Review
Adaptation of Borrelia burgdorferi in the vector and vertebrate host
Utpal Pal, Erol Fikrig *
Room 525A, Section of Rheumatology, Department of Internal Medicine,
Yale University School of Medicine, 300 Cedar Street, New Haven, CT 06520-8031, USA
Abstract
Borrelia burgdorferi sensu lato is the causative agent of Lyme disease, which afflicts both humans and some domestic animals.
B. burgdorferi, a highly evolved extracellular pathogen, uses several strategies to survive in a complex enzootic cycle involving a diverse range
of hosts. This review focuses on the unique adaptive features of B. burgdorferi, which are central to establishing a successful spirochetal
infection within arthropod and vertebrate hosts. We also discuss the regulatory mechanisms linked with the development of molecular
adaptation of spirochetes within different host environments.
© 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.
Keywords: Borrelia burgdorferi; Ticks; Adaptation
1. Introduction
Borrelia burgdorferi sensu lato is the group of spirochetes
causing Lyme disease and includes at least 10 genospecies
[1]. The three genospecies commonly associated with human
infections include B. burgdorferi sensu stricto, which is
widespread in both the USA and Europe, and B. afzelii and
B. garinii, which are primarily distributed in Europe [2]. The
clinical manifestations of Lyme borreliosis differ in North
America and Eurasia, possibly due to the genetic diversity
among different B. burgdorferi genospecies. B. garinii is
associated with neurologic diseases, while B. burgdorferi
sensu stricto and B. afzelii are more likely to cause arthritis
and cutaneous symptoms, respectively [3–5]. Different geno-
species seem to vary in their ability to survive in a given host:
for example, B. burgdorferi sensu stricto and B. garinii are
reported to persist in birds, whereas B. afzelii fails to survive
in avian hosts [6].
B. burgdorferi sensu lato is transmitted by Ixodes ticks
[7–9]. The nature of the enzootic cycle of B. burgdorferi in
specific geographic areas influences the incidence of human
infections. In the northeastern and north central United
States, B. burgdorferi sensu stricto is principally maintained
in a cycle involving larval and nymphal Ixodes scapularis
ticks and white-footed mice [2]. Occasionally, I. scapularis
nymphs or adults feed and transmit infection to a wide range
of vertebrates, including humans. Ixodes species in other
parts of the United States also harbor Lyme spirochetes, but
few ticks are infected in comparison to areas with a high
incidence of human Lyme disease [8,10]. Differences in
tick-host preferences may possibly explain the low preva-
lence of such infections. For example, I. pacificus in the west
and I. scapularis in the southeast feed on reptiles, which are
not susceptible to spirochete infection. I. neotomae, another
potential vector, feeds on rodents but rarely bites humans
[8,10]. In Europe and Asia, the Lyme disease pathogen is
generally maintained between small rodents and I. ricinus or
I. persulcatus [11].
2. Structure, genome, gene expression and regulation
The structure of B. burgdorferi sensu lato is typical of a
spirochete: a spiral or coil-shaped cell that is generally 20–30
µm in length and 0.2–0.5 µm in width. Individual spirochetes,
however, can vary in length, diameter, tightness and regular-
ity of the coils. The protoplasmic cylinder containing the
cytoplasm with its organelles and flagellar apparatus is cov-
ered by a periplasm and a lipoprotein-based outer surface
membrane [12]. Complete genome sequencing of B. burg-
dorferi sensu stricto (strain B31M1) disclosed several unique
adaptive features of the spirochete [13]. The genome size is
relatively small, approximately 1.5 megabases, consisting of
a linear chromosome of 950 kilobases and at least 21 extra-
* Corresponding author. Tel.: +1-203-785-2453;
fax: +1-203-785-7053.
E-mail address: erol.fikrig@yale.edu (E. Fikrig).
Microbes and Infection 5 (2003) 659–666
www.elsevier.com/locate/micinf
© 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.
DOI: 1 0 . 1 0 1 6 / S 1 2 8 6 - 4 5 7 9 ( 0 3 ) 0 0 0 9 7 - 2
chromosomal DNA elements or plasmids (620 kilobases
within nine linear and 12 circular plasmids) [16]. B. burgdor-
feri has the largest numbers of plasmids known for any
bacterium to date. Plasmid genes are of special interest, as
they contain many of the genes associated with spirochete
pathogenicity. The chromosome contains 853 genes coding
for a basic set of known eubacterial proteins that drive cell
cycle, growth and metabolism, with an unusual absence of
genes responsible for cellular biosynthetic reactions [13].
