Borrelia burgdorferi, the cauative agent of Lyme disease, is maintained in nature through an infectious cycle between wild mammals and ticks. Like many bacterial pathogens, B. burgdorferi must cope with a changing array of environmental conditions in order to successfully persist, proliferate and be transmitted between hosts. B. burgdorferi has an unusual genomic structure composed of a linear chromosome and a large number of linear and circular plasmids. Abundant evidence indicates that plasmid-encoded genes are critical for adaptation in the infectious cycle. A major focus of our research is to determine the contributions of individual B. burgdorferi genes and plasmids at each stage of the infectious cycle, taking a molecular genetic approach. The composition of the outer membrane of B. burgdorferi undergoes a profound change as the spirochete is transmitted from the tick vector to a mammalian host. The plasmid-encoded OspA protein is abundant on the surface of B. burgdorferi residing in the midguts of infected ticks but as ticks feed, OspA is down-regulated and replaced with OspC, which is encoded on a different plasmid. This switch in spirochetal surface proteins was hypothesized to be required for migration of B. burgdorferi from the midgut to the salivary glands of the tick or adaptation to the mammalian host after transmission. We recently directly tested this hypothesis with an investigation of the in vivo role of OspC in both the tick vector and mammalian host (Grimm et al. 2004, Stewart et al. 2006, Tilly et al. 2006). These studies utilized isogenic wild-type, ospC mutant and complemented strains that we constructed in an infectious B. burgdorferi strain B31 background. We found that B. burgdorferi ospC mutants were unable to colonize mice by needle inoculation and that this defect was independent of acquired immunity. Complementation with a wild-type copy of ospC restored infectivity, demonstrating that the mutation was responsible for the defect. In contrast to their inability to infect mice, ospC mutant spirochetes colonized the midguts of artificially infected larval ticks, persisted through the molt and migrated to the salivary glands during the nymphal blood meal. Naive mice that were fed upon by these infected ticks did not become infected, indicating that B. burgdorferi require OspC for mouse infectivity, even by tick bite. These results indicate that the switch from OspA to OspC is not required for spirochete migration from the midgut to the salivary glands of ticks, but is in preparation for transmission to the mammalian host. This represents an adaptive response in anticipation of the next host environment. We concluded from these experiments that OspC is a virulence factor required by B. burgdorferi for the initial stage of mammalian infection. However, the role of OspC in mammalian infection was still unknown. We next conducted experiments designed to distinguish between two simple models of OspC function in the mammalian host: 1.) OspC fulfills an essential physiological role for growth and host-adaptation, or 2.) OspC provides a protective role for evasion of the innate immune response. We found that the B. burgdorferi ospC mutant, previously demonstrated to be non-infectious in both immunocompetent and SCID mice, could survive in the relatively immune-privileged environment of dialysis membrane chambers (DMCs) implanted within the peritoneal cavity of a rat (Stewart et al., 2006). Growth by B. burgdorferi in DMCs was previously shown by others to require physiological functions, but not mechanisms of immune evasion. The ospC mutant also adapted to the mammalian environment, as determined by the protein profiles of the chamber-cultivated spirochetes. Therefore, OspC does not appear to provide a strictly physiological function for the survival of B. burgdorferi within the mammalian host. The second model, evasion of the innate immune system, was tested by assessing the infectivity of the ospC mutant in mice deficient for myeloid differentiation protein 88 (MyD88). These studies were done in collaboration with Dr. Janis Weis's lab at the University of Utah (Stewart et al. 2006). Previous studies by Dr. Weis's lab have shown that wild type B. burgdorferi is prevented from reaching high numbers in the mammalian host by MyD88-dependent signaling pathways. A graduate student in Dr. Weis's group, Xiaohui Wang, subsequently found that the ospC mutant was incapable of infecting MyD88-deficient mice, suggesting that the role of OspC cannot solely be related to evasion of MyD88-mediated innate immunity (Stewart et al. 2006). These results, combined with our previous data, indicate a more complex function for OspC than predicted by our simple models: although required for initial infection of mammals, OspC does not play a singular role in surviving specific branches of the innate or acquired immune system so far investigated, nor does it provide a strictly physiological function for B. burgdorferi (Stewart et al. 2006). We have recently completed a set of experiments designed to assess the timing of the requirement for OspC in the mammal (Tilly et al. 2006). Using an ospC mutant complemented with an unstable wild-type copy of the ospC gene, we confirmed that OspC is required for mammalian infection, but not for tick colonization (including natural acquisition from the mammal) or transmission. Furthermore, we established that the requirement for OspC is limited to a crucial period early in infection of the mammalian host. Using this unique system, we found that most bacteria in mice persistently infected with the initially complemented mutant strain had lost the wild-type copy of ospC (Tilly et al. 2006). Finally, we showed that OspC is even required by host-adapted spirochetes derived from mammalian tissues to establish infections in naive mice. These findings, together with previous observations regarding differential regulation of spirochetal gene expression in ticks and mammals, indicate that host adaptation not only occurs in response to disparate arthropod and mammalian environments, but also varies within each host at stages roughly corresponding to colonization, persistence and transmission.
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