The causative agent of Lyme disease, Borrelia burgdorferi, harbours a single linear chromosome and upwards of 23 linear and circular plasmids. Only a minority of these plasmids, including linear plasmid 17, are maintained with near‐absolute fidelity during extended in vitro passage, and characterisation of any putative virulence determinants they encode has only recently begun. In this work, a mutant lacking a ~4.7 kb fragment of lp17 was studied. Colonisation of murine tissues by this lp17 mutant was significantly impaired, as was the ability to induce carditis and arthritis. The deficiency in tissue colonisation was alleviated in severe combined immunodeficient (SCID) mice, implicating a role for this plasmid region in adaptive immune evasion. Through genetic complementation, the mutant phenotype could be fully attributed to a 317 bp intergenic region that corresponds to the discontinued bbd07 ORF and upstream sequence. The intergenic region was found to be transcriptionally active, and mutant spirochetes lacking this region exhibited an overall difference in the antigenic profile during infection of an immunocompetent murine host. Overall, this study is the first to provide evidence for the involvement of lp17 in colonisation of joint and heart tissues, along with the associated pathologies caused by the Lyme disease spirochete.

Efficient acquisition and transmission of Borrelia burgdorferi by the tick vector, and the ability to persistently infect both vector and host, are important elements for the life cycle of the Lyme disease pathogen. Previous work has provided strong evidence implicating the significance of the vls locus for B. burgdorferi persistence. However, studies involving vls mutant clones have thus far only utilized in vitro-grown or host-adapted spirochetes and laboratory strains of mice. Additionally, the effects of vls mutation on tick acquisition and transmission has not yet been tested. Thus, the importance of VlsE antigenic variation for persistent infection of the natural reservoir host, and for the B. burgdorferi enzootic life cycle in general, has not been examined to date. In the current work, Ixodes scapularis and Peromyscus maniculatus were infected with different vls mutant clones to study the importance of the vls locus for the enzootic cycle of the Lyme disease pathogen. The findings highlight the significance of the vls system for long-term infection of the natural reservoir host, and show that VlsE antigenic variability is advantageous for efficient tick acquisition of B. burgdorferi from the mammalian reservoir. The data also indicate that the adaptation state of infecting spirochetes influences B. burgdorferi avoidance from host antibodies, which may be in part due to its respective VlsE expression levels. Overall, the current findings provide the most direct evidence on the importance of VlsE for the enzootic cycle of Lyme disease spirochetes, and underscore the significance of VlsE antigenic variation for maintaining B. burgdorferi in nature.

ABSTRACTThe causative agent of Lyme disease,Borrelia burgdorferi, possesses a segmented genome comprised of a single linear chromosome and upwards of 23 linear and circular plasmids. Much of what is known about plasmid-borne genes comes from studying laboratory clones that have spontaneously lost one or more plasmids duringin vitropassage. Some plasmids, including the linear plasmid lp17, are never or rarely reported to be lost during routine culture; therefore, little is known about the requirement of these conserved plasmids for infectivity. In this study, the effects of deleting regions of lp17 were examined bothin vitroandin vivo. A mutant strain lacking the genesbbd16tobbd25showed no deficiency in the ability to establish infection or disseminate to the bloodstream of mice; however, colonization of peripheral tissues was delayed. Despite the ability to colonize ear, heart, and joint tissues, this mutant exhibited a defect in bladder tissue colonization for up to 56 days postinfection. This phenotype was not observed in immunodeficient mice, suggesting that bladder colonization by the mutant strain was inhibited by an adaptive immune-based mechanism. Moreover, the mutant displayed increased expression of outer surface protein Cin vitro, which was correlated with the absence of the genebbd18. To our knowledge, this is the first report involving genetic manipulation of lp17 in an infectious clone ofB. burgdorferiand reveals for the first time the effects of lp17 gene deletion during murine infection by the Lyme disease spirochete.

Prokaryote restriction modification (RM) systems serve to protect bacteria from potentially detrimental foreign DNA. Recent evidence suggests that DNA methylation by the methyltransferase (MTase) components of RM systems can also have effects on transcriptome profiles. The type strain of the causative agent of Lyme disease,Borrelia burgdorferiB31, possesses two RM systems withN6-methyladenosine (m6A) MTase activity, which are encoded by thebbe02gene located on linear plasmid lp25 andbbq67on lp56. The specific recognition and/or methylation sequences had not been identified for either of theseB. burgdorferiMTases, and it was not previously known whether these RM systems influence transcript levels. In the current study, single-molecule real-time sequencing was utilized to map genome-wide m6A sites and to identify consensus modified motifs in wild-typeB. burgdorferias well as MTase mutants lacking either thebbe02gene alone or bothbbe02andbbq67genes. Four novel conserved m6A motifs were identified and were fully attributable to the presence of specific MTases. Whole-genome transcriptome changes were observed in conjunction with the loss of MTase enzymes, indicating that DNA methylation by the RM systems has effects on gene expression. Genes with altered transcription in MTase mutants include those involved in vertebrate host colonization (e.g.,rpoSregulon) and acquisition by/transmission from the tick vector (e.g.,rrp1andpdeB). The results of this study provide a comprehensive view of the DNA methylation pattern inB. burgdorferi, and the accompanying gene expression profiles add to the emerging body of research on RM systems and gene regulation in bacteria.IMPORTANCELyme disease is the most prevalent vector-borne disease in North America and is classified by the Centers for Disease Control and Prevention (CDC) as an emerging infectious disease with an expanding geographical area of occurrence. Previous studies have shown that the causative bacterium,Borrelia burgdorferi, methylates its genome using restriction modification systems that enable the distinction from foreign DNA. Although much research has focused on the regulation of gene expression inB. burgdorferi, the effect of DNA methylation on gene regulation has not been evaluated. The current study characterizes the patterns of DNA methylation by restriction modification systems inB. burgdorferiand evaluates the resulting effects on gene regulation in this important pathogen.

Vibrio parahaemolyticus is a leading cause of seafood-borne gastroenteritis; however, its virulence mechanisms are not well understood. The identification of type III secreted proteins has provided candidate virulence factors whose functions are still being elucidated. Genotypic strain variability contributes a level of complexity to understanding the role of different virulence factors. The ability of V. parahaemolyticus to inhibit Rho family GTPases and cause cytoskeletal disruption was examined with HeLa cells. After HeLa cells were infected, intracellular Rho activation was inhibited in response to external stimuli. In vitro activation of Rho, Rac, and Cdc42 isolated from infected HeLa cell lysates was also inhibited, indicating that the bacteria were specifically targeting GTPase activation. The inhibition of Rho family GTPase activation was retained for clinical and environmental isolates of V. parahaemolyticus and was dependent on a functional chromosome I type III secretion system (CI-T3SS). GTPase inhibition was independent of hemolytic toxin genotype and the chromasome II (CII)-T3SS. Rho inhibition was accompanied by a shift in the total actin pool to its monomeric form. These phenotypes were abrogated in a mutant strain lacking the CI-T3S effector Vp1686, suggesting that the inhibiting actin polymerization may be a downstream effect of Vp1686-dependent GTPase inhibition. Although Vp1686 has been previously characterized as a potential virulence factor in macrophages, our findings reveal an effect on cultured HeLa cells. The ability to inhibit Rho family GTPases independently of the CII-T3SS and the hemolytic toxins may provide insight into the mechanisms of virulence used by strains lacking these virulence factors.