The long strange trip of Borrelia burgdorferi outer-surface protein C

Borrelia burgdorferi, the Lyme disease spirochete, accomplishes some remarkable feats during its enzootic cycle. Not only does it adjust physiologically to two markedly different host milieus, arthropod vector and small mammal reservoir, but it also exploits the windows of opportunity created during the feeding of larval and nymphal Ixodes scapularis ticks to transit seamlessly between the two (Fikrig and Narasimhan, 2006). Years after outer-surface protein C (OspC) emerged as a key player in the events that unfold when a feeding nymph transmits spirochaetes to a mammal, we still have only vague notions of how this enigmatic molecule contributes to virulence. The provocative report by Xu et al. (2008) in this issue of Molecular Microbiology deepens the mystery. It reveals that, in addition to whatever specific role OspC plays during early infection, it also has non-specific functions that can be partially replaced by seemingly unrelated borrelial outer-surface lipoproteins.

Borrelia burgdorferi is often likened to Gram-negative bacteria because of its diderm ultrastructure. This analogy belies distinctive differences between the outer membrane of B. burgdorferi and its Gram-negative counterparts. In addition to lacking lipopolysaccharide (LPS), the B. burgdorferi outer membrane is considerably more fluid, contains a much lower density of membrane-spanning proteins, and has an outer leaflet adorned with lipoproteins that comprise the bacterium's primary interface with its hosts (Radolf et al., 1994). The spirochete's genome encodes a huge number (> 150) of lipoproteins, the large majority of which lack functionally characterized orthologues (Fraser et al., 1997; Casjens et al., 2000). Differential expression of this vast lipoproteome is crucial for the ability of the spirochete to survive, multiply and migrate within both arthropod and mammalian hosts (Pal and Fikrig, 2003). This extraordinary versatility does not come without a price. Like LPS, lipoproteins are potent innate agonists that attract and activate phagocytes that, at least in vitro, are proficient at ingesting and killing spirochaetes. Moreover, lipoproteins tend to be strongly antigenic and, when surface-exposed, render the organism highly vulnerable to the antibodies they induce. Having a lipoprotein-rich outer membrane might impose other, less obvious, physical constraints on the bacterium. A minimum total density of lipoproteins might be required to maintain the structural and/or functional integrity of the outer bilayer or to shield it from membrane-lytic host defences, such as complement and antimicrobial peptides.

The central paradigm of Lyme disease research, the reciprocal regulation of OspA and OspC as spirochaetes alternate between arthropod and mammal, did not take shape overnight. OspA, the predominant lipoprotein produced by spirochaetes in vitro, was discovered soon after B. burgdorferi was isolated from deer ticks (Barbour et al., 1983). More than a decade elapsed, however, before investigators realized that high-level production of OspA by spirochaetes is a hallmark of residence in the midgut of unfed ticks, that spirochaetes downregulate its production as they replicate and migrate to salivary glands during the blood meal, and that they cease to produce it within the mammal (Fikrig and Narasimhan, 2006). Fikrig and co-workers (Pal et al., 2004a) elegantly tied these observations together by showing that OspA tethers spirochaetes to a receptor on midgut epithelium in flat ticks.

OspC has a history at least as serendipitous. North American researchers overlooked the molecule through the early 1990s because they usually studied high-passage borrelial strains that poorly express it. The immunodominance of OspC during acute infection first attracted the attention of European scientists whose strains did express the protein in vitro. In a seminal work published in 1995, Schwan et al. (1995) showed that OspC, in contrast to OspA, is not produced by spirochaetes in flat ticks, but is induced during the blood meal and in response to elevated temperature during cultivation in BSK medium. They astutely speculated that OspC is essential for infectivity in mammals, although their data also were consistent with the possibility that it promotes the dissemination of spirochaetes within the vector.

Solution of the molecule's crystal structure shed surprisingly little light on its function. OspC forms an elongated, dimeric helical bundle with presumptive binding sites at or near its membrane distal end – but for what ligand and in which host? When the OspC structure was published (Eicken et al., 2001; Kumaran et al., 2001), the periplasmic domain of the aspartate receptor of Salmonella Typhimurium was the only structurally similar protein in the databases, a result still of uncertain significance. Potentially more relevant (see below) is that OspC shares general structural features with VlsE (Eicken et al., 2002), an antigenically variable B. burgdorferi lipoprotein required for evasion of adaptive humoral immunity. Crystallographic analysis did, on the other hand, provide tantalizing indirect evidence linking OspC to spirochetal dissemination. Seinost et al. (1999) had shown that only a few of the numerous ospC alleles circulating among spirochaetes in ticks appear to be associated with disseminated borrelial infection of humans. Kumaran et al. (2001) found that OspCs from invasive clones have a much greater electrostatic potential at their distal ends, which they proposed enhances binding to positively charged extracellular matrix components.

Using improved genetic and molecular methodologies, investigators have begun to nudge the field towards its current, although still inadequate, understanding of what OspC does. In 2002, Liang et al. (2002) reported that ospC transcripts could not be detected after 2 weeks of infection and that OspC antibodies produced during this period select for OspC non-producers. Recently, this same group identified an operator sequence near the ospC promoter required for downregulation of the gene in vivo, presumably the binding site for a trans-acting repressor (Xu et al., 2007). While these results indicate that production of OspC is not only dispensable but even deleterious for the spirochete as the adaptive immune response develops, they do not unambiguously identify the host or locale in which it functions. Mutagenesis, potentially the most powerful tool in the Lyme disease researcher's expanding armamentarium, produced strikingly divergent answers to this critical question. Pal et al. (2004b) observed that an ospC-deficient mutant was unable to penetrate salivary glands and was not recovered in the skin surrounding the bite site, a result consistent with their finding in the same study that recombinant OspC bound to salivary glands. Grimm et al. (2004), in contrast, found that an ospC mutant was non-infectious by both needle and tick inoculation, but could penetrate salivary glands as well as the parental wild type. The lack of virulence by syringe injection was highly significant because this route should have bypassed the salivary gland defect observed by Pal et al. (2004b). In agreement with the work of Grimm et al. (2004), de Silva and colleagues (Ohnishi et al., 2001) showed by immunofluoresence microscopy that wild-type spirochaetes not producing OspC could be detected in both salivary glands during feeding and in skin shortly after tick inoculation.

