Regulatory roles of sRNAs in pathogenesis of Vibrio cholerae

The Gram-negative pathogen Vibrio cholerae uses variety of regulatory molecules to modulate expression of virulence factors. One important regulatory element of microorganisms is small non-coding RNAs (sRNAs), which control various cell functions such as expression of cell membrane proteins, mRNA decay and riboswitches. In this thesis studies, we demonstrated the roles of the sRNAs VrrA in regulation of outer membrane protein expression, biofilm formation and expression of ribosome binding proteins. In addition, we showed that VrrB, a newly discovered sRNA, played a role in amino acid dependent starvation survival of V. cholerae and might functioned as a riboswitch. VrrA, a 140-nt sRNAs in V. cholerae, was controlled by the alternative sigma factor σE. The outer membrane protein, OmpT is known to be regulated by environmental signals such as pH and temperature via the ToxR regulon and carbon source signals via the cAMP–CRP complex. Our studies provide new insight into the regulation of OmpT by signals received via the σE regulon through VrrA. We demonstrated that VrrA down-regulate ompT translation by base-pairing with the 5′ region of the ompT mRNA in a Hfq (RNA chaperone protein) dependent manner. V. cholerae biofilms contain three matrix proteins—RbmA, RbmC and Bap1—and exopolysaccharide. While much is known about exopolysaccharide regulation, little is known about the mechanisms by which the matrix protein components of biofilms are regulated. In our studies, we demonstrated that VrrA negatively regulated rbmC translation by pairing to the 5' untranslated region of the rbmC transcript and that this regulation was not stringently dependent on Hfq. In V. cholerae, VC0706 (Vrp) and VC2530 proteins are homologous to ribosome-associated inhibitor A (RaiA) and hibernation promoting factor (HPF) of Escherichia coli, respectively. HPF facilitates stationary phase survival through ribosome hibernation. We showed that VrrA repressed Vrp protein expression by base-pairing to the 5´ region of vrp mRNA and that this regulation required Hfq. We also showed that Vrp was highly expressed during stationary phase growth and associated with the ribosomes of V. cholerae. We further demonstrated that Vrp and VC2530 were important for V. cholerae starvation survival under nutrient-deficient conditions. While VC2530 was down-regulated in bacterial cells lacking vrrA, mutation of vrp resulted in increased expression of VC2530. Riboswitches are an important class of regulators in bacteria, which are most often located in the 5' untranslated region (5´ UTR) of bacterial mRNA. In this study, we discovered the novel non-coding sRNA, VrrB located at the 5´ UTR of a downstream gene encoding Vibrio auxotropic factor A (VafA) for phenylalanine. In V. cholerae, reduced production of VafA was observed in the presence of phenylalanine and phenylpyruvate in the culture media. Some analogs of phenylalanine and phenylpyruvate could also modulate the expression of VafA. Furthermore, bacterial cells lacking the vrrB gene exhibited high production of VafA, suggesting that VrrB might function as a riboswitch that controls VafA expression

The VrrA sRNA controls stationary phase survival factor Vrp of Vibrio cholerae

Small non-coding RNAs (sRNAs) are emerging regulatory elements in bacteria. The Vibrio cholerae sRNA VrrA has previously been shown to down-regulate outer membrane proteins (OmpA and OmpT) and biofilmmatrix protein (RbmC) by base-pairing with the 50 region of the corresponding mRNAs. In this study, we present an additional target of VrrA in V. cholerae, the mRNA coding for the ribosome binding protein Vrp. Vrp is homologous to ribosome-associated inhibitor A (RaiA) of Escherichia coli which facilitates stationary phase survival through ribosome hibernation. We show that VrrA downregulates Vrp protein synthesis by base-pairing to the 50 region of vrp mRNA and that the regulation requires the RNA chaperone protein, Hfq. We further demonstrate that Vrp is highly expressed during stationary phase growth and associates with the ribosome of V. cholerae. The effect of the Vrp protein in starvation survival is synergistic with that of the VC2530 protein, a homolog of the E. coli hibernation promoting factor HPF, suggesting a combined role for these proteins in ribosome hibernation in V. cholerae. Vrp and VC2530 are important for V. cholerae starvation survival under nutrient deficient conditions. While VC2530 is down-regulated in cells lacking vrrA, mutation of vrp results in VC2530 activation. This is the first report indicating a regulatory role for an sRNA, modulating stationary factors involved in bacterial ribosome hibernation

