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ABSTRACTBorrelia burgdorferi, the bacterium that causes Lyme disease, has a unique segmented genome consisting of numerous linear and circular plasmids and a linear chromosome. Many of these genetic elements have been found to encode factors critical for B. burgdorferito complete the infectious cycle. However, several plasmids remain poorly characterized, and their roles during infection with B. burgdorferihave not been elucidated. To more fully characterize the role of one of the four 28-kb linear plasmids, lp28-3, we generated strains specifically lacking lp28-3 and assayed the contribution of genes carried by lp28-3 to B. burgdorferiinfection. We found that lp28-3 does not carry any genes that are strictly required for infection of a mouse or tick and that lp28-3-deficient spirochetes are competent at causing a disseminated infection. Interestingly, spirochetes containing lp28-3 were at a selective advantage compared to lp28-3-deficient spirochetes when coinjected into a mouse, and this advantage was reflected in the population of spirochetes acquired by feeding ticks. Our data demonstrate that genes carried by lp28-3, although not essential, contribute to the fitness of B. burgdorferiduring infection.

ABSTRACTLinear plasmid lp54 is one of the most highly conserved and differentially expressed elements of the segmented genome of the Lyme disease spirochete Borrelia burgdorferi. We previously reported that deletion of a 4.1-kb region of lp54 (bba01to bba07[bba01-bba07]) led to a slight attenuation of tick-transmitted infection in mice following challenge with a large number of infected ticks. In the current study, we reduced the number of ticks in the challenge to more closely mimic the natural dose and found a profound defect in tick-transmitted infection of the bba01-bba07mutant relative to wild-type B. burgdorferi. We next focused on deletion of bba03as the most likely cause of this mutant phenotype, as previous studies have shown that expression of bba03is increased by culture conditions that simulate tick feeding. Consistent with this hypothesis, we demonstrated increased expression of bba03by spirochetes in fed relative to unfed ticks. We also observed that a bba03deletion mutant, although fully competent by itself, did not efficiently infect mice when transmitted by ticks that were simultaneously coinfected with wild-type B. burgdorferi. These results suggest that BBA03 provides a competitive advantage to spirochetes carrying this protein during tick transmission to a mammalian host in the natural infectious cycle.

ABSTRACTBorrelia burgdorferi, the causative agent of Lyme disease, exists in a complex enzootic cycle, transiting between its vector, Ixodesticks, and a diverse range of vertebrate hosts. B. burgdorferilinear plasmid 38 (lp38) contains several genes that are differentially regulated in response to conditions mimicking the tick or mouse environments, suggesting that these plasmid-borne genes may encode proteins important for the B. burgdorferiinfectious cycle. Some of these genes encode potential virulence factors, including hypothetical lipoproteins as well as a putative membrane transport system. To characterize the role of lp38 in the B. burgdorferiinfectious cycle, we constructed a shuttle vector to selectively displace lp38 from the B. burgdorferigenome and analyzed the resulting clones to confirm the loss of lp38. We found that, in vitro, clones lacking lp38 were similar to isogenic wild-type bacteria, both in growth rate and in antigenic protein production. We analyzed these strains in an experimental mouse-tick infectious cycle, and our results demonstrate that clones lacking lp38 are fully infectious in a mouse, can efficiently colonize the tick vector, and are readily transmitted to a naive host.

ABSTRACTBorrelia burgdorferi, the causative agent of Lyme disease, has a complex genome consisting of a linear chromosome and up to 21 linear and circular plasmids. These plasmids encode numerous proteins critical to the spirochete's infectious cycle and many hypothetical proteins whose functions and requirements are unknown. The conserved linear plasmid lp54 encodes several proteins important for survival in the mouse-tick infectious cycle, but the majority of the proteins are of unknown function and lack homologs outside the borreliae. In this study we adapted the Cre-loxrecombination system to create large deletions in the B. burgdorferigenome. Using Cre-lox, we systematically investigated the contribution of 14 adjacent genes on the left arm of lp54 to the overall infectivity of B. burgdorferi. The deletion of the region of lp54 encompassing bba07to bba14had no significant effect on the infectious cycle of B. burgdorferi. The deletion of bba01to bba07resulted in a slight growth defect but did not significantly affect the ability of B. burgdorferito complete the infectious cycle. This study demonstrated the utility of the Cre-loxsystem to efficiently explore gene requirements in B. burgdorferiand surprisingly revealed that a large number of the highly conserved proteins encoded on lp54 are not required to complete the infectious cycle.

ABSTRACTWe hypothesize a potential role for Borrelia burgdorferiOspC in innate immune evasion at the initial stage of mammalian infection. We demonstrate that B. burgdorferiis resistant to high levels (>200 μg/ml) of cathelicidin and that this antimicrobial peptide exhibits limited binding to the spirochetal outer membrane, irrespective of OspC or other abundant surface lipoproteins. We conclude that the essential role of OspC is unrelated to resistance to this component of innate immunity.