One trivial explanation for the discordant results is that the mutant used by Grimm et al. (2004) produced a truncated OspC with partial activity. The Rosa group closed this loophole by demonstrating that an ospC deletion mutant also was avirulent by both the tick and needle routes and, like its truncated predecessor, could penetrate salivary glands (Tilly et al., 2006). They also found that complemented deletion mutants recovered 6 weeks post syringe inoculation had lost the complementing plasmid, confirming that OspC becomes dispensable as infection proceeds. In a clever and meticulously executed follow-up study, they showed that ospC deletion mutants were cleared from intradermal sites within 48 h, well before the period of time (8 days) required for the wild type or complemented ospC mutant to disseminate (Tilly et al., 2007). Last, in a separate report (Stewart et al., 2006), they demonstrated that spirochaetes lacking OspC grow normally in dialysis membrane chambers implanted into the peritoneal cavities of rats and that neither severe combined immune deficiency (SCID) nor MyD88 knockout mice are susceptible to infection with ospC-deficient spirochaetes. Putting these results together, they postulated that OspC directly or indirectly protects spirochaetes against a MyD88-independent component of innate immunity during the first 48 h after transmission. A subtle but important implication of this hypothesis is that another borrelial lipoprotein might assume a protective role as production of OspC ramps down, and they proposed the structurally similar, but antibody-resistant, VlsE as the OspC replacement. It is essential to point out, however, that their interpretation of these collective data seems overly restrictive, given that rapid clearance of spirochaetes deficient in OspC would obscure its role in facilitating dissemination at subsequent time points.

Indeed, the current communication from the Liang group (Xu et al., 2008) supports the notion that OspC has dual early protective and dissemination-promoting functions. It does so, however, in a paradigm-bending fashion. At the outset, they compared the recovery of ospC mutants complemented with either ospC or genes for four other lipoproteins (OspA, VlsE, OspE and DbpA) constitutively expressed from a flaB promoter 24–72 h following intradermal syringe inoculation into SCID mice. Amazingly, all five protected the OspC-deficient spirochaetes from early elimination. They next assessed the ability of the complemented mutants to disseminate. Again, contrary to expectations, three of the four heterologous lipoproteins (OspA, VlsE and OspE) promoted dissemination to ear, heart and joint in SCID mice, although with lower efficiency than OspC. Interestingly, mutants complemented with DbpA remained ‘stuck’ at the skin inoculation site, presumably too tightly bound to decorin to disseminate, but were not cleared. OspC was particularly important for dissemination to hearts, as opposed to skin and joints, based on measurement of spirochetal burdens by quantitative PCR. When they compared complementing plasmids in which ospC or ospA were expressed from the full-length (and, hence, downregulatable) ospC promoter, they found that the ospC-complemented spirochaetes disseminated more quickly in SCID mice, but that the ID₅₀ values of the ospA-complemented clones at 4 weeks were only marginally greater. Surprisingly, the requirement for ospC for colonization of distant sites was greater in immunocompetent than in SCID mice. In the former background, the same ospA construct had a 60-fold greater ID₅₀ value at 6 weeks and a much diminished capacity for persistence in hearts and joints at 4 months.

Clearly, devising a mechanistic explanation for these largely counter-intuitive results presents a challenge. The authors proposed that OspC stabilizes the B. burgdorferi outer membrane against innate immune clearance mechanisms at the site of inoculation, and that other lipoproteins can provide this stabilizing function when produced at high enough levels. They seemed more at a loss to explain how three of the lipoproteins partially replaced OspC in promoting dissemination. The findings for VlsE might be explained by its loose structural relatedness to OspC, but this explanation is unlikely for OspE and is certainly untenable for OspA, which has extensive beta sheet structure (Li et al., 1997). By any standard, therefore, these perplexing results run counter to prevailing orthodoxies of bacterial pathogenesis.

An undeniable concern with the Xu et al. (2008) study is that it utilized spirochaetes subjected to an extreme form of genetic manipulation. Although admittedly unconventional, the innovative experimental strategy devised by these investigators enabled them to separate the protective from the disseminating aspects of OspC function, both of which could be discerned, but not clearly distinguished, in previous work. In so doing, the study has brought to light some obvious but remediable deficiencies in our current understanding of critical early events in Lyme disease pathogenesis. First and foremost is whether OspC also functions within the tick or solely within the mouse; until this issue is resolved, there will be lingering uncertainty as to whether needle inoculation will suffice for further delineating the molecule's function. The second relates to our knowledge of the basic membrane biology of B. burgdorferi and its relationship to the survival and dissemination strategies of spirochaetes following deposition by ticks at the feeding site. Last, we need to learn more about the interactions of spirochaetes with matrix components and resident innate immune cells following inoculation. Behavioural characterization of individual wild type and mutated spirochaetes in situ should be possible using fluorescent reporters in conjunction with recent advances in real-time intravital microscopy. We predict that such studies will reveal that the protective and dissemination-promoting functions of OspC are actually two sides of the same coin and that the molecule has been finely tuned to fulfil these dual biological roles.