Identification of an ospC operator critical for immune evasion of Borrelia burgdorferi

Timely expression of the outer surface protein C (OspC) is crucial for the pathogenic strategy of the Lyme disease spirochete Borrelia burgdorferi. The pathogen abundantly expresses OspC during initial infection when the antigen is required, but downregulates when its presence poses a threat to the spirochetes once the anti-OspC humoral response has developed. Here, we show that a large palindromic sequence immediately upstream of the ospC promoter is essential for the repression of ospC expression during murine infection and for the ability of B. burgdorferi to evade specific OspC humoral immunity. Deletion of the sequence completely diminished the ability of B. burgdorferi to avoid clearance by transferred OspC antibody in SCID mice. B. burgdorferi lacking the regulatory element was able to initiate infection but unable to persist in immunocompetent mice. Taken together, the regulatory element immediately upstream of the ospC promoter serves as an operator that may interact with an unidentified repressor(s) to negatively regulate ospC expression and is essential for the immune evasion of B. burgdorferi.     

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.     

The critical role of the linear plasmid lp36 in the infectious cycle of Borrelia burgdorferi

Borrelia burgdorferi, the aetiological agent of Lyme disease, follows a life cycle that involves passage between the tick vector and the mammalian host. To investigate the role of the 36 kb linear plasmid, lp36 (also designated the B. burgdorferi K plasmid), in the infectious cycle of B. burgdorferi, we examined a clone lacking this plasmid, but containing all other plasmids known to be required for infectivity. Our results indicated that lp36 was not required for spirochete survival in the tick, but the clone lacking lp36 demonstrated low infectivity in the mammal. Restoration of lp36 to the mutant strain confirmed that the infectivity defect was due to loss of lp36. Moreover, spirochetes lacking lp36 exhibited a nearly 4-log increase in ID(50) relative to the isogenic lp36(+) clone. The infectivity defect of lp36-minus spirochetes was localized, in part, to loss of the bbk17 (adeC) gene, which encodes an adenine deaminase. This work establishes a vital role for lp36 in the infectious cycle of B. burgdorferi and identifies the bbk17 gene as a component of this plasmid that contributes to mammalian infectivity.     

The role of VlsE antigenic variation in the Lyme disease spirochete: persistence through a mechanism that differs from other pathogens

The linear plasmid, lp28-1, is required for persistent infection by the Lyme disease spirochete, Borrelia burgdorferi. This plasmid contains the vls antigenic variation locus, which has long been thought to be important for immune evasion. However, the role of the vls locus as a virulence factor during mammalian infection has not been clearly defined. We report the successful removal of the vls locus through telomere resolvase-mediated targeted deletion, and demonstrate the absolute requirement of this lp28-1 component for persistence in the mouse host. Moreover, successful infection of C3H/HeN mice with an lp28-1 plasmid in which the left portion was deleted excludes participation of other lp28-1 non-vls genes in spirochete virulence, persistence and the process of recombinational switching at vlsE. Data are also presented that cast doubt on an immune evasion mechanism whereby VlsE directly masks other surface antigens similar to what has been observed for several other pathogens that undergo recombinational antigenic variation.     

Borrelia burgdorferi sensu stricto invasiveness is correlated with OspC-plasminogen affinity

Borrelia burgdorferi sensu lato, the causative agent of Lyme borreliosis, is transmitted through tick bite. Lyme borreliosis evolves in two stages: a primary red skin lesion called erythema migrans; later on, invasive bacteria disseminate to distant sites inducing secondary manifestations (neuropathies, arthritis, carditis, late skin disorders). It has been previously suggested that the ospC gene could be associated with invasiveness in humans depending on its sequence. Here, we confirm the pattern of invasiveness, according to B. burgdorferi sensu stricto (B. b. ss) ospC group, using the mouse as an experimental host of B. b. ss. As it has been shown that the host plasminogen activation system is used by B. burgdorferi to disseminate throughout the host, we studied the interaction of plasminogen with OspC proteins from invasive and non-invasive groups of B. b. ss. Using two methods, ELISA and surface plasmon resonance, we demonstrate that indeed OspC is a plasminogen-binding protein. Moreover, significant differences in binding affinity for plasminogen are correlated with different invasiveness patterns in mice. These results suggest that the correlation between ospC polymorphism and Borrelia invasiveness in humans is linked, at least in part, to differences in OspC affinity for plasminogen.     