Plasmids encode another 535 genes, and 90% of the genes
have no convincing similarity to genes outside Borrelia ge-
nus, suggesting that they perform specialized functions pos-
sibly related to spirochete adaptation. Further experiments
demonstrated that some of the plasmids can be lost during
propagation of the bacteria in vitro, and loss of infectivity in
mice often parallels the loss of specific plasmids [14–18].
Most of the spirochete genome consist of linear DNA with
covalently closed hairpin ends or telomeres. B. burgdorferi
has evolved highly efficient enzymes to replicate such telo-
meric DNA structures. Recently ResT, a new class of telom-
ere resolvase, has been identified to be encoded by B. burg-
dorferi locus bbb03, which performs a highly efficient but
complex two-step DNA transesterification during replication
to generate covalently closed hairpin telomeres [19].
B. burgdorferi has evolved remarkable abilities to survive
in a wide range of organisms such as arthropods, and verte-
brates like birds and mammals. In contrast to other patho-
genic bacteria, B. burgdorferi has devoted a large portion of
its genome (more than 8% of coding genes or 150 genes)
towards producing lipoproteins [13,16]. Studies have dem-
onstrated the ability of the bacterium to alter its surface
structure by differential lipoprotein gene expression at vari-
ous stages of its life cycle in mammals and ticks, which is
likely to aid in host adaptation and immune evasion [20–27].
A few host molecules were also identified that are involved in
interactions with spirochete ligands and thus aid in bacterial
survivability in diverse host environments (Table 1). In addi-
tion to the transcriptional activation of selective genes, events
such as variable recombination also contribute to the alter-
ations of spirochete structure. As an example, the B.
burgdirferi vlsE locus consists of an active telomeric expres-
sion site flanked by a number of upstream silent vls cassettes
[28]. Recombination events between the vlsE gene and vls
cassettes occur in mammals [29] and most likely in feeding
ticks [30], producing a genetically diverse population of
spirochetes. Apart from controlling transcription or recombi-
nation events, spirochetes were also reported to generate
antigenetically diverse populations by modulating intracellu-
lar (surface vs. periplasmic) translocation of lipoproteins
[31].
A series of in vitro studies have identified a number of
environmental signals that are thought to regulate gene ex-
pression in spirochetes and includes temperature [27,32], pH
[33], cell density [34,35] and host factors [36,37]. Interest-
ingly, many of these regulated genes are differentially ex-
pressed in vivo and include hsp, mlp, dbpA, bbk 32, erp
(ospE/F -related), ospC and ospA [21,22,36,38–44]. Induc-
tion of lipoproteins belonging to large gene families such as
Mlp [45], Erp [44] as well as OspC [27,43] at higher tem-
perature may mimic temperature-induced changes in gene
expression that occur during tick feeding, when the spiro-
chetes are exposed to warm host blood. Likewise, lowered
pH changes the expression of a few spirochete genes in vitro
[33,46], and these genes may represent regulated genes dur-
ing the tick blood meal, because the pH of the tick gut drops
during the feeding process. However, the regulation seems
complex and is likely to be multifactorial. For example,
temperature alone does not appear to be a sufficient signal for
ospC induction, because unfed ticks exposed to higher tem-
peratures do not induce ospC expression [27]. A recent study
suggested that the same environmental stimuli seem to co-
regulate spirochete proteins like OspA and OspC in vitro,
suggesting that regulatory pathways of differentially ex-
pressed genes are interlinked [46]. However, little is known
about the mechanism or key regulatory networks that govern
spirochete gene regulation in vivo. The B. burgdorferi ge-
nome surprisingly contained relatively few homologs of
known eubacterial regulatory proteins [13]. For example,
although B. burgdorferi displays a classical heat-shock re-
sponse, the genome does not contain a known heat-shock
sigma factor [13]. A recent microarray analysis also failed to
identify significant evidence of adaptive changes in clusters
of spirochete regulatory genes under different host or growth
conditions, whereas the same conditions caused significant
changes in lipoprotein gene expression [37]. It is proposed
that B. burgdorferi adapts to exploit relatively minor changes
in the expression of regulatory genes in order to affect down-
stream target gene expression, so that relatively small alter-
ation in regulatory gene expression can control production of
large numbers of target lipoproteins [37]. B. burgdorferi
Table 1
Examples of differentially expressed B. burgdorferi lipoprotein gene products with suggested functions
Gene product Expression Receptor Function Reference
DbpA and DbpB Mammal Decorin Colonization [40]
Bgp Mammal GAGs Colonization [102]
P66 Mammal Integrins Colonization [72]
Erp (OspE/F) Mammal Factor H Host defense [62]
ErpT (Arp) Mammal ? Colonization? [103]
P47 (Bbk32) Mammal Fibronectin Colonization [68]
OspC Mammal/tick ? Transmission [51]
OspA Tick A gut protein Colonization [91]
660 U. Pal, E. Fikrig / Microbes and Infection 5 (2003) 659–666
constitutively expresses RpoN, a sigma subunit, which is
regulated via a post-transcriptional mechanism [47]. RpoN
controls the expression of RpoS, an alternative sigma factor,
which in turn, is thought to regulate the transcription of
several lipoproteins like OspC, OspF, Mlp-8 and DbpA
[46,48]. An external or environmental signaling event is
proposed to induce an RpoN activator protein which is likely
to bind to an enhancer region upstream of where RpoN
complexed with the RNA polymerase holoenzyme (–24/–12
region), leading to the synthesis of rpoS mRNA. RpoS then
mediates the synthesis of target lipoproteins of B. burgdor-
feri [48].
