criteria
Data for this review were identified by searches of Medline
and references from relevant articles. Many articles were
identified through searches of the files of the authors. Search
terms were “Lyme borreliosis”, “outer surface proteins AND
Borrelia”, “Borrelia burgdorferi AND immune evasion”,
“spirochetal persistence”, “Erp proteins AND Borrelia
burgdorferi”, “CRASP AND Borrelia burgdorferi”, “Borrelia
burgdorferi AND quorum sensing”, “antigenic variation AND
Borrelia”, “genetic recombination AND Borrelia”, “Borrelia
AND environment”, “OspC AND Borrelia”, “OspA AND
Borrelia”, “tick borne diseases”, “vector and host interaction
AND borrelia”, “intracellular persistence AND borrelia”,
“extracellular persistence AND borrelia”, “environmental
adaptation AND Borrelia burgdorferi”, “Gene regulation AND
Borrelia burgdorferi”, “host adaptation AND Borrelia
burgdorferi”, “spirochetal pathogenicity”, “gene transfer AND
Borrelia burgdorferi”, AND “protein polymorphism AND
Borrelia burgdorferi”. English language papers were reviewed.
of plasmid-encoded gene expression are because of changes
in DNA supercoiling.119,120 The rise in culture temperature
can reduce DNA twists, whereas a temperature fall can
enhance DNA twists.121 Thus, environmental factors that
differ between ticks and mammals, like temperature,
influence DNA supercoiling of B burgdorferi.122–124 The study
done by Alverson and colleagues,124 showed that the
temperature-induced changes in the expression of genes
ospA and ospC are a result of changes in DNA supercoiling.
This phenomenon suggested that DNA supercoiling is a
component of the transcriptional regulatory network.
Conclusion
B burgdorferi has a strong potential for acclimatisation in
the invertebrate as well as in the vertebrate host. During
this process it adopts different molecular strategies needed
for the survival in these different environments. The Lyme
spirochete B burgdorferi challenges the host immune system
through a broad variety of molecular mechanisms that
include complex regulation of differential gene expression.
The knowledge of mechanisms of spirochetal gene
regulation influencing spirochete/host interactions will
open new doors in the understanding of the molecular
pathogenesis of Lyme disease. Strategies in the treatment of
chronic, antibiotic-resistant Lyme disease might evolve
based on future research in this respect.
Acknowledgments
We acknowledge the continuous support of J Hacker, M Frosch,
C Faber, H Morbach, and CP Speer during the preparation of this
manuscript. We would particularly like to thank Gundula Girschick for
carefully reading the manuscript. Financial support by IZKF grant A26
of the University of Würzburg, Germany is acknowledged.
Conflicts of interest
None declared.
Wikel SK. Tick modulation of host immunity:
an important factor in pathogen transmission.
Int J Parasitol 1999; 29: 851–59.
Hughes CA, Kodner CB, Johnson RC. DNA analysis
of Borrelia burgdorferi NCH-1, the first northcentral
Infectious Diseases Vol 4 September 2004
http://infection.thelancet.com
4
US human Lyme disease isolate. J Clin Microbiol
1992; 30: 698–703.
Charon NW, Goldstein SF. Genetics of motility and
chemotaxis of a fascinating group of bacteria: the
spirochetes. Annu Rev Genet 2002; 36: 47–73.
581
For personal use. Only reproduce with permission from Elsevier Ltd.
Review
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
Goldstein SF, Buttle KF, Charon NW. Structural
analysis of the Leptospiraceae and Borrelia
burgdorferi by high-voltage electron microscopy.
J Bacteriol 1996; 178: 6539–545.
Pal U, Fikrig E. Adaptation of Borrelia burgdorferi in
the vector and vertebrate host. Microbes Infect 2003;
5: 659–66.
Casjens S, Palmer N, van Vugt R, et al. A bacterial
genome in flux: the twelve linear and nine circular
extrachromosomal DNAs in an infectious isolate of
the Lyme disease spirochete Borrelia burgdorferi.
