Similarities in murine infection and immune response to Borrelia bissettii and Borrelia burgdorferi sensu stricto.

In 1982, Borrelia burgdorferi sensu stricto (ss) was identified as the aetiological agent of Lyme disease. Since then an increasing number of Borrelia burgdorferi sensu lato (sl) species have been isolated in the United States. To date, many of these species remain understudied despite mounting evidence associating them with human illness. Borrelia bissettii is a spirochaete closely related to B. burgdorferi that has been loosely associated with human illness. Using an experimental murine infection model, we compared the infectivity and humoral immune response with a North American isolate of B. bissettii and B. burgdorferi using culture, molecular and serological methods. The original B. bissettii cultures were unable to infect immunocompetent mice, but were confirmed to be infectious after adaptation in immunodeficient animals. B. bissettii infection resulted in spirochaete burdens similar to B. burgdorferi in skin, heart and bladder whereas significantly lower burdens were observed in the joint tissues. B. bissettii induced an antibody response similar to B. burgdorferi as measured by both immunoblotting and the C6 ELISA. Additionally, this isolate of B. bissettii was sequenced on the Ion Torrent PGM, which successfully identified many genes orthologous to mammalian virulence factors described in B. burgdorferi. Similarities seen between both infections in this well-characterized murine model contribute to our understanding of the potential pathogenic nature of B. bissettii. Infection dynamics of B. bissettii, and especially the induced humoral response, are similar to B. burgdorferi, suggesting this species may contribute to the epidemiology of human borreliosis.

Borrelia burgdorferi oxidative stress regulator BosR directly represses lipoproteins primarily expressed in the tick during mammalian infection.

Differential gene expression is a key strategy adopted by the Lyme disease spirochaete, Borrelia burgdorferi, for adaptation and survival in the mammalian host and the tick vector. Many B. burgdorferi surface lipoproteins fall into two distinct groups according to their expression patterns: one group primarily expressed in the tick and the other group primarily expressed in the mammal. Here, we show that the Fur homologue in this bacterium, also known as Borrelia oxidative stress regulator (BosR), is required for repression of outer surface protein A (OspA) and OspD in the mammal. Furthermore, BosR binds directly to sequences upstream of the ospAB operon and the ospD gene through recognition of palindromic motifs similar to those recognized by other Fur homologues but with a 1 bp variation in the spacer length. Putative BosR binding sites have been identified upstream of 156 B. burgdorferi genes. Some of these genes share the same expression pattern as ospA and ospD. Most notably, 12 (67%) of the 18 genes previously identified in a genome-wide microarray study to be most significantly repressed in the mammal are among the putative BosR regulon. These data indicate that BosR may directly repress transcription of many genes that are downregulated in the mammal.

Detection of Lyme Borrelia in questing Ixodes scapularis (Acari: Ixodidae) and small mammals in Louisiana.

Lyme borreliosis is caused by spirochetes from the Borrelia burgdorferi sensu lato species complex. In the United States, B. burgdorferi sensu stricto (s.s.; Johnson, Schmid, Hyde, Steigerwalt, and Brenner) is the most common cause of human Lyme borreliosis. With >25,000 cases reported annually, it is the most common vector-borne disease in the United States. Although approximately 90% of cases are contained to the northeastern and Great Lake states, areas in Canada and some southern states are reporting rises in the number of human disease cases. Louisiana records a few cases of Lyme each year. Although some are most certainly the result of travel to more endemic areas, there exists evidence of locally acquired cases. Louisiana has established populations of the vector tick, Ixodes scapularis (Say), and a wide variety of potential reservoir animals, yet Lyme Borrelia has never been described in the state. Using culture and polymerase chain reaction, we investigated the presence of Lyme Borrelia in both mammals and questing ticks at a study site in Louisiana. Although culture was mostly unsuccessful, we did detect the presence of B. burgdorferi s.s. DNA in 6.3% (11 of 174) of ticks and 22.7% (five of 22) of animal samples. To our knowledge, this is among the first evidence documenting B. burgdorferi s.s. in Louisiana. Further investigations are required to determine the significance these findings have on human and animal health.

Inactivation of a putative flagellar motor switch protein FliG1 prevents Borrelia burgdorferi from swimming in highly viscous media and blocks its infectivity.

