Antigenic variation with a twist--the Borrelia story.

Abstract

A common mechanism of immune evasion in pathogenic bacteria and protozoa is antigenic variation, in which genetic or epigenetic changes result in rapid, sequential shifts in a surface-exposed antigen. In this issue of Molecular Microbiology, Dai et al. provide the most complete description to date of the vlp/vsp antigenic variation system of the relapsing fever spirochaete, Borrelia hermsii. This elaborate, plasmid-encoded system involves an expression site that can acquire either variable large protein (vlp) or variable small protein (vsp) surface lipoprotein genes from 59 different archival copies. The archival vlp and vsp genes are arranged in clusters on at least five different plasmids. Gene conversion occurs through recombination events at upstream homology sequences (UHS) found in each gene copy, and at downstream homology sequences (DHS) found periodically among the vlp/vsp archival genes. Previous studies have shown that antigenic variation in relapsing fever Borrelia not only permits the evasion of host antibody responses, but can also result in changes in neurotropism and other pathogenic properties. The vlsE antigenic variation locus of Lyme disease spirochaetes, although similar in sequence to the relapsing fever vlp genes, has evolved a completely different antigenic variation mechanism involving segmental recombination from a contiguous array of vls silent cassettes. These two systems thus appear to represent divergence from a common precursor followed by functional convergence to create two distinct antigenic variation processes.

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Molecular Microbiology (2006)doi:10.1111/j.1365-2958.2006.05204.x
© 2006 The Author
Journal compilation © 2006 Blackwell Publishing Ltd
Blackwell Publishing LtdOxford, UKMMIMolecular Microbiology0950-382X© 2006 The Author; Journal compilation © 2006 Blackwell Publishing Ltd
? 2006
?
?••••
Commentary
Antigenic variation in BorreliaS. J. Norris
Accepted 18 April, 2006. *For correspondence. E-mail Steven.J.
Norris@uth.tmc.edu; Tel. (
+
1) 713 500 5338; Fax (
+
1) 713 500 0730.
MicroCommentary
Antigenic variation with a twist – the Borrelia story
Steven J. Norris
Departments of Pathology & Laboratory Medicine and
Microbiology & Molecular Genetics, University of Texas
Medical School at Houston, PO Box 20708, Houston, TX
77225-0708, USA.
*
Summary
A common mechanism of immune evasion in patho-
genic bacteria and protozoa is antigenic variation, in
which genetic or epigenetic changes result in rapid,
sequential shifts in a surface-exposed antigen. In this
issue of Molecular Microbiology
most complete description to date of the
genic variation system of the relapsing fever spiro-
chaete, Borrelia hermsii. This elaborate, plasmid-
encoded system involves an expression site that can
acquire either variable large protein (
small protein (
vsp ) surface lipoprotein genes from 59
different archival copies. The archival
genes are arranged in clusters on at least five dif-
ferent plasmids. Gene conversion occurs through
recombination events at
sequences (UHS) found in each gene copy, and at
downstream homology sequences (DHS) found peri-
odically among the
vlp/ vsp
studies have shown that antigenic variation in relaps-
ing fever Borrelia not only permits the evasion of host
antibody responses, but can also result in changes in
neurotropism and other pathogenic properties. The
vlsE antigenic variation locus of Lyme disease
spirochaetes, although similar in sequence to the
relapsing fever vlp genes, has evolved a completely
different antigenic variation mechanism involving
segmental recombination from a contiguous array of
vls silent cassettes. These two systems thus appear
to represent divergence from a common precursor
followed by functional convergence to create two dis-
tinct antigenic variation processes.