Mol Microbiol 2000; 35: 490–516.
Fraser CM, Casjens S, Huang WM, et al. Genomic
sequence of a Lyme disease spirochaete, Borrelia
burgdorferi. Nature 1997; 390: 580–86.
Li H, Dunn JJ, Luft BJ, Lawson CL. Crystal structure
of Lyme disease antigen outer surface protein A
complexed with an Fab. Proc Natl Acad Sci USA
1997; 94: 3584–89.
Wilske B, Preac-Mursic V, Jauris S, et al.
Immunological and molecular polymorphisms of
OspC, an immunodominant major outer surface
protein of Borrelia burgdorferi. Infect Immun 1993;
61: 2182–911.
Norris SJ, Carter CJ, Howell JK, Barbour AG. Lowpassage-associated proteins of Borrelia burgdorferi
B31: characterization and molecular cloning of
OspD, a surface-exposed, plasmid-encoded
lipoprotein. Infect Immun 1992; 60: 4662–72.
Lam TT, Nguyen TP, Montgomery RR, Kantor FS,
Fikrig E, Flavell RA. Outer surface proteins E and F
of Borrelia burgdorferi, the agent of Lyme disease.
Infect Immun 1994; 62: 290–98.
Barbour AG. Antigenic variation of surface proteins
of Borrelia species. Rev Infect Dis 1988; 10 (suppl 2):
S399–402.
Marconi RT, Samuels DS, Garon CF. Transcriptional
analyses and mapping of the ospC gene in Lyme
disease spirochetes. J Bacteriol 1993; 175: 926–32.
Jauris-Heipke S, Liegl G, Preac-Mursic V, et al.
Molecular analysis of genes encoding outer surface
protein C (OspC) of Borrelia burgdorferi sensu lato:
relationship to ospA genotype and evidence of
lateral gene exchange of ospC. J Clin Microbiol 1995;
33: 1860–66.
Wilske B, Jauris-Heipke S, Lobentanzer R, et al.
Phenotypic analysis of outer surface protein C
(OspC) of Borrelia burgdorferi sensu lato by
monoclonal antibodies: relationship to genospecies
and OspA serotype. J Clin Microbiol 1995; 33:
103–09.
Schwan TG, Piesman J, Golde WT, Dolan MC,
Rosa PA. Induction of an outer surface protein on
Borrelia burgdorferi during tick feeding. Proc Natl
Acad Sci USA 1995; 92: 2909–13.
Hefty PS, Jolliff SE, Caimano MJ, Wikel SK,
Akins DR. Changes in temporal and spatial patterns
of outer surface lipoprotein expression generate
population heterogeneity and antigenic diversity in
the Lyme disease spirochete, Borrelia burgdorferi.
Infect Immun 2002; 70: 3468–78.
Wallich R, Brenner C, Kramer MD, Simon MM.
Molecular cloning and immunological
characterization of a novel linear-plasmid-encoded
gene, pG, of Borrelia burgdorferi expressed only in
vivo. Infect Immun 1995; 63: 3327–35.
Takayama K, Rothenberg RJ, Barbour AG. Absence
of lipopolysaccharide in the Lyme disease
spirochete, Borrelia burgdorferi. Infect Immun 1987;
55: 2311–13.
Ma Y, Weis JJ. Borrelia burgdorferi outer surface
lipoproteins OspA and OspB possess B-cell
mitogenic and cytokine-stimulatory properties.
Infect Immun 1993; 61: 3843–53.
Akins DR, Caimano MJ, Yang X, Cerna F,
Norgard MV, Radolf JD. Molecular and evolutionary
analysis of Borrelia burgdorferi 297 circular plasmidencoded lipoproteins with OspE- and OspF-like
leader peptides. Infect Immun 1999; 67: 1526–32.