The flagellar motor switch complex protein FliG plays an essential role in flagella biosynthesis and motility. In most motile bacteria, only one fliG homologue is present in the genome. However, several spirochete species have two putative fliG genes (referred to as fliG1 and fliG2) and their roles in flagella assembly and motility remain unknown. In this report, the Lyme disease spirochete Borrelia burgdorferi was used as a genetic model to investigate the roles of these two fliG homologues. It was found that fliG2 encodes a typical motor switch complex protein that is required for the flagellation and motility of B. burgdorferi. In contrast, the function of fliG1 is quite unique. Disruption of fliG1 did not affect flagellation and the mutant was still motile but failed to translate in highly viscous media. GFP-fusion and motion tracking analyses revealed that FliG1 asymmetrically locates at one end of cells and the loss of fliG1 somehow impacted one bundle of flagella rotation. In addition, animal studies demonstrated that the fliG1- mutant was quickly cleared after inoculation into the murine host, which highlights the importance of the ability to swim in highly viscous media in the infectivity of B. burgdorferi and probably other pathogenic spirochetes.

Unexpected failure of Ixodes scapularis nymphs to transmit a North American Borrelia bissettiae strain.

Globally, the Borrelia burgdorferi (sensu lato) complex comprises more than 21 species of spirochetes. Although the USA is home to a diverse fauna of Lyme disease group Borrelia species, only two are considered responsible for human clinical disease: Borrelia burgdorferi (sensu stricto) and Borrelia mayonii. However, evidence has implicated additional B. burgdorferi (s.l.) species in human illness elsewhere. While much research has focused on the B. burgdorferi (s.s.)-tick interface, tick vectors for most of the other North American Lyme disease group Borrelia species remain experimentally unconfirmed. In this report we document the ability of Ixodes scapularis to acquire but not transmit a single strain of Borrelia bissettiae, a potential human pathogen, in a murine infection model. Pathogen-free I. scapularis larvae were allowed to feed on mice with disseminated B. burgdorferi (s.s.) or B. bissettiae infections. Molted infected nymphs were then allowed to feed on naïve mice to assess transmission to a susceptible host through spirochete culture and qPCR throughout in ticks collected at various developmental stages (fed larvae and nymphs, molted nymphs, and adults). In this study, similar proportions of I. scapularis larvae acquired B. bissettiae and B. burgdorferi (s.s.) but transstadial passage to the nymphal stage was less effective for B. bissettiae. Furthermore, B. bissettiae-infected nymphs did not transmit B. bissettiae infection to naïve susceptible mice as determined by tissue culture and serology. In the tick, B. bissettiae spirochete levels slightly increased from fed larvae to molted and then fed nymphs, yet the bacteria were absent in molted adults. Moreover, in contrast to B. burgdorferi (s.s.), B. bissettiae failed to exponentially increase in upon completion of feeding in our transmission experiment. In this specific model, I. scapularis was unable to support B. bissettiae throughout its life-cycle, and while live spirochetes were detected in B. bissettiae-infected ticks fed on naïve mice, there was no evidence of murine infection. These data question the vector competence of Ixodes scapularis for B. bissettiae. More importantly, this specific B. bissettiae-I. scapularis model may provide a tool for researchers to delineate details on mechanisms involved in Borrelia-tick compatibility.

BB0250 of Borrelia burgdorferi is a conserved and essential inner membrane protein required for cell division.

The gene bb0250 of Borrelia burgdorferi is a homolog of the dedA family, encoding integral inner membrane proteins that are present in nearly all species of bacteria. To date, no precise function has been attributed to any dedA gene. Unlike many bacterial species, such as Escherichia coli, which has eight dedA genes, B. burgdorferi possesses only one, annotated bb0250, providing a unique opportunity to investigate the functions of the dedA family. Here, we show that bb0250 is able to restore normal growth and cell division to a temperature-sensitive E. coli mutant with simultaneous deletions of two dedA genes, yqjA and yghB, and encodes a protein that localizes to the inner membrane of E. coli. The bb0250 gene could be deleted from B. burgdorferi only after introduction of a promoterless bb0250 under the control of an inducible lac promoter, indicating that it is an essential gene in this organism. Growth of the mutant in the absence of isopropyl-ß-d-thiogalactopyranoside resulted in cell death, preceded by cell division defects characterized by elongated cells and membrane bulges, demonstrating that bb0250 is required for proper cell division and envelope integrity. Finally, we show that BB0250 depletion leads to imbalanced membrane phospholipid composition in borrelia. These results demonstrate a strong conservation of function of the dedA gene family across diverse species of Gram-negative bacteria and a requirement for this protein family for normal membrane lipid composition and cell division.

