Borrelia burgdorferi, the causative agent of Lyme disease, is maintained in nature through an infectious cycle that alternates between various species of small mammals and a tick vector. Like many bacterial pathogens, B. burgdorferi must adapt to a changing array of environmental conditions in order to successfully persist, proliferate and be transmitted between hosts. B. burgdorferi has an unusual segmented genome that includes a large number of linear and circular plasmids. Increasing evidence indicates that plasmid-encoded genes are critical for successful adaptation by B. burgdorferi to the different environments that the spirochete encounters during its infectious cycle. A major focus of this project is to determine how and why the Lyme disease spirochete maintains such a unique genomic structure, and the specific contributions of individual plasmids and genes at each stage of the infectious cycle. In FY2013, Dr. Dan Dulebohn, a post-doctoral fellow, investigated the role of a 28 kilobase (kb) linear plasmid, designated lp28-3, in the B. burgdorferi infectious cycle (1). Analysis of the nucleotide sequence of lp28-3 indicates that this plasmid is highly conserved among B. burgdorferi strains and that the predicted functions of some lp28-3 encoded gene products are consistent with a role in the infectious cycle. Dan characterized lp28-3 deficient strains obtained by two independent methods and analyzed these B. burgdorferi variants during in vitro growth and the entire mouse-tick infectious cycle. Dan found that lp28-3 does not carry any genes that are strictly required for B. burgdorferi infection of a mouse or tick, and that spirochetes lacking lp28-3 can cause a disseminated and persistent infection. However, these lp28-3-minus spirochetes were at a selective dis-advantage relative to wild type spirochetes when both were co-injected into a mouse, and this attenuation was reflected in the relative proportion of lp28-3 deficient to wild type spirochetes acquired by feeding ticks. These data demonstrate that lp28-3-encoded genes, although not essential, contribute to the fitness of B. burgdorferi during infection of the mammalian host (1). This study demonstrates the utility of using a co-infection model to analyze B. burgdorferi mutants for which there is no obvious phenotype when assayed individually (1). This strategy facilitates determining the strict requirement for a genetic element during infection, while also providing a quantitative assessment of the contribution to the fitness of Borrelia in vivo. This approach could be especially useful for studying other genetic elements of B. burgdorferi that would appear to be important, but have been shown to be dispensable in an experimental infectious cycle. Our data suggest that although many B. burgdorferi genes are not strictly required for completion of an experimental mouse-tick-mouse infectious cycle, they can make substantial contributions in the competitive environment of a mixed infection, which is frequently encountered in a natural infectious cycle. As described above, Borrelia burgdorferi alternates between ticks and mammals, requiring variable gene expression and protein production to adapt to these diverse niches. These adaptations include switching between the major outer surface lipoproteins OspA, OspC, and VlsE at different stages of the infectious cycle. Although this pattern of lipoprotein succession has been described, the functions of these outer surface proteins remain undefined. Previous work by other investigators suggested that several B. burgdorferi lipoproteins, including OspA and VlsE, could substitute for OspC at the initial stage of mouse infection, when OspC is transiently but absolutely required. In FY2013, Dr. Kit Tilly and Aaron Bestor assessed whether complementation with the vlsE or ospA gene could restore infectivity to an ospC mutant, and found that neither gene product effectively compensated for the absence of OspC during early infection (2). In contrast, Kit and Aaron determined that OspC production was required by B. burgdorferi for persistent infection of an immunodeficient mouse when the vlsE gene was absent. Together, these results indicate that OspC can substitute for VlsE on B. burgdorferi when antigenic variation is unnecessary, but that these two abundant outer surface lipoproteins are optimized for their related but specific roles during early and persistent mammalian infection by B. burgdorferi. A simple model to explain these and other results is that these major B. burgdorferi outer surface lipoproteins serve a common basic function at different stages of the bacterial mouse-tick life cycle. In this model, similar roles are played by OspC during the initial stage of mammalian infection, by VlsE during persistent infection, and by OspA during the tick stage of the life cycle. The seemingly non-specific nature of that function may be protection against some aspect of mammalian and tick immune defenses, or structural stabilization of the outer membrane. The FY2013 study described above was designed to further test the model that OspC and VlsE play similar roles at different stages of mammalian infection (2). Because it appears that OspC and VlsE are not fully interchangeable, we propose that these two proteins require both appropriate context and appropriate timing to be fully functional. By context, we encompass the roles of other proteins produced at the same time and also variations in membrane composition and arrangement, which are influenced by lipid availability and temperature. In fiscal year 2013 we reported the culminations of two long-standing collaborations with the laboratories of Dr. Md Motaleb at East Carolina University and Dr. Mollie Jewett at the University of Central Florida (3, 4). Dr. Syed Sultan, a post-doctoral fellow in Dr. Motalebs lab, completed a genetic study investigating the requirement for motility in the mouse-tick infectious cycle of B. burgdorferi (3). This study demonstrated that motility driven by the spirochete's periplasmic flagella is required for optimal survival in and transmission between the tick vector and the mammalian host. Tish Choudry Ellis, a graduate student in Dr. Jewetts lab, reported the development and application of an In Vivo Expression Technology system (IVET) for identification of B. burgdorferi genes that are expressed during infection of the mammalian host (4). Using this approach, they have identified a number of genes that are expressed during active infection, including a gene that appears to contribute to the ability of the spirochete to persist in an immune-competent host. Our contributions to both of these studies were discrete but significant at their inceptions.

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Rego, Ryan O M; Bestor, Aaron; Stefka, Jan et al. (2014) Population bottlenecks during the infectious cycle of the Lyme disease spirochete Borrelia burgdorferi. PLoS One 9:e101009
Dulebohn, Daniel P; Hayes, Beth M; Rosa, Patricia A (2014) Global repression of host-associated genes of the Lyme disease spirochete through post-transcriptional modulation of the alternative sigma factor RpoS. PLoS One 9:e93141
Hayes, Beth M; Dulebohn, Daniel P; Sarkar, Amit et al. (2014) Regulatory protein BBD18 of the lyme disease spirochete: essential role during tick acquisition? MBio 5:e01017-14
Tilly, Kit; Bestor, Aaron; Rosa, Patricia A (2013) Lipoprotein succession in Borrelia burgdorferi: similar but distinct roles for OspC and VlsE at different stages of mammalian infection. Mol Microbiol 89:216-27
Dulebohn, Daniel P; Bestor, Aaron; Rosa, Patricia A (2013) Borrelia burgdorferi linear plasmid 28-3 confers a selective advantage in an experimental mouse-tick infection model. Infect Immun 81:2986-96
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Lawrence, Kevin A; Jewett, Mollie W; Rosa, Patricia A et al. (2009) Borrelia burgdorferi bb0426 encodes a 2'-deoxyribosyltransferase that plays a central role in purine salvage. Mol Microbiol 72:1517-29
Sarkar, Amit; Tilly, Kit; Stewart, Philip et al. (2009) Borrelia burgdorferi resistance to a major skin antimicrobial peptide is independent of outer surface lipoprotein content. Antimicrob Agents Chemother 53:4490-4
Stewart, Philip E; Rosa, Patricia A (2008) Transposon mutagenesis of the lyme disease agent Borrelia burgdorferi. Methods Mol Biol 431:85-95