Barbour AG, Tessier SL, Todd WJ. Lyme disease
spirochetes and ixodid tick spirochetes share a
common surface antigenic determinant defined by a
monoclonal antibody. Infect Immun 1983; 41:
795–804.
Anguita J, Hedrick MN, Fikrig E. Adaptation of
Borrelia burgdorferi in the tick and the mammalian
host. FEMS Microbiol Rev 2003; 27: 493–504.
de Silva AM, Telford SR, 3rd, Brunet LR,
Barthold SW, Fikrig E. Borrelia burgdorferi OspA is
an arthropod-specific transmission-blocking Lyme
disease vaccine. J Exp Med 1996; 183: 271–75.
Pal U, de Silva AM, Montgomery RR, Fish D,
Anguita J, Anderson JF, et al. Attachment of Borrelia
burgdorferi within Ixodes scapularis mediated by
outer surface protein A. J Clin Invest 2000; 106:
561–69.
582
Molecular survival strategies of Borrelia burgdorferi
27 Barthold SW, Fikrig E, Bockenstedt LK, Persing DH.
Circumvention of outer surface protein A immunity
by host-adapted Borrelia burgdorferi. Infect Immun
1995; 63: 2255–61.
28 Akins DR, Bourell KW, Caimano MJ, Norgard MV,
Radolf JD. A new animal model for studying Lyme
disease spirochetes in a mammalian host-adapted
state. J Clin Invest 1998; 101: 2240–50.
29 Akin E, McHugh GL, Flavell RA, Fikrig E, Steere AC.
The immunoglobulin (IgG) antibody response to
OspA and OspB correlates with severe and
prolonged Lyme arthritis and the IgG response to
P35 correlates with mild and brief arthritis. Infect
Immun 1999; 67: 173–81.
30 Gilmore RD Jr., Piesman J. Inhibition of Borrelia
burgdorferi migration from the midgut to the
salivary glands following feeding by ticks on OspCimmunized mice. Infect Immun 2000; 68: 411–14.
31 Fingerle V, Hauser U, Liegl G, Petko B,
Preac-Mursic V, Wilske B. Expression of outer
surface proteins A and C of Borrelia burgdorferi in
Ixodes ricinus. J Clin Microbiol 1995; 33: 1867–69.
32 Montgomery RR, Malawista SE, Feen KJ,
Bockenstedt LK. Direct demonstration of antigenic
substitution of Borrelia burgdorferi ex vivo:
exploration of the paradox of the early immune
response to outer surface proteins A and C in Lyme
disease. J Exp Med 1996; 183: 261–69.
33 Indest KJ, Ramamoorthy R, Philipp MT.
Transcriptional regulation in spirochetes.
J Mol Microbiol Biotechnol 2000; 2: 473–81.
34 Ohnishi J, Piesman J, de Silva AM. Antigenic and
genetic heterogeneity of Borrelia burgdorferi
populations transmitted by ticks. Proc Natl Acad Sci
USA 2001; 98: 670–75.
35 Sadziene A, Wilske B, Ferdows MS, Barbour AG.
The cryptic ospC gene of Borrelia burgdorferi B31 is
located on a circular plasmid. Infect Immun 1993;
61: 2192–95.
36 Stevenson B, Tilly K, Rosa PA. A family of genes
located on four separate 32-kilobase circular
plasmids in Borrelia burgdorferi B31. J Bacteriol 1996;
178: 3508–16.
37 Suk K, Das S, Sun W, et al. Borrelia burgdorferi genes
selectively expressed in the infected host. Proc Natl
Acad Sci USA 1995; 92: 4269–73.
38 Stevenson B, Miller JC. Intra- and interbacterial
genetic exchange of Lyme disease spirochete erp
genes generates sequence identity amidst diversity.
J Mol Evol 2003; 57: 309–24.
39 Hefty PS, Jolliff SE, Caimano MJ, Wikel SK,
Radolf JD, Akins DR. Regulation of OspE-related,
OspF-related, and Elp lipoproteins of Borrelia
burgdorferi strain 297 by mammalian host-specific
signals. Infect Immun 2001; 69: 3618–27.