Molecular adaptation of Borrelia burgdorferi in the murine host.

An analysis of expression of 137 lipoprotein genes on the course of murine infection revealed a two-step molecular adaptation by Borrelia burgdorferi, the Lyme disease spirochete. For the first step, regardless whether the initial inocula of B. burgdorferi expressed either all (cultured spirochetes) or less than 40 (host-adapted spirochetes) of the 137 lipoprotein genes, the spirochetes were modulated to transcribe 116 of the genes within 10 d after being introduced to the murine host. This step of adaptation was induced by the microenvironment of the host tissue. During the second step, which was forced by host immune selection pressure and occurred between 17 and 30 d after infection, B. burgdorferi down-regulated most of the lipoprotein genes and expressed less than 40 of the 137 genes. This novel adaptation mechanism could be a critical step for B. burgdorferi to proceed to chronic infection, as the pathogen would be cleared at the early stage of infection if the spirochetes failed to undergo this process.

An immune evasion mechanism for spirochetal persistence in Lyme borreliosis.

Borrelia burgdorferi, the Lyme disease spirochete, persistently infects mammalian hosts despite the development of strong humoral responses directed against the pathogen. Here we describe a novel mechanism of immune evasion by B. burgdorferi. In immunocompetent mice, spirochetes that did not express ospC (the outer-surface protein C gene) were selected within 17 d after inoculation, concomitantly with the emergence of anti-OspC antibody. Spirochetes with no detectable OspC transcript that were isolated from immunocompetent mice reexpressed ospC after they were either cultured in vitro or transplanted to naive immunocompetent mice, but not in OspC-immunized mice. B. burgdorferi persistently expressed ospC in severe combined immune-deficient (SCID) mice. Passive immunization of B. burgdorferi-infected SCID mice with an anti-OspC monoclonal antibody selectively eliminated ospC-expressing spirochetes but did not clear the infection. OspC-expressing spirochetes reappeared in SCID mice after the anti-OspC antibody was eliminated. We submit that selection of surface-antigen nonexpressers is an immune evasion mechanism that contributes to spirochetal persistence.

Two regulatory elements required for enhancing ospA expression in Borrelia burgdorferi grown in vitro but repressing its expression during mammalian infection.

During cycling between the tick vector and a mammal, the Lyme disease spirochaete Borrelia burgdorferi must coordinate expression of outer-surface proteins (Osps) A and B to quickly respond to environmental changes. The pathogen abundantly produces OspA/B in the tick, but represses their expression during mammalian infection. This paper reports a regulatory structure, consisting of two sequences flanking the ospAB promoter, that is required for enhancing ospA expression in B. burgdorferi grown in vitro, but repressing its expression during murine infection. Deletion or replacement of either the upstream or downstream sequence of the ospAB promoter caused a significant decrease in ospA expression in vitro, but a dramatic increase during murine infection. Fusion of either sequence with the flaB reporter promoter led to increased expression of an ospA reporter gene in vitro, but a decrease in the murine host. Furthermore, simultaneous fusion of both sequences with the reporter promoter showed a synergistic effect in enhancing expression of the ospA reporter in vitro, but repressing its expression during murine infection. Taken together, the results demonstrate that the regulatory structure functions oppositely in the two different environments and potentially provides B. burgdorferi with a molecular mechanism to quickly adapt to the distinct environments during its enzootic life cycle.

Essential protective role attributed to the surface lipoproteins of Borrelia burgdorferi against innate defences.

To initiate infection, a microbial pathogen must be able to evade innate immunity. Here we show that the Lyme disease spirochete Borrelia burgdorferi depends on its surface lipoproteins for protection against innate defences. The deficiency for OspC, an abundantly expressed surface lipoprotein during early infection, led to quick clearance of B. burgdorferi after inoculation into the skin of SCID mice. Increasing expression of any of the four randomly chosen surface lipoproteins, OspA, OspE, VlsE or DbpA, fully protected the ospC mutant from elimination from the skin tissue of SCID mice; moreover, increased OspA, OspE or VlsE expression allowed the mutant to cause disseminated infection and restored the ability to effectively colonize both joint and skin tissues, albeit the dissemination process was much slower than that of the mutant restored with OspC expression. When the ospC mutant was modified to express OspA under control of the ospC regulatory elements, it registered only a slight increase in the 50% infectious dose than the control in SCID mice but a dramatic increase in immunocompetent mice. Taken together, the study demonstrated that the surface lipoproteins provide B. burgdorferi with an essential protective function against host innate elimination.