, Dai et al. provide the
vlp/ vsp anti-
vlp) or variable
vlp and vsp
upstream homology
archival genes. Previous
Many, if not most, microbial pathogens have developed
mechanisms for evading the immune response, including
the invasion of immunoprivileged sites, the inhibition of
immune responses, blocking of engulfment or intracellular
killing by phagocytes, masking of surface antigens, phase
variation and antigenic variation. Phase variation and anti-
genic variation are perhaps the most complex, involving
genetic or epigenetic changes that occur more frequently
than the basal mutation rate. Many of these systems are
mediated by specialized cis-
acting factors. Phase variation refers to a switch between
two phenotypes, i.e. turning gene expression on or off (as
exemplified by the surface protein gene
gonorrhoeae
) or toggling between the production of two
different gene products (such as the switching between
H1 and H2 flagellar antigen types in
rium
). Antigenic variation involves the sequential expres-
sion of multiple different forms of an antigenic surface
protein, permitting the organism to keep one step ahead
of immune response. A large number of phase variation
and antigenic variation systems have been described in
bacterial and protozoal pathogens. Indeed, a possible
point of view is that if one of these systems has not been
found in a persistent pathogen, it is because we have not
looked hard enough. In this issue of
ogy
, Dai et al . (2006) provide a detailed analysis of the
vlp/ vsp antigenic variation system of the relapsing fever
spirochaete, Borrelia hermsii
of bacterial antigenic variation.
Relapsing fever is an arthropod-transmitted bacterial
infection caused by any of a number of
The epidemic form of relapsing fever is caused by
recurrentis and is transmitted by the human body louse,
Pudiculus humanus. Found in areas with crowding and
poor sanitation, epidemic relapsing fever caused out-
breaks involving hundreds of thousands of soldiers and
civilians during World War I and after World War II. Soft-
bodied ticks of the genus Ornithodoros transmit the
endemic form of relapsing fever. Sporadic human cases
of this epizootic disease result from exposure to infected
ticks acquired from animals. There is host specificity
between the Borrelia species and the tick; for example,
B. hermsii is transmitted by Ornithodoros hermsi
ing fever is characterized by several spikes of high tem-
perature accompanied by phases of bacteraemia, in
which the number of Borrelia
(Fig. 1A). Each cycle of infection is caused by spirocha-
acting elements and trans-
opa of Neisseria
Salmonella typhimu-
Molecular Microbiol-
, one of the prime examples
Borrelia species.
Borrelia
. Relaps-
in blood can reach 10
8
ml
−
1

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2 S. J. Norris
© 2006 The Author
Molecular Microbiology
Journal compilation © 2006 Blackwell Publishing Ltd,
etes producing a different form of the Vlp or Vsp surface
lipoprotein (Fig. 1B). Clearance occurs when the host pro-
duces sufficient antibodies specific for that serotype to
result in elimination. vlp/vsp
quency (10
to 10 recombination events per cell per
generation), and each new wave of infection is predomi-
nated by a bacterial clone producing one particular form
of Vlp or Vsp. Bacteraemia in animals is thereby able to
variants occur at high fre-
−
4
−
3
persist long enough to permit transmission to feeding
ticks, thus completing the tenuous infection cycle.
Lyme disease spirochaetes (e.g.
B. garinii and B. afzelii) are transmitted by hard-bodied
ticks of the genus Ixodes . Lyme disease is characterized
by a localized infection (erythema migrans) at the site of
the tick bite, followed by disseminated and chronic stages
with neurological, cardiological, dermatological and
arthritic manifestations. In contrast to relapsing fever,
Lyme disease Borrelia never achieve high blood concen-
trations but, instead, establish low levels of infection in
multiple tissues that can persist for months to years. Lyme
disease Borrelia possess the
tem (Zhang et al ., 1997), in which segmental recombina-
tion results in the establishment of a large repertoire of
variants in the surface lipoprotein VlsE (Fig. 1C). The
degree of variation is so high that each clone examined
from a single mouse skin biopsy 28 days post infection
had a different
vlsE sequence (Zhang and Norris, 1998).
Thus, the relapsing fever system favours the predomi-
nance of a single Vlp/Vsp variant at any one time, whereas
Lyme disease infection results in a mosaic of bacterial
clones each producing a different VlsE variant.
