SummaryBorrelia burgdorferi , the aetiological agent of Lyme disease, utilizes multiple adhesins to interact with both the arthropod vector and mammalian hosts it colonizes. One such adhesive molecule is a surfaceexposed fibronectin-binding lipoprotein, designated BBK32. Previous characterization of BBK32-mediated fibronectin binding has been limited to biochemical analyses due to the difficulty in mutagenizing infectious isolates of B. burgdorferi . Here we report an alternative method to inactivate bbk32 via allelic exchange through use of a low-passage variant of B. burgdorferi strain B31 that is more readily transformed. The resulting mutant does not synthesize BBK32, exhibits reduced fibronectin binding in solid phase assays and manifests decreased interactions with mouse fibroblast cells relative to both the infectious parent and genetic complement. Furthermore, the bbk32 knockout was significantly attenuated in the murine model of Lyme disease, whereas a genetically complemented control was not, indicating that BBK32 is necessary for maximal B. burgdorferi infection in the mouse. To our knowledge this is the first mutational analysis of a surface exposed, functional borrelial lipoprotein adhesin whose activity is associated with the mammalian host environment. By analogy with other pathogens that utilize fibronectin binding as an important virulence determinant, the borrelial fibronectin-BBK32 interaction is likely to be important in B. burgdorferi-specific pathogenic mechanisms, particularly in the context of dissemination, secondary colonization and/or persistence.

The 25-kb linear plasmid lp25 and one of the 28-kb linear plasmids (lp28-1) are required for experimental infection in Borrelia burgdorferi, the etiologic agent of Lyme disease. The loss of these plasmids either eliminates infectivity (lp25) or significantly increases the 50% infective dose during a 2-week infection period (lp28-1). This study assessed the kinetics of bacterial dissemination in C3H/HeN mice infected with B. burgdorferi lacking either lp25 or lp28-1, as well as their wild-type parent, and tracked the development of specific borrelial antibodies over a 3-week period. The results indicated that the wild type and the lp28-1 ؊ strains were able to disseminate throughout the host, whereas the lp25 ؊ strain was cleared within 48 h of inoculation. While the wild-type B. burgdorferi persisted in tissues for the duration of the study, the lp28-1 ؊ mutant began clearing at day 8, with no detectable bacteria present by day 18. As expected, the wild-type strain persisted in C3H/HeN mice despite a strong humoral response; however, the lp28-1 ؊ mutant was cleared coincidently with the development of a modest immunoglobulin M response. The lp28-1 ؊ mutant was able to disseminate and persist in C3H-scid mice at a level indistinguishable from that of wild-type cells, confirming that acquired immunity was required for clearance in C3H/HeN mice. Thus, within an immunocompetent host, lp28-1-encoded proteins are not required for dissemination but are essential for persistence associated with Lyme borreliosis.Lyme disease or Lyme borreliosis is a multisystemic disorder transmitted by ticks of the genus Ixodes infected with the spirochetal bacterium Borrelia burgdorferi sensu lato, composed of B. burgdorferi sensu stricto, B. garinii, and B. afzelii (1,8,16). Lyme disease is the leading zoonotic infection in the United States, accounting for 95% of all reported vector-borne illness (10). The completed B. burgdorferi sensu stricto strain B31 annotated sequence revealed an unusual genome containing a linear chromosome of 910 kb in size as well as several extrachromosomal elements, including 12 linear plasmids of sizes ranging from 5 to 56 kb and 9 circular plasmids with sizes between 9 and 32 kb (9,14).Early studies had demonstrated a correlation between plasmid content and infectivity but were hindered by the plethora of plasmid species in B. burgdorferi and the inherent difficulty in resolving the many linear and circular species (24, 29-31). As such, prior to the information gained from the B. burgdorferi strain B31 genome sequence, it was difficult to definitively link a specific plasmid to a defect in infectivity. Exploiting the completed genome sequence of B. burgdorferi, we designed oligonucleotide primers that could be used in conjunction with PCR to specifically catalog the presence of plasmids from clonal isolates of B. burgdorferi (17). In our quest to link infectivity to any of the plasmids, we determined, along with others, that two linear plasmids, 25 and 28 kb in size (designated lp25 and lp28-1, respectiv...

The immunopathogenesis of

SummaryThe RpoS transcription factor of Borrelia burgdorferi is a 'gatekeeper' because it activates genes required for spirochaetes to transition from tick to vertebrate hosts. However, it remains unknown how RpoS becomes repressed to allow the spirochaetes to transition back from the vertebrate host to the tick vector. Here we show that a putative carbohydrate-responsive regulatory protein, designated BadR (Borrelia host adaptation Regulator), is a transcriptional repressor of rpoS. BadR levels are elevated in B. burgdorferi cultures grown under in vitro conditions mimicking unfed-ticks and badR-deficient strains are defective for growth under these same conditions. Microarray and immunoblot analyses of badR-deficient strains showed upregulation of rpoS and other factors important for virulence in vertebrate hosts, as well as downregulation of putative tick-specific determinants (e.g. linear plasmid 28-4 genes). DNA-binding assays revealed BadR binds to upstream regions of rpoS. Site-directed mutations in BadR and the presence of phosphorylated sugars affected BadR's binding to the rpoS promoters. badR-deficient B. burgdorferi were unable to colonize mice. Several putative tick-specific targets have been identified. Our study identified a novel regulator, BadR, and provides a link between nutritional environmental cues utilized by spirochaetes to adaptation to disparate conditions found in the tick and vertebrate hosts.