3. Adaptation in vertebrates
B. burgdorferi is transmitted to vertebrates, mostly to
mammals, during tick feeding. An Ixodes tick takes approxi-
mately 3–4 d to complete the engorgement process, during
which a pronounced multiplication of spirochetes takes place
in the gut of the tick [49]. The spirochete numbers are
reported to increase several hundredfold [50], and the differ-
ential gene expression as well as the variable recombination
may contribute to the production of new molecules on the
spirochete surface [30]. The newly synthesized proteins are
believed to aid in the transmission from tick gut via salivary
gland to the dermis of the host [51]. Although spirochetes
evolve their own mechanism to fight host immune defense,
the transmission via tick saliva provides them with certain
adaptive advances. A feeding tick secretes molecules that
influence the host immune system. For example, tick saliva
inactivates the host complement system [52] and inhibits
phagocyte function [53], which in turn, could help the adap-
tation of the spirochete to its new environment. Recently, an
I. scapularis salivary protein, Salp15, has been shown to
modulate CD4+ T cells [54]. After transmission, B. burgdor-
feri remains localized into the host skin for several days. The
spirochetes are reported to invade distant skin sites or a
number of organs and are found in high concentrations in the
spleen, urinary bladder, joints and heart [8]. Spirochetes can
cross the blood-brain barrier in several experimental models,
most notably primates, and colonize the nervous system
[55–57]. It is interesting to note that the bacteria are able to
establish a chronic infection even in the face of the sophisti-
cated immune system that exists in mammals. How does the
pathogen adapt itself to this challenging environment?
Like many other invasive pathogens, B. burgdorferi uses a
variety of mechanisms for protection against components of
the host innate immune system. The alternate pathway of the
complement system is a major primary host defense [58].
The pathway activates with an initial deposition of the C3
protein to the pathogen surface, followed by several amplifi-
cation loops, ultimately resulting in the formation of mem-
brane attack complex, which kills the pathogen. Some of the
Erp (OspE/F-related) proteins have been reported to be syn-
thesized by B. burgdorferi during early mammalian infection
[59–61]. Studies have revealed that Erps contribute to the
ability of B. burgdorferi to infect mammals by blocking host
complement-mediated killing [62,63]. The spirochetes are
capable of producing different Erps on the surface, and each
Erp is reported to exhibit different relative affinities for the
complement inhibitors of various potential vertebrate hosts
[64]. Therefore, the presence of multiple Erps on the surface
can allow for a single B. burgdorferi to resist complement-
mediated killing in a wide range of hosts. All known B. burg-
dorferi strains can bind C3, although the deposition of the
downstream components of complement system like C5b to
C9 varies in different strains, resulting in a vast number of
strains being resistant to complement attack [65]. The
mechanism of resistance to complement is mediated by the
binding of two host-derived complement control proteins:
factor H and factor H-like protein-1 / reconectin [62,66].
B. burgdorferi surface protein OspE has been identified to
bind the complement regulatory factor H [62]. OspE inter-
acts with carboxy-terminal of factor H; therefore, the amino-
terminal domain of the complement inhibitor remains free to
exert its regulatory activities. The spirochete has also evolved
other interesting survival strategies against innate immune
attack. B. burgdorferi was shown to bypass the need for
physiological iron for its growth and survival [67]; many of
the iron-containing enzymes are well-known targets for oxi-
dative host defenses against pathogens.