40 Stevenson B, Zuckert, WR, Akins DR. Repetition,
conservation and variation: the multiple cp32
plasmids of Borrelia species. In: Saier MH, GarciaLara, J, eds. The spirochetes: molecular and cellular
biology. Oxford: Horizon press, 2001: 87–100.
41 Stevenson B. Borrelia burgdorferi erp (ospE-related)
gene sequences remain stable during mammalian
infection. Infect Immun 2002; 70: 5307–11.
42 Marconi RT, Sung SY, Hughes CA, Carlyon JA.
Molecular and evolutionary analyses of a variable
series of genes in Borrelia burgdorferi that are related
to ospE and ospF, constitute a gene family, and
share a common upstream homology box.
J Bacteriol 1996; 178: 5615–26.
43 El-Hage N, Babb K, Carroll JA, et al. Surface
exposure and protease insensitivity of Borrelia
burgdorferi Erp (OspEF-related) lipoproteins.
Microbiology 2001; 147(Pt 4): 821–30.
44 Stevenson B, Casjens S, Rosa P. Evidence of past
recombination events among the genes encoding the
Erp antigens of Borrelia burgdorferi. Microbiology
1998; 144 (Pt 7): 1869–79.
45 Stevenson B, Zuckert WR, Akins DR. Repetition,
conservation, and variation: the multiple cp32
plasmids of Borrelia species. J Mol Microbiol
Biotechnol 2000; 2: 411–22.
46 Barbour AG, Restrepo BI. Antigenic variation in
vector-borne pathogens. Emerg Infect Dis 2000; 6:
449–57.
47 Zhang JR, Hardham JM, Barbour AG, Norris SJ.
Antigenic variation in Lyme disease borreliae by
promiscuous recombination of VMP-like sequence
cassettes. Cell 1997; 89: 275–85.
48 Anguita J, Thomas V, Samanta S, Persinski R,
Hernanz C, Barthold SW, et al. Borrelia burgdorferiinduced inflammation facilitates spirochete
adaptation and variable major protein-like
sequence locus recombination. J Immunol 2001; 167:
3383–90.
49 Liang FT, Jacobs MB, Philipp MT. C-terminal
invariable domain of VlsE may not serve as target
for protective immune response against Borrelia
burgdorferi. Infect Immun 2001; 69: 1337–43.
50 Wang G, van Dam AP, Dankert J. Analysis of a VMPlike sequence (vls) locus in Borrelia garinii and Vls
homologues among four Borrelia burgdorferi sensu
lato species. FEMS Microbiol Lett 2001; 199: 39–45.
51 Labandeira-Rey M, Skare JT. Decreased infectivity in
Borrelia burgdorferi strain B31 is associated with loss
of linear plasmid 25 or 28-1. Infect Immun 2001; 69:
446–55.
52 Purser JE, Norris SJ. Correlation between plasmid
content and infectivity in Borrelia burgdorferi.
Proc Natl Acad Sci USA 2000; 97: 13865–870.
53 Zhang JR, Norris SJ. Gentic variation of the Borrelia
burgdorferi gene vlse involves cassette specific,
segmental gene conversion. Infect Immun 1998; 66:
3698–3704.
54 Eicken C, Sharma V, Klabunde T, et al. Crystal
structure of Lyme disease variable surface antigen
VlsE of Borrelia burgdorferi. J Biol Chem 2002; 277:
21691–96.
55 McDowell JV, Sung SY, Hu LT, Marconi RT.
Evidence that the variable regions of the central
domain of VlsE are antigenic during infection with
lyme disease spirochetes. Infect Immun 2002; 70:
4196–203.
56 Eggers CH, Casjens S, Hayes SF, et al. Bacteriophages
of spirochetes. J Mol Microbiol Biotechnol 2000; 2:
365–73.
