Project description:Borrelia burgdorferi, the causative agent of Lyme disease, is transmitted to vertebrate hosts by Ixodes ticks. As it moves from tick to host, B. burgdorferi must adapt to survive in a vastly different environment. During the tick bloodmeal, which lasts several days, B. burgdorferi is primed for mammalian infection, growing increasingly virulent as it senses cues from its surroundings in the tick. This conditioning is dependent on key transcriptional regulators; however, the downstream transcriptional changes occurring inside of the tick that promote B. burgdorferi transmission and infection are poorly understood due to technical difficulties in sequencing the B. burgdorferi transcriptome from inside of ticks. We developed a protocol to enrich and sequence B. burgdorferi from inside the tick, and we measured global transcriptional changes occurring in feeding ticks. We identified 192 genes that change expression twofold over the course of the tick bloodmeal, which were predominantly located on the plasmids of the genome. The majority of the upregulated genes encode proteins found at the cell envelope or proteins of unknown function, including 45 upregulated genes encoding outer surface lipoproteins. These genes that increase during feeding are candidates for future functional studies, which can help identify new targets for methods that aim to control the spread of Lyme disease.
Project description:The bacterial stringent response is triggered by deficiencies of available nutrients andother environmental stresses. It is mediated by 5'-triphosphate-guanosine-3'- diphosphate and 5'-diphosphate-guanosine-3'-diphosphate (collectively (p)ppGpp) and generates global changes in gene expression and metabolism that enable bacteria to adapt to and survive these challenges. Borrelia burgdorferi encounters multiple stressors in its cycling between ticks and mammals that could trigger the stringent response. We have previously shown that the B. burgdorferi stringent response is mediated by a single enzyme, RelBbu, with both (p)ppGpp synthase and hydrolase activities, and that a B. burgdorferi 297 relBbu null deletion mutant was defective in adapting to stationary phase, incapable of down-regulating synthesis of rRNA and could not infect mice. We have now used this deletion mutant and microarray analysis to identify genes comprising the rel regulon in B. burgdorferi cultured at 34°C, and found that transcription of genes involved in glycerol metabolism is induced by relBbu. Culture of the wild-type parental strain, the relBbu deletion mutant and its complemented derivative at 34°C and 25°C in media containing glucose or glycerol as principal carbon sources revealed a growth defect in the mutant, most evident at the lower temperature. Transcriptional analysis of the glp operon for glycerol uptake and metabolism in these three strains confirmed that relBbu was necessary and sufficient to increase transcription of this operon in the presence of glycerol at both temperatures, and particularly at 25°C. These results confirm and extend previous findings regarding the stringent response in B. burgdorferi. They also demonstrate that the stringent response regulates glycerol metabolism in this organism and is likely crucial for its optimal growth in ticks.
Project description:Borrelia burgdorferi, the causative agent of Lyme disease, is transmitted to vertebrate hosts by Ixodes ticks. As it moves from tick to host, B. burgdorferi must adapt to survive in a vastly different environment. During the tick bloodmeal, which lasts several days, B. burgdorferi is primed for mammalian infection, growing increasingly virulent as it senses cues from its surroundings in the tick. This conditioning is dependent on key transcriptional regulators; however, the downstream transcriptional changes occurring inside of the tick that promote B. burgdorferi transmission and infection are poorly understood due to technical difficulties in sequencing the B. burgdorferi transcriptome from inside of ticks. We developed a protocol to enrich and sequence B. burgdorferi from inside the tick, and we measured global transcriptional changes occurring in feeding ticks. We identified 192 genes that change expression twofold over the course of the tick bloodmeal, which were predominantly located on the plasmids of the genome. The majority of the upregulated genes encode proteins found at the cell envelope or proteins of unknown function, including 45 upregulated genes encoding outer surface lipoproteins. These genes that increase during feeding are candidates for future functional studies, which can help identify new targets for methods that aim to control the spread of Lyme disease.