The Borrelia burgdorferi circular plasmid cp26: conservation of plasmid structure and targeted inactivation of the ospC gene

The 26 to 28 kb circular plasmid of B. burgdorferi sensu lato (cp26) is ubiquitous among bacteria of this group and contains loci implicated in the mouse–tick transmission cycle. Restriction mapping and Southern hybridization indicated that the structure of cp26 is conserved among isolates from different origins and culture passage histories. The cp26 ospC gene encodes an outer surface protein whose synthesis within infected ticks increases when the ticks feed, and whose synthesis in culture increases after a temperature upshift. Previous studies of ospC coding sequences showed them to have stretches of sequence apparently derived from the ospC genes of distantly related isolates by homologous recombination after DNA transfer. We found conservation of the promoter regions of the ospC and guaA genes, which are divergently transcribed. We also demonstrated that the increase in OspC protein after a temperature upshift parallels increases in mRNA levels, as expected if regulatory regions adjoin the conserved sequences in the promoter regions. Finally, we used directed insertion to inactivate the ospC gene of a non-infectious isolate. This first example of directed gene inactivation in B. burgdorferi shows that the OspC protein is not required for stable maintenance of cp26 or growth in culture.     

Analysis of the ospC regulatory element controlled by the RpoN-RpoS regulatory pathway in Borrelia burgdorferi

Outer surface lipoprotein C (OspC) is a key virulence factor of Borrelia burgdorferi. ospC is differentially regulated during borrelial transmission from ticks to rodents, and such regulation is essential for maintaining the spirochete in its natural enzootic cycle. Recently, we showed that the expression of ospC in B. burgdorferi is governed by a novel alternative sigma factor regulatory network, the RpoN-RpoS pathway. However, the precise mechanism by which the RpoN-RpoS pathway controls ospC expression has been unclear. In particular, there has been uncertainty regarding whether ospC is controlled directly by RpoS (sigma(s)) or indirectly through a transactivator (induced by RpoS). Using deletion analyses and genetic complementation in an OspC-deficient mutant of B. burgdorferi, we analyzed the cis element(s) required for the expression of ospC in its native borrelial background. Two highly conserved upstream inverted repeat elements, previously implicated in ospC regulation, were not required for ospC expression in B. burgdorferi. Using similar approaches, a minimal promoter that contained a canonical -35/-10 sequence necessary and sufficient for sigma(s)-dependent regulation of ospC was identified. Further, targeted mutagenesis of a C at position -15 within the extended -10 region of ospC, which is postulated to function like the strategic C residue important for Esigma(s) binding in Escherichia coli, abolished ospC expression. The minimal ospC promoter also was responsive to coumermycin A(1), further supporting its sigma(s) character. The combined data constitute a body of evidence that the RpoN-RpoS regulatory network controls ospC expression by direct binding of sigma(s) to a sigma(s)-dependent promoter of ospC. The implication of our findings to understanding how B. burgdorferi differentially regulates ospC and other ospC-like genes via the RpoN-RpoS regulatory pathway is discussed.     

Evolution of the Borrelia burgdorferi outer surface protein OspC.