57 Eggers CH, Samuels DS. Molecular evidence for a
new bacteriophage of Borrelia burgdorferi. J Bacteriol
1999; 181: 7308–13.
58 Balmelli T, Piffaretti JC. Analysis of the genetic
polymorphism of Borrelia burgdorferi sensu lato by
multilocus enzyme electrophoresis. Int J Syst Bacteriol
1996; 46: 167–72.
59 Wang G, van Dam AP, Schwartz I, Dankert J.
Molecular typing of Borrelia burgdorferi sensu lato:
taxonomic, epidemiological, and clinical
implications. Clin Microbiol Rev 1999; 12: 633–53.
60 Azulay RD, Azulay-Abulafia L, Sodre CT, Azulay DR,
Azulay MM. Lyme disease in Rio de Janeiro, Brazil.
Int J Dermatol 1991; 30: 569–71.
61 Kirstein F, Rijpkema S, Molkenboer M, Gray JS.
Local variations in the distribution and prevalence of
Borrelia burgdorferi sensu lato genomospecies in
Ixodes ricinus ticks. Appl Environ Microbiol 1997; 63:
1102–6.
62 Sung SY, McDowell JV, Carlyon JA, Marconi RT.
Mutation and recombination in the upstream
homology box-flanked ospE- related genes of the
Lyme disease spirochetes result in the development of
new antigenic variants during infection. Infect
Immun 2000; 68: 1319–27.
63 Nordstrand A, Barbour AG, Bergstrom S. Borrelia
pathogenesis research in the post-genomic and postvaccine era. Curr Opin Microbiol 2000; 3: 86–92.
64 Wang IN, Dykhuizen DE, Qiu W, Dunn JJ,
Bosler EM, Luft BJ. Genetic diversity of ospC in a
local population of Borrelia burgdorferi sensu stricto.
Genetics 1999; 151: 15–30.
65 Sung SY, McDowell JV, Carlyon JA, Marconi RT.
Mutation and recombination in the upstream
homology box-flanked ospE-related genes of the
Lyme disease spirochetes result in the development of
new antigenic variants during infection. Infect
Immun 2000; 68: 1319–27.
66 Akins DR, Porcella SF, et al. Evidence for in vivo but
not in vitro expression of a Borrelia burgdorferi outer
surface protein F (OspF) homologue. Mol Microbiol
1995; 18: 507–20.
67 Champion CI, Blanco DR, Skare JT, et al. A 9.0kilobase-pair circular plasmid of Borrelia burgdorferi
encodes an exported protein: evidence for expression
only during infection. Infect Immun 1994; 62: 2653–61.
68 Zipfel PF, Skerka C. FHL-1/reconectin: a human
complement and immune regulator with celladhesive function. Immunol Today 1999; 20: 135–40.
69 Kraiczy P, Skerka C, Brade V, Zipfel PF. Further
characterisation of complement regulator-acquiring
surface proteins of Borrelia burgdorferi. Infect Immun
2001; 69: 7800-809.
70 Zipfel PF, Skerka C, Hellwage J, et al. Factor H family
proteins: on complement, microbes and human
diseases. Biochem Soc Trans 2002; 30 (Pt 6): 971–78.
71 Kraiczy P, Skerka C, Kirschfink M, Brade V,
Zipfel PF. Immune evasion of Borrelia burgdorferi by
acquisition of human complement regulators FHL1/reconectin and Factor H. Eur J Immunol 2001; 31:
1674–84.
72 Stevenson B, Bono JL, Schwan TG, Rosa P. Borrelia
burgdorferi erp proteins are immunogenic in
mammals infected by tick bite, and their synthesis is
inducible in cultured bacteria. Infect Immun 1998; 66:
2648–54.
Infectious Diseases Vol 4 September 2004
http://infection.thelancet.com
For personal use. Only reproduce with permission from Elsevier Ltd.