Both relapsing fever and Lyme disease
large number of linear and circular plasmids, representing
about a third of the genome. By sequencing
the
B. hermsii HS1 plasmids, Dai
the genome contains at least 21
gene segments, each of which comprises a complete
open reading frame (ORF) and ribosome binding site with-
out a promoter. The vlp and vsp
proteins have no homology to one another (other than a
shared sequence in the 5
′
region; see below) and, thus,
appear to have evolved separately. The
subdivided into
α
,
β
,
γ
and
δ
sequence identity within each group. Amplification of
and vsp sequences using specific primers indicated that
the 59 alleles identified represent
ber present. The
vlp and vsp alleles are arranged together
in 10 clusters containing 2–14 gene segments each. Each
vlp and vsp allele contains a
sequence (UHS) that comprises the region immediately
upstream of the ORF and the first 27 nt of the ORF;
divergence at two central nucleotides provided a marker
for the location of recombination cross-over events. Thir-
teen copies of a 214 bp downstream homology sequence
(DHS) are interspersed among the variable gene segment
alleles. The expression site with a promoter sequence is
at one end of a 28 kb linear plasmid, lp28-1.
The recombination events occurring during relapses
were then determined by characterizing 83 variants that
arose following infection of mice with
expressing either vlp7 or vlp17
expressed sequences could be attributed to a gene con-
Borrelia burgdorferi,
vlsE antigenic variation sys-
Borrelia have a
∼
445 kb of
et al
vsp
. (2006) found that
and 38 vlp silent
sequences and encoded
vlp alleles are
≥
groups based on the 60%
vlp
∼
90% of the total num-
∼
60 bp upstream homology
B. hermsii HS1
. In all cases, the newly
Fig. 1.
disease
A. Pattern of infection antigenic variation during mammalian infection
with the relapsing fever spirochaete,
phase of infection, a predominant serotype (in this case, Vlp7) is
expressed. Concentrations of organisms in the blood can reach
10
ml. The first serotype is rapidly eliminated by serotype-specific
antibodies, and subsequent relapses are caused by spirochaetes that
have undergone antigenic variation to different Vlp or Vsp serotypes
(e.g. Vsp24 and Vlp18).
B. The principal mechanism of antigenic variation in
gene conversion involving recombination at the upstream homology
sequences (UHS) and the downstream homology sequences (DHS),
resulting in replacement of the
vlp or
C. Antigenic variation in the Lyme disease spirochaete
dorferi
utilizes a different mechanism, in which segments within the
vlsE cassette region are replaced by sections of varied length and
location from the silent cassettes. In this hypothetical example,
has undergone three sequential gene conversion events with silent
cassettes
vls6, vls8 and vls7. In contrast to relapsing fever, mammals
infected with Lyme disease
Borrelia
different VlsE variants rather than a single predominant serotype.
Antigenic variation mechanisms in relapsing fever and Lyme
Borrelia.
Borrelia hermsii. During the initial
8
−
1
B. hermsii is
vsp gene at the expression site.
Borrelia burg-
vlsE
appear to harbour thousands of

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Antigenic variation in Borrelia3
© 2006 The Author
Journal compilation © 2006 Blackwell Publishing Ltd,
Molecular Microbiology
version event involving 17 of the 59 available
silent (archival) sequences. In 68 of the 83 variants, both
upstream and downstream sequences were available to
evaluate the locations of recombination. The 5
recombination events occurred primarily within the 3
of the UHS, as determined by examining the polymor-
phisms occurring in this region. Only one of these 5
recombinations occurred beyond the UHS, resulting in a
chimeric vlp7/ vlp18 gene. With few exceptions, the 3
recombination event could be traced to a pairing of DHS
regions, with 83% of the recombinations occurring within
the distal half of the DHS. In a few examples where more
than one DHS was available downstream of the archival
gene, the most distant DHS was selected. Overall, the
vast majority of relapse serotypes resulted from recombi-
nation at the UHS and DHS sites, replacing the interven-
ing DNA with the archival ‘donor’ sequence (Fig. 1B); the
remaining scenarios are presented in detail by Dai
(2006). The most frequently used archival sequences
(
α
vlp18, vsp24, vsp2 , vsp1 and
immediately downstream of the silent gene segment, indi-
cating that this arrangement is favourable to gene conver-
sion. Interestingly, a small number of variants utilized
archival sequences that lacked a DHS, implicating other
sequences in the downstream cross-over event. The
frequencies of archival vls and
be addressed in a separate article (A.G. Barbour, Q. Dai,
B.I. Restrepo and S.A. Frank, submitted).
