<p> Small non-coding RNAs (sRNAs) are being increasingly recognized as critical regulators of a wide variety of processes in bacteria. To investigate the contribution of unknown sRNAs to virulence gene regulation in <i>Vibrio cholerae,</i> we undertook a screen to identify previously uncharacterized sRNAs under the control of the major virulence gene activator in <i> V. cholerae,</i> ToxT. Using a combination of direct sRNA cloning and sequencing together with a genome-wide ToxT <i>in vitro</i> binding assay, we identified 18 putative ToxT-regulated sRNAs. Two of these ToxT regulated sRNAs were located within the <i>Vibrio</i> Pathogenicity Island-1 (VPI-1), the genetic element that encodes ToxT and the Toxin Co-regulated Pilus (TCP). We verified regulation of these sRNAs by ToxT and showed that deletion of one of them, now designated <i>tarB</i>, caused a variable colonization phenotype when competed against the parental strain in an infant mouse model of <i>V. cholerae</i> infection. Infections progressing for 18 hours or less showed the Δ<i>tarB</i> strain was out-competed by the wild type strain, while those carried out longer, showed Δ<i> tarB</i> out-competing the wild type. Additionally, if inoculated from a resource poor environment the Δ<i>tarB</i> strain also showed decreased colonization relative to wild type. Using a bioinformatic approach, we identified that <i>tarB</i>-mediated regulation of the gene <i> tcpF</i> was primarily responsible for the <i>tarB</i> mutant's <i> in vivo</i> colonization phenotype. Further investigation of genes regulated by <i>tarB</i> using genome-wide transcriptional profiling of a <i> tarB</i> over-expressing strain revealed that <i>tarB</i> also directly regulates genes involved in iron and amino acid uptake. We determined that <i>tarB</i> has a repressive effect on many genes within the VPI-1, but has an activating effect on <i>tcpP/tcpH,</i> encoding regulators upstream of ToxT. Taken together, the data suggest that <i> tarB</i> plays an important role in regulating virulence and metabolic genes early after <i>V. cholerae</i> infection, but that this repressive effect on virulence genes later in infection may lead to reduced replication <i> in vivo.</i></p>
| Identifer | oai:union.ndltd.org:PROQUEST/oai:pqdtoai.proquest.com:3624938 |
| Date | 20 August 2014 |
| Creators | Bradley, Evan |
| Publisher | Sackler School of Graduate Biomedical Sciences (Tufts University) |
| Source Sets | ProQuest.com |
| Language | English |
| Detected Language | English |
| Type | thesis |
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