The genes coding for outer surface protein OspC from 22 Borrelia burgdorferi strains isolated from patients with Lyme borreliosis were cloned and sequenced. For reference purposes, the 16S rRNA genes from 17 of these strains were sequenced after being cloned. The deduced OspC amino acid sequences were aligned with 12 published OspC sequences and revealed the presence of 48 conserved amino acids. On the basis of the alignment, OspC could be divided into an amino-terminal relatively conserved region and a relatively variable region in the central portion. The distance tree obtained divided the ospC sequences into three groups. The first group contained ospC alleles from all (n = 13) sensu stricto strains, the second group contained ospC alleles from seven Borrelia afzelii strains, and the third group contained ospC alleles from five B. afzelii and all (n = 9) Borrelia garinii strains. The ratio of the mean number of synonymous (dS) and nonsynonymous (dN) nucleotide substitutions per site calculated for B. burgdorferi sensu stricto, B. garinii, and B. afzelii ospC alleles suggested that the polymorphism of OspC is due to positive selection favoring diversity at the amino acid level in the relatively variable region. On the basis of the comparison of 16S rRNA gene sequences, Borrelia hermsii is more closely related to B. afzelii than to B. burgdorferi sensu stricto and B. garinii. In contrast, the phylogenetic tree obtained for the B. hermsii variable major protein, Vmp33, and 18 OspC amino acid sequences suggested that Vmp33 and OspC from B. burgdorferi sensu stricto strains share a common evolutionary origin.     

Role of Borrelia burgdorferi linear plasmid 25 in infection of Ixodes scapularis ticks

The tick-borne bacterium Borrelia burgdorferi has over 20 different circular and linear plasmids. Some B. burgdorferi plasmids are readily lost during in vitro culture or genetic manipulation. Linear plasmid 25, which is often lost in laboratory strains, is required for the infection of mice. Strains missing linear plasmid 25 (lp25(-)) are able to infect mice if the BBE22 gene on lp25 is provided on a shuttle vector. In this study, we examined the role of lp25 and BBE22 in tick infections. We tested the hypothesis that complementation with BBE22 in spirochetes lacking lp25 would restore the ability of spirochetes to infect ticks. A natural tick infection cycle was performed by feeding larvae on mice injected with the parental, lp25(-), or lp25(-) BBE22-complemented spirochete strains. In addition, larvae and nymphs were artificially infected with different strains to study tick infections independent of mouse infections. B. burgdorferi missing lp25 was significantly impaired in its ability to infect larval and nymphal ticks. When an lp25(-) strain was complemented with BBE22, the ability to infect ticks was partially restored. Complementation with BBE22 allowed spirochetes lacking lp25 to establish short-term infections in ticks, but in most cases the infection prevalence was lower than that of the wild-type strain. In addition, the number of infected ticks decreased over time, suggesting that another gene(s) on lp25 is required for long-term persistence in ticks and completion of a natural infection cycle.     

Plasmid location of Borrelia purine biosynthesis gene homologs.

The Lyme disease spirochete Borrelia burgdorferi must survive in both its tick vector and its mammalian host to be maintained in nature. We have identified the B. burgdorferi guaA gene encoding GMP synthetase, an enzyme involved in de novo purine biosynthesis that is important for the survival of bacteria in mammalian blood. This gene encodes a functional product that will complement an Escherichia coli GMP synthetase mutant. The gene is located on a 26-kb circular plasmid, adjacent to and divergent from the gene encoding the outer surface protein C (OspC). The guaB gene homolog encoding IMP dehydrogenase, another enzyme in the purine biosynthetic pathway, is adjacent to guaA. In Borrelia hermsii, a tick-borne relapsing fever spirochete, the guaA and guaB genes are located on a linear plasmid. These are the first genes encoding proteins of known function to be mapped to a borrelial plasmid and the only example of genes encoding enzymes involved in the de novo purine biosynthesis pathway to be mapped to a plasmid in any organism. The unique plasmid location of these and perhaps other housekeeping genes may be a consequence of the segmented genomes in borreliae and reflect the need to adapt to both the arthropod and mammalian environments.     

OspC is potent plasminogen receptor on surface of Borrelia burgdorferi

Host-derived proteases are crucial for the successful infection of vertebrates by several pathogens, including the Lyme disease spirochete bacterium, Borrelia burgdorferi. B. burgdorferi must traverse tissue barriers in the tick vector during transmission to the host and during dissemination within the host, and it must disrupt immune challenges to successfully complete its infectious cycle. It has been proposed that B. burgdorferi can accomplish these tasks without an endogenous extra-cytoplasmic protease by commandeering plasminogen, the highly abundant precursor of the vertebrate protease plasmin. However, the molecular mechanism by which B. burgdorferi immobilizes plasminogen to its surface remains obscure. The data presented here demonstrate that the outer surface protein C (OspC) of B. burgdorferi is a potent plasminogen receptor on the outer membrane of the bacterium. OspC-expressing spirochetes readily bind plasminogen, whereas only background levels of plasminogen are detectable on OspC-deficient strains. Furthermore, plasminogen binding by OspC-expressing spirochetes can be significantly reduced using anti-OspC antibodies. Co-immunofluorescence staining assays demonstrate that wild-type bacteria immobilize plasminogen only if they are actively expressing OspC regardless of the expression of other surface proteins. The co-localization of plasminogen and OspC on OspC-expressing spirochetes further implicates OspC as a biologically relevant plasminogen receptor on the surface of live B. burgdorferi.     