Molecular survival strategies of Borrelia burgdorferi
73 Alitalo A, Meri T, Ramo L, et al. Complement
evasion by Borrelia burgdorferi: serum-resistant
strains promote C3b inactivation. Infect Immun
2001; 69: 3685–91.
74 Hellwage J, Meri T, Heikkila T, et al. The
complement regulator factor H binds to the surface
protein OspE of Borrelia burgdorferi. J Biol Chem
2001; 276: 8427–35.
75 Stevenson B, El-Hage N, Hines MA, Miller JC,
Babb K. Differential binding of host complement
inhibitor factor H by Borrelia burgdorferi Erp surface
proteins: a possible mechanism underlying the
expansive host range of Lyme disease spirochetes.
Infect Immun 2002; 70: 491–97.
76 Kurtenbach K, De Michelis S, Etti S, et al. Host
association of Borrelia burgdorferi sensu lato—the key
role of host complement. Trends Microbiol 2002; 10:
74–79.
77 Dorward DW, Fischer ER, Brooks DM. Invasion and
cytopathic killing of human lymphocytes by
spirochetes causing Lyme disease. Clin Infect Dis
1997; 25 (suppl 1): S2–8.
78 Grab DJ, Salim M, Chesney J, Bucala R, Lanners HN.
A role for peripheral blood fibrocytes in Lyme
disease? Med Hypotheses 2002; 59: 1–10.
79 Grab DJ, Lanners H, Martin LN, et al. Interaction
of Borrelia burgdorferi with peripheral blood
fibrocytes, antigen-presenting cells with the
potential for connective tissue targeting. Mol Med
1999; 5: 46-54.
80 Georgilis K, Peacocke M, Klempner MS. Fibroblasts
protect the Lyme disease spirochete, Borrelia
burgdorferi, from ceftriaxone in vitro. J Infect Dis
1992; 166: 440–44.
81 Girschick HJ, Huppertz HI, Russmann H, Krenn V,
Karch H. Intracellular persistence of Borrelia
burgdorferi in human synovial cells. Rheumatol Int
1996; 16: 125-32.
82 Girschick HJ, Meister S, Karch H, Huppertz HI.
Borrelia burgdorferi downregulates ICAM-1 on
human synovial cells in vitro. Cell Adhes Commun
1999; 7: 73–83.
83 Ma Y, Sturrock A, Weis JJ. Intracellular localisation
of Borrelia burgdorferi within human endothelial cell.
Infect Immunol 1991; 59: 671–78.
84 Montgomery RR, Nathanson MH, Malawista SE.
The fate of Borrelia burgdorferi, the agent for Lyme
disease, in mouse macrophages. Destruction,
survival, recovery. J Immunol 1993; 150: 909–15.
85 Pachner AR, Basta J, Delaney E, Hulinska D.
Localisation of Borrelia burgdorferi in murine Lyme
borreliosis by electron microscopy. Am J Trop Med
Hyg 1995; 52: 128–33.
86 Chary-Valckenaere I, Jaulhac B, Champigneulle J,
Piemont Y, Mainard D, Pourel J. Ultrastructural
demonstration of intracellular localisation of Borrelia
burgdorferi in Lyme arthritis. Br J Rheumatol 1998;
37: 468–70.
87 Dejmkova H, Hulinska D, Tegzova D, Pavelka K,
Gatterova J, Vavrik P. Seronegative Lyme arthritis
caused by Borrelia garinii. Clin Rheumatol 2002; 21:
330–34.
88 Haupl T, Hahn G, Rittig M, et al. Persistence of
Borrelia burgdorferi in ligamentous tissue from a
patient with chronic Lyme borreliosis. Arthritis
Rheum 1993; 36: 1621–26.
89 Franz JK, Fritze O, Rittig M, et al. Insights from a
novel three-dimensional in vitro model of lyme
arthritis: standardized analysis of cellular and
molecular interactions between Borrelia burgdorferi
and synovial explants and fibroblasts. Arthritis
Rheum 2001; 44: 151–62.