Campylobacter jejuni are a common cause of human diarrheal illness. Previous work has demonstrated that C. jejuni synthesize a novel set of proteins upon coculturing with epithelial cells, some of which are secreted. The secreted proteins have been collectively referred to as Campylobacter invasion antigens (Cia proteins). Metabolic labeling experiments revealed that Cia protein synthesis and secretion are separable and that secretion is the rate-limiting step of these processes. Additional work indicated that Cia protein synthesis is induced in response to bile salts and various eukaryotic host cell components. Host cell components also can induce Cia protein secretion. Culturing C. jejuni on plates supplemented with the bile salt deoxycholate retarded the inhibitory effect of chloramphenicol on C. jejuni invasion, as judged by the gentamicin-protection assay. These data suggest that the coordinate expression of the genes encoding the Cia proteins is subject to environmental regulation.

SummaryBorrelia burgdorferi, the causative agent of Lyme disease, has a limited set of genes to combat oxidative/nitrosative stress encountered in its tick vector or mammalian hosts. We inactivated the gene encoding for superoxide dismutase A (sodA, bb0153), an enzyme mediating the dismutation of superoxide anions and examined the in vitro and in vivo phenotype of the mutant. There were no significant differences in the in vitro growth characteristics of the sodA mutant compared with the control strains. Microscopic analysis of viability of spirochaetes revealed greater percentage of cell death upon treatment of sodA mutant with superoxide generators compared with its controls. Infectivity analysis in C3H/HeN mice following intradermal needle inoculation of 10 3 or 10 5 spirochaetes per mouse revealed complete attenuation of infectivity for the sodA mutant compared with control strains at 21 days post infection. The sodA mutant was more susceptible to the effects of activated macrophages and neutrophils, suggesting that its in vivo phenotype is partly due to the killing effects of activated immune cells. These studies indicate that SodA plays an important role in combating oxidative stress and is essential for the colonization and dissemination of B. burgdorferi in the murine model of Lyme disease.

The Lyme disease spirochete, Borrelia burgdorferi, encounters many environmental signals as it cycles between the arthropod vector and mammalian hosts, including temperature, pH, and other host factors. To test the possibility that dissolved oxygen modulates gene expression in B. burgdorferi, spirochetes were exposed to differential levels of dissolved oxygen, and distinct alterations were observed at both the transcriptional and translational levels. Specifically NapA, a Dps/Dpr homologue involved in the oxidative stress response in other bacteria, was reduced when B. burgdorferi was grown under oxygen-limiting conditions. In contrast, several immunoreactive proteins were altered when tested with infection-derived sera from different hosts. Specifically, OspC, DbpA, and VlsE were synthesized at greater levels when cells were grown in limiting oxygen, whereas VraA was reduced. The levels of oxygen in the medium did not affect OspA production. Real-time reverse transcription-PCR analysis of RNA isolated from infectious isolates of strains B31 and cN40 indicated that the expression of ospC, dbpA, and vlsE increased while napA expression decreased under dissolved-oxygen-limiting conditions, whereas flaB was not affected. The reverse transcription-PCR results corroborated the immunoblot analyses and indicated that the increase in OspC, DbpA, and VlsE was due to regulation at the transcriptional level of the genes encoding these antigens. These results indicate that dissolved oxygen modulates gene expression in B. burgdorferi and imply that the redox environment may be an additional regulatory cue that spirochetes exploit to adapt to the disparate niches that they occupy in nature.

Borrelia burgdorferi, the agent of Lyme disease, differentially expresses numerous genes and proteins as it cycles between mammalian hosts and tick vectors. Insights on regulatory mechanisms have been provided by earlier studies that examined B. burgdorferi gene expression patterns during cultivation. However, prior studies examined bacteria at only a single time point of cultivation, providing only a snapshot of what is likely a dynamic transcriptional program driving B. burgdorferi adaptations to changes during culture growth phases. To address that concern, we performed RNA sequencing (RNA-Seq) analysis of B. burgdorferi cultures at early-exponential, mid-exponential, and early-stationary phases of growth. We found that expression of nearly 18% of annotated B. burgdorferi genes changed significantly during culture maturation. Moreover, genome-wide mapping of the B. burgdorferi transcriptome in different growth phases enabled insight on transcript boundaries, operon structures, and identified numerous putative non-coding RNAs. These RNA-Seq data are discussed and presented as a resource for the community of researchers seeking to better understand B. burgdorferi biology and pathogenesis.