Project description:As vector-borne pathogens transit between the arthropod and vertebrate, adaptation is key for survival as each host varies and initiates unique defense mechanisms. An environmental signal that relapsing fever (RF) and Lyme causing spirochetes detect is the change of temperature between vector and mammal, yet incomplete genomes have hindered progress in understanding the genetic constituents expressed during tick colonization. We conducted a combined transcriptional and genomic sequence analysis to further assemble the ~150 kb linear plasmid (lp150) of Borrelia turicatae, a causative agent of RF borreliosis. Contiguous sequences (contigs), which were originally generated by Sanger sequencing, contained open reading frames (ORFs) identified to be up-regulated by the spirochetes when grown under tick-like conditions compared to the mammal. To aid in assembling the contigs, a PacBio RS I Single Molecule Real-Time DNA sequencing approach was used, given extended nucleotide reads over several thousand base pairs. A 36 kb locus was identified toward the 3‘ end of lp150, and expression of the ORFs was verified in the tick and mammal. We report the most complete version of lp150, and this study indicates that the plasmid likely facilitates vector colonization and establishing early mammalian infection
Project description:Spirochetes are long, thin, motile, helical or flat wave bacteria that are unique among the prokaryotes by having flagella or axial filaments confined to an internal periplasmic space. These flagella are complex organelles that can play major roles in bacterial pathogenicity and are used as propellers allowing bacteria to move through liquids, viscous environments or along surfaces. While most bacteria species use transcriptional regulatory cascades to regulate the synthesis and assembly of their flagella, spirochetes employ an unusual post-transcriptional mechanism. Using next generation sequencing, we characterized a natural mutant of the relapsing fever spirochete Borrelia hermsii lacking the flagellar export apparatus protein FliH. The mutant was non-motile, uncoiled and straight compared to the wild-type spirochetes. The B. hermsii fliH mutant produced only a residual amount of the major flagellin protein FlaB, which was correlated with a reduced number of periplasmic flagella. The amounts of flaB transcript were comparable in the fliH mutant and the wild-type strain. The synthesis of FlaB, the motility and the infectivity of the fliH mutant were rescued by trans-complementation. This report reveals a new function for FliH, and we propose that this regulator of the flagellar export apparatus influences the post-transcriptional processing of the flagella, motility and virulence of the relapsing fever spirochete Borrelia hermsii. Spirochetes are bacteria characterized in part by rotating periplasmic flagella that impart their flat-wave morphology and motility. While other bacteria rely on a transcriptional cascade to regulate the expression of motility genes, spirochetes employ posttranscriptional mechanism(s) that are only partially known. In the present study, we characterize a non-motile mutant of the relapsing fever spirochete Borrelia hermsii that was straight, non-motile and deficient in flagella. We used next generation DNA sequencing of the mutant’s genome, which when compared to the wild-type genome identified a 142 bp deletion in the chromosomal gene encoding the flagellar export apparatus protein FliH. Immunoblot and transcriptome analyses showed that the mutant phenotype was linked to the posttranscriptional deficiency in the synthesis of the major flagellar filament core protein FlaB. The turnover of the residual pool of FlaB produced by the fliH mutant was comparable to the wild-type spirochete while the amount of FlaA was similar to the wild-type level. The non-motile mutant was not infectious in mice and its inoculation did not induce an antibody response. Trans-complementation of the mutant with an intact fliH gene restored the synthesis of FlaB, a normal morphology, motility and infectivity in mice. Therefore, we propose that the flagellar export apparatus protein regulates motility of B. hermsii at the posttranscriptional level by influencing the synthesis of FlaB. Borrelia hermsii wild type vs. motility mutant
Project description:Lyme disease (LD) is one of the most important human vector-borne diseases in North America. Since transmission of Borrelia burgdorferi (Bb), the causative agent of LD, is influenced by yet unknown tick saliva proteins (TSP), the discovery and characterization of such TSPs are highly sought after for their potential as tick-antigen based vaccine targets. We validated a novel non-invasive approach of collecting sufficient amounts of saliva from unfed, partially fed (12, 24, 36, 48, 60, and 72h), and replete fed Ixodes scapularis nymphs for identification of tick saliva proteins using the LC-MS/MS approach. Our data has described TSPs that might be injected into the host within few hours of the tick starting to feed and are likely associated with manipulating host immunity to facilitate transmitted Bb to colonize the host. Thus, these data will serve as a source for Bb-infection associated TSPs that might be targeted in tick-antigen based vaccines to prevent LD. Equally important, the non-invasive method to collect nymph tick saliva is likely to provide transformative impact on research to understand transmission of other TBD agents.