90 Priem S, Burmester GR, Kamradt T, Wolbart K,
Rittig MG, Krause A. Detection of Borrelia
burgdorferi by polymerase chain reaction in synovial
membrane, but not in synovial fluid from patients
with persisting Lyme arthritis after antibiotic
therapy. Ann Rheum Dis 1998; 57: 118–21.
91 Cadavid D, Pachner AR, Estanislao L, Patalapati R,
Barbour AG. Isogenic serotypes of Borrelia turicatae
show different localisation in the brain and skin of
mice. Infect Immun 2001; 69: 3389–397.
92 Cadavid D, O'Neill T, Schaefer H, Pachner AR.
Localisation of Borrelia burgdorferi in the nervous
system and other organs in a nonhuman primate
model of lyme disease. Lab Invest 2000; 80: 1043–54.
93 Yang X, Goldberg MS, Popova TG, et al.
Interdependence of environmental factors
influencing reciprocal patterns of gene expression in
virulent Borrelia burgdorferi. Mol Microbiol 2000; 37:
1470–79.
94 Fikrig E, Feng W, Barthold SW, Telford SR 3rd,
Flavell RA. Arthropod- and host-specific Borrelia
burgdorferi bbk32 expression and the inhibition of
spirochete transmission. J Immunol 2000; 164:
5344–51.
95 Schwan TG, Piesman J. Temporal changes in outer
surface proteins A and C of the lyme diseaseassociated spirochete, Borrelia burgdorferi, during the
chain of infection in ticks and mice. J Clin Microbiol
2000; 38: 382–88.
96 Revel AT, Talaat AM, Norgard MV. DNA
microarray analysis of differential gene expression in
Borrelia burgdorferi, the Lyme disease spirochete.
Proc Natl Acad Sci USA 2002; 99: 1562–67.
97 Carroll JA, Garon CF, Schwan TG. Effects of
environmental pH on membrane proteins in Borrelia
burgdorferi. Infect Immun 1999; 67: 3181–87.
98 Babb K, El-Hage N, Miller JC, Carroll JA, Stevenson
B. Distinct regulatory pathways control expression of
Borrelia burgdorferi infection-associated OspC and
Erp surface proteins. Infect Immun 2001; 69:
4146–53.
99 Fikrig E, Chen M, Barthold SW, et al. Borrelia
burgdorferi erpT expression in the arthropod vector
and murine host. Mol Microbiol 1999; 31: 281–90.
100 Wang XG, Lin B, Kidder JM, Telford S, Hu LT.
Effects of environmental changes on expression of
the oligopeptide permease (opp) genes of Borrelia
burgdorferi. J Bacteriol 2002; 184: 6198–206.
101 Bono JL, Tilly K, Stevenson B, Hogan D, Rosa P.
Oligopeptide permease in Borrelia burgdorferi:
putative peptide-binding components encoded by
both chromosomal and plasmid loci. Microbiology
1998; 144 ( Pt 4): 1033–44.
102 Roberts DM, Theisen M, Marconi RT. Analysis of
the cellular localization of Bdr paralogs in Borrelia
burgdorferi, a causative agent of lyme disease:
evidence for functional diversity. J Bacteriol 2000;
182: 4222–26.
103 Carlyon JF, Roberts DM, Marconi RT. Evolutionary
and molecular analyses of the Borrelia bdr super
gene family: delineation of distinct sub-families and
demonstration of the genus wide conservation of
putative functional domains, structural properties
and repeat motifs. Microb Pathog 2000; 28: 89–105.
104 Zuckert WR, Meyer J, Barbour AG. Comparative
analysis and immunological characterisation of the
Borrelia Bdr protein family. Infect Immun 1999; 67:
3257–66.