ABSTRACTBorrelia burgdorferisynthesizes a variety of differentially regulated outer surface lipoproteins in the tick vector and in vertebrate hosts. Among these is OspD, a protein that is highly induced in vitro by conditions that mimic the tick environment. Using genetically engineered strains in which ospDis deleted, we demonstrate that this protein is not required for B. burgdorferisurvival and infectivity in either the mouse or the tick. However, examination of both transcript levels and protein expression indicates that OspD expression is limited to a discrete window of time during B. burgdorferireplication within the tick. This time frame corresponds to tick detachment from the host following feeding, and expression of OspD continues during tick digestion of the blood meal but is low or undetectable after the tick has molted. The high level of OspD production correlates to the highest cell densities that B. burgdorferiis known to reach in vivo. Although OspD is nonessential to the infectious cycle of B. burgdorferi, the tight regulation of expression suggests a beneficial contribution of OspD to the spirochete during bacterial replication within the tick midgut.

ABSTRACTThis study demonstrates a strict temporal requirement for a virulence determinant of the Lyme disease spirochete Borrelia burgdorferiduring a unique point in its natural infection cycle, which alternates between ticks and small mammals. OspC is a major surface protein produced by B. burgdorferiwhen infected ticks feed but whose synthesis decreases after transmission to a mammalian host. We have previously shown that spirochetes lacking OspC are competent to replicate in and migrate to the salivary glands of the tick vector but do not infect mice. Here we assessed the timing of the requirement for OspC by using an ospCmutant complemented with an unstable copy of the ospCgene and show that B. burgdorferi's requirement for OspC is specific to the mammal and limited to a critical early stage of mammalian infection. By using this unique system, we found that most bacterial reisolates from mice persistently infected with the initially complemented ospCmutant strain no longer carried the wild-type copy of ospC. Such spirochetes were acquired by feeding ticks and migrated to the tick salivary glands during subsequent feeding. Despite normal behavior in ticks, these ospCmutant spirochetes did not infect naive mice. ospCmutant spirochetes from persistently infected mice also failed to infect naive mice by tissue transplantation. We conclude that OspC is indispensable for establishing infection by B. burgdorferiin mammals but is not required at any other point of the mouse-tick infection cycle.

ABSTRACTPlasmid vectors and fluorescent protein reporter systems are commonly used in the study of bacterial pathogenesis. Here we show that they can impair the ability of Salmonella entericaserovar Typhimurium to productively infect either cultured mammalian cells or mice. This has significant implications for studies that rely on these systems.

ABSTRACTIn many bacteria, the FtsH protease and its modulators, HflK and HflC, form a large protein complex that contributes to both membrane protein quality control and regulation of the cellular response to environmental stress. Both activities are crucial to the Lyme disease pathogen Borrelia burgdorferi, which depends on membrane functions, such as motility, protein transport, and cell signaling, to respond to rapid changes in its environment. Using an inducible system, we demonstrate that FtsH production is essential for both mouse and tick infectivity and for in vitrogrowth of B. burgdorferi. FtsH depletion in B. burgdorfericells resulted in membrane deformation and cell death. Overproduction of the protease did not have any detectable adverse effects on B. burgdorferigrowth in vitro, suggesting that excess FtsH does not proteolytically overwhelm its substrates. In contrast, we did not observe any phenotype for cells lacking the protease modulators HflK and HflC (ΔHflK/C), although we examined morphology, growth rate, growth under stress conditions, and the complete mouse-tick infectious cycle. Our results demonstrate that FtsH provides an essential function in the life cycle of the obligate pathogen B. burgdorferibut that HflK and HflC do not detectably affect FtsH function.IMPORTANCELyme disease is caused by Borrelia burgdorferi, which is maintained in nature in an infectious cycle alternating between small mammals and Ixodesticks. B. burgdorferiproduces specific membrane proteins to successfully infect and persist in these diverse organisms. We hypothesized that B. burgdorferihas a specific mechanism to ensure that membrane proteins are properly folded and biologically active when needed and removed if improperly folded or dysfunctional. Our experiments demonstrate that FtsH, a protease that fulfills this role in other microorganisms, is essential to B. burgdorferiviability. Cells depleted of FtsH do not survive in laboratory culture medium and cannot colonize mice or ticks, revealing an absolute requirement for this protease. However, the loss of two potential modulators of FtsH activity, HflK and HflC, does not detectably affect B. burgdorferiphysiology. Our results provide the groundwork for the identification of FtsH substrates that are critical for the bacterium’s viability.