In Lyme disease Borrelia, antigenic variation involves
segmental gene conversion between the
site and one of a series of
vls silent cassettes correspond-
ing to the middle region of vlsE (Fig. 1C). In B. burgdorferi
B31, vlsE and the contiguous array of vls silent cassettes
are located adjacent to one another in the linear plasmid
lp28-1 (Zhang et al., 1997). The vls silent cassettes are
arranged in a similar manner in the other B. burgdorferi,
B. garinii and B. afzelii strains examined to date (Kawa-
bata et al., 1998; Wang et al., 2001; 2003; Glöckner et al.,
2004). Gene conversion events involve replacement of
random, variable length segments of the central cassette
region of vlsE with corresponding regions from any of the
silent cassettes. The recombination events have been
detected as early as 4 days after infection of mice and
appear to occur continuously during infection. As a result,
mammals could harbour thousands of different variants at
any one time, resulting in altered epitopes (McDowell
et al., 2002) and confounding efforts by the immune
response to keep up with the sequence variation. The
sequence changes primarily affect six variable regions
that are localized in the most exposed, membrane distal
portion of VlsE (Eicken et al., 2002). Interestingly, a strong
antibody response is mounted against conserved regions
(particularly invariant region 6) of VlsE, and VlsE-based
recombinant proteins or synthetic peptides are now being
vlp and vsp
′
end of the
′
half
′
′
et al.
vsp6) all have a DHS
vlp gene utilization will
vlsE expression
used for the immunodiagnosis of Lyme disease (Bacon
et al., 2003; Schulte-Spechtel et al., 2003). These highly
antigenic regions are ‘buried’ in subsurface regions of
VlsE and are apparently inaccessible to antibodies. Vari-
ation in vlsE has not been detected during in vitro culture,
in ticks, or even in dialysis membrane chambers implanted
in animals, indicating that as yet uncharacterized host
factor(s) trigger the recombination process.
Although the functions of Vlp, Vsp and VlsE lipoproteins
are not known, there are indications that they are involved
in host–pathogen interactions independent of their anti-
genic variation properties. Their production is highly reg-
ulated, resulting in higher abundance during mammalian
infection. Production of the B. hermsii ‘bloodstream’ pro-
teins Vlp or Vsp in mammals alternates with the induced
production of the ‘tick’ protein Vtp (also called Vsp33) in
ticks and in vitro culture (Schwan and Hinnebusch, 1998;
Barbour, 2003; Porcella et al., 2005). Vtp is closely related
to the Vsps. In addition, the Lyme disease Borrelia protein
OspC is homologous to the Vsps, is induced during tick
feeding and is vital to transmission of B. burgdorferi from
ticks to mammals (Grimm et al., 2004; Pal et al., 2004).
Variants of the relapsing fever organism Borrelia turicatae
producing two different Vsp proteins, VspA and VspB,
differ markedly in their pattern of pathogenesis in mice:
VspA production results in infection of the central nervous
system, whereas VspB is associated with 10-fold higher
levels in the blood, severe arthritis, myocarditis and
vestibular dysfunction (Pennington et al., 1997; Cadavid
et al., 2001). The involvement of Vlps and the related
Lyme disease protein VlsE in pathogenesis has not been
studied in great detail, but a study by Crother et al. (2003)
indicates that VlsE levels are higher in bacteria in the joint
and skin tissue than in heart tissue of B. burgdorferi-
infected mice. Taken together, these results point towards
a role of the Borrelia antigenic variation proteins in tissue
tropism and varied patterns of pathogenesis.