Infectious Diseases Vol 4 September 2004
http://infection.thelancet.com
Review
105 Roberts DM, Caimano M, McDowell J, et al.
Environmental regulation and differential
production of members of the Bdr protein family of
Borrelia burgdorferi. Infect Immun 2002; 70: 7033–41.
106 Barbour AG, Hayes SF. Biology of Borrelia species.
Microbiol Rev 1986; 50: 381–400.
107 Brorson O, Brorson SH. Transformation of cystic
forms of Borrelia burgdorferi to normal, mobile
spirochetes. Infection 1997; 25: 240–6.
108 Alban PS, Johnson PW, Nelson DR. Serumstarvation-induced changes in protein synthesis and
morphology of Borrelia burgdorferi. Microbiology
2000; 146 (Pt 1): 119–27.
109 Garon CF, Dorward DW, Corwin MD. Structural
features of Borrelia burgdorferi—the Lyme disease
spirochete: silver staining for nucleic acids. Scanning
Microsc Suppl 1989; 3: 109–15.
110 Concepcion MB, Nelson DR. Expression of spoT in
Borrelia burgdorferi during serum starvation.
J Bacteriol 2003; 185: 444–52.
111 Wosten MM. Eubacterial sigma-factors. FEMS
Microbiol Rev 1998; 22: 127–50.
112 Hubner A, Yang X, Nolen DM, Popova TG, Cabello
FC, Norgard MV. Expression of Borrelia burgdorferi
OspC and DbpA is controlled by a RpoN-RpoS
regulatory pathway. Proc Natl Acad Sci USA 2001;
98: 12724–729.
113 Yang XF, Hubner A, Popova TG, Hagman KE,
Norgard MV. Regulation of expression of the
paralogous Mlp family in Borrelia burgdorferi. Infect
Immun 2003; 71: 5012–20.
114 Stevenson B, Babb K. LuxS-mediated quorum
sensing in Borrelia burgdorferi, the lyme disease
spirochete. Infect Immun 2002; 70: 4099–105.
115 Taga ME, Bassler BL. Chemical communication
among bacteria. Proc Natl Acad Sci USA 2003; 100
(suppl 2): 14549–554.
116 Schauder S, Bassler BL. The languages of bacteria.
Genes Dev 2001; 15: 1468–80.
117 Miller JC, Stevenson B. Increased expression of
Borrelia burgdorferi factor H-binding surface
proteins during transmission from ticks to mice. Int
J Med Microbiol 2004; 293 (suppl 37): 120–5.
118 Stevenson B, von Lackum K, Wattier RL,
McAlister JD, Miller JC, Babb K. Quorum sensing by
the Lyme disease spirochete. Microbes Infect 2003; 5:
991–7.
119 Samuels DS, Garon CF. Coumermycin A1 inhibits
growth and induces relaxation of supercoiled
plasmids in Borrelia burgdorferi, the Lyme disease
agent. Antimicrob Agents Chemother 1993; 37: 46–50.
120 El-Hage N, Stevenson B. Simultaneous coexpression
of Borrelia burgdorferi Erp proteins occurs through a
specific, erp locus-directed regulatory mechanism.
J Bacteriol 2002; 184: 4536–43.
121 Rohde JR, Fox JM, Minnich SA. Thermoregulation
in Yersinia enterocolitica is coincident with changes
in DNA supercoiling. Mol Microbiol 1994; 12:
187–99.
122 Tse-Dinh YC, Qi H, Menzel R. DNA supercoiling
and bacterial adaptation: thermotolerance and
thermoresistance. Trends Microbiol 1997; 5: 323–6.
123 Lopez-Garcia P, Forterre P. DNA topology and the
thermal stress response, a tale from mesophiles and
hyperthermophiles. Bioessays 2000; 22: 738–46.
124 Alverson J, Bundle SF, Sohaskey CD, Lybecker MC,
Samuels DS. Transcriptional regulation of the ospAB
and ospC promoters from Borrelia burgdorferi. Mol
Microbiol 2003; 48: 1665–77.
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