The VrrA sRNA controls a stationary phase survival factor Vrp of <i>Vibrio cholerae</i>

<div><p>Small non-coding RNAs (sRNAs) are emerging regulatory elements in bacteria. The <i>Vibrio cholerae</i> sRNA VrrA has previously been shown to down-regulate outer membrane proteins (OmpA and OmpT) and biofilm matrix protein (RbmC) by base-pairing with the 5′ region of the corresponding mRNAs. In this study, we present an additional target of VrrA in <i>V. cholerae</i>, the mRNA coding for the ribosome binding protein Vrp. Vrp is homologous to ribosome-associated inhibitor A (RaiA) of <i>Escherichia coli</i> which facilitates stationary phase survival through ribosome hibernation. We show that VrrA down-regulates Vrp protein synthesis by base-pairing to the 5′ region of <i>vrp</i> mRNA and that the regulation requires the RNA chaperone protein, Hfq. We further demonstrate that Vrp is highly expressed during stationary phase growth and associates with the ribosome of <i>V. cholerae</i>. The effect of the Vrp protein in starvation survival is synergistic with that of the VC2530 protein, a homolog of the <i>E. coli</i> hibernation promoting factor HPF, suggesting a combined role for these proteins in ribosome hibernation in <i>V. cholerae</i>. Vrp and VC2530 are important for <i>V. cholerae</i> starvation survival under nutrient deficient conditions. While VC2530 is down-regulated in cells lacking <i>vrrA</i>, mutation of <i>vrp</i> results in VC2530 activation. This is the first report indicating a regulatory role for an sRNA, modulating stationary factors involved in bacterial ribosome hibernation.</p></div

Vibrio cholerae Utilizes Direct sRNA Regulation in Expression of a Biofilm Matrix Protein

Vibrio cholerae biofilms contain exopolysaccharide and three matrix proteins RbmA, RbmC and Bap1. While much is known about exopolysaccharide regulation, little is known about the mechanisms by which the matrix protein components of biofilms are regulated. VrrA is a conserved, 140-nt sRNA of V. cholerae, whose expression is controlled by sigma factor sigma(E). In this study, we demonstrate that VrrA negatively regulates rbmC translation by pairing to the 5' untranslated region of the rbmC transcript and that this regulation is not stringently dependent on the RNA chaperone protein Hfq. These results point to VrrA as a molecular link between the sigma(E)-regulon and biofilm formation in V. cholerae. In addition, VrrA represents the first example of direct regulation of sRNA on biofilm matrix component, by-passing global master regulators

Sub-lethal AMP treatment and OMVs formation of <i>V. cholerae</i>.

<p>(A) Growth curves of A1552 in normal LB (square), LB supplemented with 12.5 µg/ml of PmB (diamond) and LB supplemented with 12.5 µg/ml of LL-37 (triangles). (B) Electron micrographs of OMVs from A1552 control cultures without AMPs (panel a) or cultures with a sub-lethal concentration of LL-37 (pannel b) or PmB (pannel c). Bars: 50 nm.</p

Immunoblot detection of OmpT and Bap1 in OMV samples and in total cell extracts from whole cell bacteria.

<p>(A) Detection of Bap1 in OMVs from A1552 grown with PmB (lane 1), OMVs from A1552 grown without OMVs(lane 2), OMVs from Δ<i>ompT</i> mutant grown with PmB (lane 3) and OMVs from Δ<i>toxR</i> mutant grown with PmB (lane 4). (B) Detection of OmpT in OMVs from A1552 grown without PmB (lane 1), OMVs from A1552 grown with PmB (lane 2), whole cell extract of A1552 grown without PmB (lane 3) and whole cell extract of A1552 grown with PmB (lane 4). (C) Detection of OmpT and Bap1 in control-OMVs isolated from Δ<i>ompT</i>/pBR-<i>ompT</i> (lane 1), in PmB-OMVs isolated from Δ<i>ompT</i>/pBR-<i>ompT</i> (lane 2), incontrol-OMVs from Δ<i>ompT</i>/pBR-<i>ompT</i>* (LDV mutant) (lane 3) and PmB-OMVs from Δ<i>ompT</i>/pBR-<i>ompT</i>* (LDV mutant) (lane 4).</p