Both decorin-binding proteins A and B are critical for the overall virulence of Borrelia burgdorferi.

Both decorin-binding proteins (DbpA and DbpB) of the Lyme disease spirochete Borrelia burgdorferi bind decorin and glycosaminoglycans, two important building blocks of proteoglycans that are abundantly found in the extracellular matrix (ECM) and connective tissues as well as on cell surfaces of mammals. As an extracellular pathogen, B. burgdorferi resides primarily in the ECM and connective tissues and between host cells during mammalian infection. The interactions of B. burgdorferi with these host ligands mediated by DbpA and DbpB potentially influence various aspects of infection. Here, we show that both DbpA and DbpB are critical for the overall virulence of B. burgdorferi in the murine host. Disruption of the dbpBA locus led to nearly a 10(4)-fold increase in the 50% infectious dose (ID50). Complementation of the mutant with either dbpA or dbpB reduced the ID50 from over 10(4) to roughly 10(3) organisms. Deletion of the dbpBA locus affected colonization in all tissues of infected mice. The lack of dbpA alone precluded the pathogen from colonizing the heart tissue, and B. burgdorferi deficient for DbpB was recovered only from 42% of the heart specimens of infected mice. Although B. burgdorferi lacking either dbpA or dbpB was consistently grown from joint specimens of almost all infected mice, it generated bacterial loads significantly lower than the control. The deficiency in either DbpA or DbpB did not reduce the bacterial load in skin, but lack of both significantly did. Taken together, the study results indicate that neither DbpA nor DbpB is essential for mammalian infection but that both are critical for the overall virulence of B. burgdorferi.

Antigenicity and recombination of VlsE, the antigenic variation protein of Borrelia burgdorferi, in rabbits, a host putatively resistant to long-term infection with this spirochete.

Borrelia burgdorferi, the Lyme disease pathogen, employs several immune-evasive strategies to survive in mammals. Unlike mice, major reservoir hosts for B. burgdorferi, rabbits are considered to be nonpermissive hosts for persistent infection. Antigenic variation of the VlsE molecule is a probable evasion strategy known to function in mice. The invariable region 6 (IR6) and carboxyl-terminal domain (Ct) of VlsE elicit dominant antibody responses that are not protective, perhaps to function as decoy epitopes that protect the spirochete. We sought to determine if either of these characteristics of VlsE differed in rabbit infection, contributing to its reputed nonpermissiveness. VlsE recombination was observed in rabbits that were given inoculations with either cultured or host-adapted spirochetes. Early observations showed a lack of anti-C6 (a peptide encompassing the IR6 region) response in most rabbits, so the anti-Ct and anti-C6 responses were monitored for 98 weeks. Anti-C6 antibody appeared as late as 20 weeks postinoculation, and the anti-Ct response, evident within the first 2 weeks, oscillated for prolonged periods of time. These observations, together with the recovery of cultivable spirochetes from tissue of one animal at 98 weeks postinoculation, challenge the notion that the rabbit cannot harbour a long-term B. burgdorferi infection.

BosR functions as a repressor of the ospAB operon in Borrelia burgdorferi.

The Lyme disease spirochete, Borrelia burgdorferi, must abundantly produce outer surface lipoprotein A (OspA) in the tick vector but downregulate OspA in mammals in order to evade the immune system and maintain its natural enzootic cycle. Here, we show that BosR binds two regulatory elements of the ospAB operon and that increasing BosR expression leads to downregulation of OspA. Both regulatory sequences, cisI and cisII, showed strong BosR-binding and cisII bound much tighter than cisI. A promoterless bosR gene fused with an inducible promoter was introduced into an rpoS mutant and a wild-type strain to assess RpoS-independent and -dependent downregulation of OspA by BosR. With the induction of BosR expression, OspA expression was reduced more significantly in the RpoS-deficient than wild-type background, but not completely repressed. In the presence of constitutive expression of OspC, DbpA and DbpB, increasing BosR production resulted in complete repression of OspA in the RpoS mutant. Taken together, the study clearly demonstrated BosR serves as a repressor that binds both regulatory elements of the ospAB operon and shuts off expression.

Detection of human bacterial pathogens in ticks collected from Louisiana black bears (Ursus americanus luteolus).