A large number of genes have been identified in B. burg-
dorferi which are selectively expressed, when spirochetes are
inside a mammalian host milieu. The differentially expressed
genes are likely to contribute to spirochete dissemination and
colonization of target tissues. For example, B. burgdorferi
synthesizes DbpA, DbpB and BBK 32 early in mammalian
infection, which bind to host extracellular matrix proteins
like decorin or fibronectin [40,68,69]. Spirochete adhesins
such as DbpA or BBK 32 are thought to play critical roles in
the early stage of Lyme disease by mediating the tissue
adherence of B. burgdorferi. Local adherence ability in the
skin extracellular matrix proteins could facilitate the survival
of extremely small numbers of spirochetes (estimated to be
between 1 and 10 [70]), which first enter the host at the site of
tick feeding. B. burgdorferi is thought to replicate in the skin
before endovascular dissemination towards distant organs.
Theoretically, binding of soluble extracellular matrix pro-
teins by spirochetes may also provide this organism with a
mechanism for establishing persistent infection in wide host
tissues. As shown with B. crocidurae [71], the ability of
B. burgdorferi to bind bulky, host-derived proteins, such as
fibronectin, may also mask recognition of the spirochete by
the host immune system. B. burgdorferi surface molecule
P66 has been shown to bind b3-chain integrins [72], which
are expressed in a variety of host locations. Thus, multiple
adhesin mechanisms through such widespread host receptors
as decorin, fibronectin or integrins contribute to the virulence
of the organism and aid in the ability of the pathogen to
establish chronic infection in multiple tissues. However, little
is known about specific host receptors expressed in a local-
661U. Pal, E. Fikrig / Microbes and Infection 5 (2003) 659–666
ized or tissue-specific manner and that bind to B. burgdor-
feri. The spirochetes are reported to use host-derived plasmin
to facilitate invasion, as demonstrated in cell cultures and in
mice genetically deficient in plasminogen [73,74]. Identifi-
cation of the guaA gene encoding GMP synthetase in
B. burgdorferi, an enzyme responsible for de novo purine
biosynthesis, has also been implicated in the survival of
bacteria in mammalian blood [75].
A number of recent studies involving microarray analysis
of spirochete gene expression within chamber-implants [76]
or in hosts at initial or chronic phases of murine infection
address the question of how B. burgdorferi evades adaptive
immunity in mammals [36,37]. Studies indicated that
B. burgdorferi is able to generate multiple phenotypes during
the transmission and earlier phases of murine infection
[30,77]. Transcription and recombination events have been
shown to occur in the feeding tick gut to generate a diverse
population of spirochetes with antigenic and genetic variabil-
ity [30]. Antibodies generated in the infected host selectively
eliminate the targeted immunodominant phenotypes of spi-
rochetes, while the adapted spirochetes continue to survive
by downregulation of a selected set of proteins targeted by
host antibody. A recent study demonstrated that B. burgdor-
feri expresses a set of approximately 116 lipoprotein genes
during early mammalian infection and, as a part of adaptive
immune response, will downregulate more than 80 of these
genes [36]. This adaptation is proposed to be fundamental for
B. burgdorferi to survive in mammals and establish persis-
tent infection. In addition, B. burgdorferi has been proposed
to develop alternate strategies to remain hidden from the
immune system, such as masking immunodominant surface
antigens [78] or persisting in close association with cells at
immune privilege sites [79]. B. burgdorferi has been specu-
lated to generate specific mechanisms to inhibit phagocytosis
or ingestion by host cells [80]. For long-term persistence in
host tissues, B. burgdorferi has also been proposed to modu-
late a wide range of host cytokines such as TNF-alpha [81],
IFN-gamma [82,83], IL-6 [84,85], and IL-12 [86]. IFN-
gamma-mediated events have actually been shown to pro-
mote vls locus recombination to generate a diverse popula-
tion of spirochetes [87], which could potentially help to
evade host immune response.
4. Adaptation in ticks
B. burgdorferi faces an entirely different environment
when transmitted from mammals to Ixodes ticks. Studies
have shown that although ticks usually feed for 96 h, both
larval and nymphal ticks rapidly acquire spirochetes during
the first 24 h of attachment [43], even before ticks are en-
gorged with significant amounts of blood. The number of
organisms within fed ticks continues to increase during and
after the blood meal, possibly by continual entry of spiro-
chetes during the blood meal and by replication [43,88]. The
bacterium must avoid being digested with the tick blood meal
and has to survive in significant temperature extremities of
the poikilothermic organism, and also periods of limited
nutritional resources and metabolic activity. During a subse-
quent blood meal, spirochetes must also have to sense the
appropriate stimuli, and cross the gut barrier to travel to the
salivary gland at the right time for transmission to a new
vertebrate host.