In summary, the relapsing fever and Lyme disease Bor-
relia have evolved complex, yet distinct, antigenic variation
systems that are based on two discrete gene families
encoding Vlp and Vsp homologues. Although these
two branches of the Borrelia genus are closely related (i.e.
are about as similar as are Escherichia coli and
S. typhimurium), it is likely that they diverged a million or
more years ago. The common Borrelia progenitor carried
vlp and vsp precursors that underwent duplication and
divergence in sequence, expression patterns and func-
tion. A chicken or egg (or mammal or tick) question is
whether an antigenic variation system already existed in
this common precursor, or the vlp/vsp and vlsE variation
systems evolved after the split between relapsing fever
and Lyme borreliosis organisms occurred. Most of the
evidence points to the latter possibility. In the relapsing
fever vlp/vls system, recombination occurs predominantly

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4S. J. Norris
© 2006 The Author
Journal compilation © 2006 Blackwell Publishing Ltd, Molecular Microbiology
within the intragenic UHS and extragenic DHS
sequences, and results in replacement of the entire gene
(except for part of the UHS), whereas the Lyme borreliosis
system results in the replacement of random, seemingly
unanchored segments within the central cassette region
of vlsE. In this respect, the relapsing fever system most
closely resembles gene conversion in trypanosomes,
while the Lyme borreliosis mechanism is similar to the
N. gonorrhoeae pilE system (Criss et al., 2005). The pro-
moterless gene copies are loosely arranged in 10 clusters
in the B. hermsii system; the vls silent cassettes in the
Lyme disease strains examined comprise only the central
region of the expression site and are packed cheek to jowl
in a contiguous array. Finally, the relapsing fever system
has the unique ability to alternate between two distinct
gene families (expressing Vlps and Vsps). Thus, these
remarkable antigenic variation systems, further revealed
by the work of Dai et al. (2006), may represent a rare
example of divergent evolution leading to convergent
function in terms of antigenic variation and immune
evasion.
Acknowledgements
Studies related to this Microcommentary in the authors’ lab-
oratory were supported by US Public Health Service Grant
R01 AI37277.
References
Bacon, R.M., Biggerstaff, B.J., Schriefer, M.E., Gilmore, R.D.,
Jr, Philipp, M.T., Steere, A.C., et al. (2003) Serodiagnosis
of Lyme disease by kinetic enzyme-linked immunosorbent
assay using recombinant VlsE1 or peptide antigens of
Borrelia burgdorferi compared with 2-tiered testing using
whole-cell lysates. J Infect Dis 187: 1187–1199.
Barbour, A.G. (2003) Antigenic variation in Borrelia: relapsing
fever and Lyme borreliosis. In Antigenic Variation. Craig,
A., and Scherf, A. (eds). London: Academic Press, pp.
319–356.
Cadavid, D., Pachner, A.R., Estanislao, L., Patalapati, R.,
and Barbour, A.G. (2001) Isogenic serotypes of Borrelia
turicatae show different localization in the brain and skin of
mice. Infect Immun 69: 3389–3397.
Criss, A.K., Kline, K.A., and Seifert, H.S. (2005) The fre-
quency and rate of pilin antigenic variation in Neisseria
gonorrhoeae. Mol Microbiol 58: 510–519.
Crother, T.R., Champion, C.I., Wu, X.Y., Blanco, D.R., Miller,
J.N., and Lovett, M.A. (2003) Antigenic composition of
Borrelia burgdorferi during infection of SCID mice. Infect
Immun 71: 3419–3428.
Dai, Q., Restrepo, B.I., Porcella, S.F., Raffel, S.J., Schwan,
T.G., and Barbour, A.G. (2006) Antigenic variation by
Borrelia hermsii occurs through recombination between
extragenic repetitive elements on linear plasmids. Mol
Microbiol doi:10.1111/j.1365-2958.2006.05177.