There are 4 major human-biting tick species in the northeastern United States, which include: Amblyomma americanum, Amblyomma maculatum, Dermacentor variabilis, and Ixodes scapularis. The black bear is a large mammal that has been shown to be parasitized by all the aforementioned ticks. We investigated the bacterial infections in ticks collected from Louisiana black bears (Ursus americanus subspecies luteolus). Eighty-six ticks were collected from 17 black bears in Louisiana from June 2010 to March 2011. All 4 common human-biting tick species were represented. Each tick was subjected to polymerase chain reaction (PCR) targeting select bacterial pathogens and symbionts. Bacterial DNA was detected in 62% of ticks (n=53). Rickettsia parkeri, the causative agent of an emerging spotted fever group rickettsiosis, was identified in 66% of A. maculatum, 28% of D. variabilis, and 11% of I. scapularis. The Lyme disease bacterium, Borrelia burgdorferi, was detected in 2 I. scapularis, while one A. americanum was positive for Borrelia bissettii, a putative human pathogen. The rickettsial endosymbionts Candidatus Rickettsia andeanae, rickettsial endosymbiont of I. scapularis, and Rickettsia amblyommii were detected in their common tick hosts at 21%, 39%, and 60%, respectively. All ticks were PCR-negative for Anaplasma phagocytophilum, Ehrlichia spp., and Babesia microti. This is the first reported detection of R. parkeri in vector ticks in Louisiana; we also report the novel association of R. parkeri with I. scapularis. Detection of both R. parkeri and B. burgdorferi in their respective vectors in Louisiana demands further investigation to determine potential for human exposure to these pathogens.

Increasing RpoS expression causes cell death in Borrelia burgdorferi.

RpoS, one of the two alternative σ factors in Borrelia burgdorferi, is tightly controlled by multiple regulators and, in turn, determines expression of many critical virulence factors. Here we show that increasing RpoS expression causes cell death. The immediate effect of increasing RpoS expression was to promote bacterial division and as a consequence result in a rapid increase in cell number before causing bacterial death. No DNA fragmentation or degradation was observed during this induced cell death. Cryo-electron microscopy showed induced cells first formed blebs, which were eventually released from dying cells. Apparently blebbing initiated cell disintegration leading to cell death. These findings led us to hypothesize that increasing RpoS expression triggers intracellular programs and/or pathways that cause spirochete death. The potential biological significance of induced cell death may help B. burgdorferi regulate its population to maintain its life cycle in nature.

RpoS regulates essential virulence factors remaining to be identified in Borrelia burgdorferi.

BACKGROUND: Since the RpoN-RpoS regulatory network was revealed in the Lyme disease spirochete Borrelia burgdorferi a decade ago, both upstream and downstream of the pathway have been intensively investigated. While significant progress has been made into understanding of how the network is regulated, most notably, discovering a relationship of the network with Rrp2 and BosR, only three crucial virulence factors, including outer surface protein C (OspC) and decorin-binding proteins (Dbps) A and B, are associated with the pathway. Moreover, for more than 10 years no single RpoS-controlled gene has been found to be critical for infection, raising a question about whether additional RpoS-dependent virulence factors remain to be identified. METHODOLOGY/PRINCIPAL FINDINGS: The rpoS gene was deleted in B. burgdorferi; resulting mutants were modified to constitutively express all the known virulence factors, OspC, DbpA and DbpB. This genetic modification was unable to restore the rpoS mutant with infectivity. CONCLUSIONS/SIGNIFICANCE: The inability to restore the rpoS mutant with infectivity by simultaneously over-expressing all the three virulence factors allows us to conclude RpoS also regulates essential genes that remain to be identified in B. burgdorferi.

Outer surface protein C is a dissemination-facilitating factor of Borrelia burgdorferi during mammalian infection.

BACKGROUND: The Lyme disease spirochete Borrelia burgdorferi dramatically upregulates outer surface protein C (OspC) in response to fresh bloodmeal during transmission from the tick vector to a mammal, and abundantly produces the antigen during early infection. As OspC is an effective immune target, to evade the immune system B. burgdorferi downregulates the antigen once the anti-OspC humoral response has developed, suggesting an important role for OspC during early infection. METHODOLOGY/PRINCIPAL FINDINGS: In this study, a borrelial mutant producing an OspC antigen with a 5-amino-acid deletion was generated. The deletion didn't significantly increase the 50% infectious dose or reduce the tissue bacterial burden during infection of the murine host, indicating that the truncated OspC can effectively protect B. burgdorferi against innate elimination. However, the deletion greatly impaired the ability of B. burgdorferi to disseminate to remote tissues after inoculation into mice. CONCLUSIONS/SIGNIFICANCE: The study indicates that OspC plays an important role in dissemination of B. burgdorferi during mammalian infection.