Expression and sequence of outer surface protein C among North American isolates of Borrelia burgdorferi

Abstract The expression of outer surface protein C (OspC) was determined for North American Borrelia burgdorferi isolates HB19, DN127c19-2, 25015 and both low and high culture passage B31. A monoclonal antibody detected the presence of OspC protein in only two isolates, while polyclonal antiserum identified this protein in all five isolates. The ospC gene was cloned and sequenced for isolates HB19, DN127c19-2 and 25015, and compared with the published ospC sequences of other Lyme disease spirochetes. Bothe the nucleotide and amino acid sequences were found to vary as much among isolates from the same geographic area as between isolates of different species.     

Interdependence of environmental factors influencing reciprocal patterns of gene expression in virulent Borrelia burgdorferi

The paradigm for differential antigen expression in Borrelia burgdorferi, the agent of Lyme disease, is the reciprocal expression of its outer surface (lipo)proteins (Osp) A and C; as B. burgdorferi transitions from its arthropod vector into mammalian tissue, ospC is upregulated, and ospA is downregulated. In the current study, using B. burgdorferi cultivated under varying conditions in BSK-H medium, we found that a decrease in pH, in conjunction with increases in temperature (e.g. 34 degrees C or 37 degrees C) and cell density, acted interdependently for the reciprocal expression of ospC and ospA. The lower pH (6.8), which induced the reciprocal expression of ospC and ospA in BSK-H medium, correlated with a drop in pH from 7.4 to 6.8 of tick midgut contents during tick feeding. In addition to ospC and ospA, other genes were found to be regulated in reciprocal fashion. Such genes were either ospC-like (e.g. ospF, mlp-8 and rpoS) (group I) or ospA-like (lp6.6 and p22) (group II); changes in expression occurred at the mRNA level. That the expression of rpoS, encoding a putative stress-related alternative sigma factor (sigma(s)), was ospC-like suggested that the expression of some of the group I genes may be controlled through sigma(s). The combined results prompt a model that allows for predicting the regulation of other B. burgdorferi genes that may be involved in spirochaete transmission, virulence or mammalian host immune responses.     

Mapping of essential replication functions of the linear plasmid lp17 of B. burgdorferi by targeted deletion walking

The genome of the Lyme disease pathogen Borrelia burgdorferi strain B31 MI includes one linear chromosome, 10 circular and 12 linear plasmids. Members of four paralogous gene families, revealed by genome sequencing, have been suggested as replication/partition functions for both the linear and circular plasmids. Some of these genes have been experimentally shown to be essential for the replication of the B. burgdorferi replicons that encode them. In this study, we located the region essential for replication of lp17, the second smallest linear plasmid in B. burgdorferi. We used a novel in vivo method, targeted deletion walking, to systematically delete DNA from either the left or right end of lp17. We report that the region essential for replication of lp17 is 1.8 kb (bp 7946-9766) and contains only one intact open reading frame (BBD14). Expression of BBD14 is required for the replication, suggesting that it is the replication initiator for lp17. The BBD14 protein is a member of paralogous family (PF) 62 and we present the first experimental evidence for the role of a PF 62 member. Adjacent non-coding sequences are also required, suggesting that the origin lies at least partially outside the coding region. Surprisingly, deletion of BBD21, the ParA orthologue (PF 32), had little effect upon plasmid stability or incompatibility. Finally, data are presented suggesting that lp17 replication occurs preferentially on a linear rather than a circular DNA molecule.