Eicken, C., Sharma, V., Klabunde, T., Lawrenz, M.B.,
Hardham, J.M., Norris, S.J., and Sacchettini, J.C. (2002)
Crystal structure of Lyme disease variable surface antigen
VlsE of Borrelia burgdorferi. J Biol Chem 277: 21691–
21696.
Glöckner, G., Lehmann, R., Romualdi, A., Pradella, S.,
Schulte-Spechtel, U., Schilhabel, M., et al. (2004) Compar-
ative analysis of the Borrelia garinii genome. Nucleic Acids
Res 32: 6038–6046.
Grimm, D., Tilly, K., Byram, R., Stewart, P.E., Krum, J.G.,
Bueschel, D.M., et al. (2004) Outer-surface protein C of
the Lyme disease spirochete: a protein induced in ticks for
infection of mammals. Proc Natl Acad Sci USA 101: 3142–
3147.
Kawabata, H., Myouga, F., Inagaki, Y., Murai, N., and
Watanabe, H. (1998) Genetic and immunological analyses
of Vls (VMP-like sequences) of Borrelia burgdorferi. Microb
Pathog 24: 155–166.
McDowell, J.V., Sung, S.Y., Hu, L.T., and Marconi, R.T.
(2002) Evidence that the variable regions of the central
domain of VlsE are antigenic during infection with Lyme
disease spirochetes. Infect Immun 70: 4196–4203.
Pal, U., Yang, X., Chen, M., Bockenstedt, L.K., Anderson,
J.F., Flavell, R.A., et al. (2004) OspC facilitates Borrelia
burgdorferi invasion of Ixodes scapularis salivary glands. J
Clin Invest 113: 220–230.
Pennington, P.M., Allred, C.D., West, C.S., Alvarez, R., and
Barbour, A.G. (1997) Arthritis severity and spirochete bur-
den are determined by serotype in the Borrelia turicatae-
mouse model of Lyme disease. Infect Immun 65: 285–292.
Porcella, S.F., Raffel, S.J., Anderson, D.E., Jr, Gilk, S.D.,
Bono, J.L., Schrumpf, M.E., and Schwan, T.G. (2005) Vari-
able tick protein in two genomic groups of the relapsing
fever spirochete Borrelia hermsii in western North America.
Infect Immun 73: 6647–6658.
Schulte-Spechtel, U., Lehnert, G., Liegl, G., Fingerle, V.,
Heimerl, C., Johnson, B.J., and Wilske, B. (2003) Signifi-
cant improvement of the recombinant Borrelia-specific
immunoglobulin G immunoblot test by addition of VlsE and
a DbpA homologue derived from Borrelia garinii for diag-
nosis of early neuroborreliosis. J Clin Microbiol 41: 1299–
1303.
Schwan, T.G., and Hinnebusch, B.J. (1998) Bloodstream-
versus tick-associated variants of a relapsing fever bacte-
rium. Science 280: 1938–1940.
Wang, D., Botkin, D.J., and Norris, S.J. (2003) Characteriza-
tion of the vls antigenic variation loci of the Lyme disease
spirochaetes Borrelia garinii Ip90 and Borrelia afzelii ACAI.
Mol Microbiol 47: 1407–1417.
Wang, G., van Dam, A.P., and Dankert, J. (2001) Analysis of
a VMP-like sequence (vls) locus in Borrelia garinii and Vls
homologues among four Borrelia burgdorferi sensu lato
species. FEMS Microbiol Lett 199: 39–45.
Zhang, J.R., and Norris, S.J. (1998) Kinetics and in vivo
induction of genetic variation of vlsE in Borrelia burgdorferi.
Infect Immun 66: 3689–3697.
Zhang, J.R., Hardham, J.M., Barbour, A.G., and Norris, S.J.
(1997) Antigenic variation in Lyme disease borreliae by
promiscuous recombination of VMP-like sequence cas-
settes. Cell 89: 275–285.