Modification of Borrelia burgdorferi to overproduce OspA or VlsE alters its infectious behaviour.

The surface lipoproteins of the Lyme disease spirochaete Borrelia burgdorferi directly interact with tissue microenvironments during mammalian infection, and thus potentially affect various aspects of infection. To investigate the influence of surface antigen synthesis on infectious behaviour, B. burgdorferi was modified to constitutively produce the well-characterized surface lipoproteins OspA and invariant VlsE. Although increasing OspA or VlsE production did not significantly affect synthesis of other surface lipoproteins or spirochaetal growth in vitro, overexpressing vlsE resulted in increased ospA but decreased ospC expression, and overexpressing ospA led to decreased ospC and vlsE expression in severe combined immunodeficient (SCID) mice. Increasing the expression of either ospA or vlsE did not alter the ID(50), but affected spirochaetal dissemination and significantly reduced tissue spirochaete loads in SCID mice. In immunocompetent mice, increased vlsE expression resulted in quick clearance of infection, while constitutive ospA expression led to a substantial ID(50) increase and severely impaired dissemination. Furthermore, B. burgdorferi with constitutive ospA expression persisted in the skin tissue but was cleared from both heart and joints of chronically infected immunocompetent mice. Taken together, the study indicates that increasing production of OspA or invariant VlsE influences lipoprotein gene expression in the murine host and alters the infectious behaviour of B. burgdorferi.

Verification and dissection of the ospC operator by using flaB promoter as a reporter in Borrelia burgdorferi.

The Lyme disease spirochete Borrelia burgdorferi must repress expression of outer surface protein C (OspC) to effectively evade specific humoral immunity and to establish persistent infection. This ability largely relies upon a regulatory element, the only operator that has been reported in spirochetal bacteria. Immediately upstream of the ospC promoter, two sets of inverted repeats (IRs) constitute small and large palindromes, in which the right IR of the large palindrome contains the left IR of the small one, and may collectively function as the ospC operator. In the study, the large palindrome with or without the small IR was fused with an flaB promoter, which was used to drive expression of a promoterless ospC copy as a reporter gene, and introduced into OspC-deficient B. burgdorferi. The presence of the large palindrome alone significantly reduced ospC expression driven by the fused flaB promoter in the joint tissue of severe combined immunodeficiency (SCID) mice, and rescued spirochetes from elimination by passively transferred OspC antibody in infected SCID mice and specific immune responses elicited in immunocompetent mice, confirming a function of the IRs as an operator. Inclusion of the small IR further enhanced the ability of the large palindrome to reduce the activity of the fused flaB promoter, indicating that the small IR is a part of the operator. Taken together, the study led to successful verification and dissection of the ospC operator.

Common and unique contributions of decorin-binding proteins A and B to the overall virulence of Borrelia burgdorferi.

As an extracellular bacterium, the Lyme disease spirochete Borrelia burgdorferi resides primarily in the extracellular matrix and connective tissues and between host cells during mammalian infection, where decorin and glycosaminoglycans are abundantly found, so its interactions with these host ligands potentially affect various aspects of infection. Decorin-binding proteins (Dbps) A and B, encoded by a 2-gene operon, are outer surface lipoproteins with similar molecular weights and share approximately 40% identity, and both bind decorin and glycosaminoglycans. To investigate how DbpA and DbpB contribute differently to the overall virulence of B. burgdorferi, a dbpAB mutant was modified to overproduce the adhesins. Overproduction of either DbpA or DbpB resulted in restoration of the infectivity of the mutant to the control level, measured by 50% infectious dose (ID(50)), indicating that the two virulence factors are interchangeable in this regard. Overproduction of DbpA also allowed the mutant to disseminate to some but not all distal tissues slightly slower than the control, but the mutant with DbpB overproduction showed severely impaired dissemination to all tissues that were analyzed. The mutant with DbpA overproduction colonized all tissues, albeit generating bacterial loads significantly lower than the control in heart and joint, while the mutant overproducing DbpB remained severely defective in heart colonization and registered bacterial loads substantially lower than the control in joint. Taken together, the study indicated that DbpA and DbpB play a similar role in contribution to infectivity as measured by ID(50) value but contribute differently to dissemination and tissue colonization.