Discovery and characterization of small non-coding RNAs in Vibrio cholerae that contribute to gene regulation during infection.
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 Vibrio cholerae, we undertook a screen to identify
previously uncharacterized sRNAs under the control of the major virulence gene activator
in V. cholerae, ToxT. Using a com... read morebination of direct sRNA cloning and sequencing together
with a genome-wide ToxT in vitro binding assay, we identified 18 putative ToxT-regulated
sRNAs. Two of these ToxT regulated sRNAs were located within the Vibrio 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 TarB, caused a variable colonization phenotype when competed
against the parental strain in an infant mouse model of V. cholerae infection.
Infections progressing for 18 hours or less showed the ΔtarB strain was
out-competed by the wild type strain, while those carried out longer, showed ΔtarB
out-competing the wild type. Additionally, if inoculated from a resource poor
environment the ΔtarB strain also showed decreased colonization relative to wild
type. Using a bioinformatic approach, we identified that TarB-mediated regulation of the
gene tcpF was primarily responsible for the TarB mutant's in vivo colonization
phenotype. Further investigation of genes regulated by TarB using genome-wide
transcriptional profiling of a TarB over-expressing strain revealed that TarB also
directly regulates genes involved in iron and amino acid uptake. We determined that TarB
has a repressive effect on many genes within the VPI-1, but has an activating effect on
tcpP/tcpH, encoding regulators upstream of ToxT. Taken together, the data suggest that
TarB plays an important role in regulating virulence and metabolic genes early after V.
cholerae infection, but that this repressive effect on virulence genes later in
infection may lead to reduced replication in
Thesis (Ph.D.)--Tufts University, 2012.
Submitted to the Dept. of Molecular Microbiology.
Advisor: Andrew Camilli.
Committee: Ralph Isberg, Michael Malamy, and Linden Hu.
Keywords: Microbiology, and Molecular biology.read less