A new Vibrio cholerae sRNA modulates colonization and affects release of outer membrane vesicles

We discovered a new small non-coding RNA (sRNA) gene, vrrA of Vibrio cholerae O1 strain A1552. A vrrA mutant overproduces OmpA porin, and we demonstrate that the 140 nt VrrA RNA represses ompA translation by base-pairing with the 5′ region of the mRNA. The RNA chaperone Hfq is not stringently required for VrrA action, but expression of the vrrA gene requires the membrane stress sigma factor, σE, suggesting that VrrA acts on ompA in response to periplasmic protein folding stress. We also observed that OmpA levels inversely correlated with the number of outer membrane vesicles (OMVs), and that VrrA increased OMV production comparable to loss of OmpA. VrrA is the first sRNA known to control OMV formation. Moreover, a vrrA mutant showed a fivefold increased ability to colonize the intestines of infant mice as compared with the wild type. There was increased expression of the main colonization factor of V. cholerae, the toxin co-regulated pili, in the vrrA mutant as monitored by immunoblot detection of the TcpA protein. VrrA overproduction caused a distinct reduction in the TcpA protein level. Our findings suggest that VrrA contributes to bacterial fitness in certain stressful environments, and modulates infection of the host intestinal tract

Lipoarabinomannan biosynthesis in Corynebacterineae: the interplay of two α(1→2)-mannopyranosyltransferases MptC and MptD in mannan branching

Lipomannan (LM) and lipoarabinomannan (LAM) are key Corynebacterineae glycoconjugates that are integral components of the mycobacterial cell wall, and are potent immunomodulators during infection. LAM is a complex heteropolysaccharide synthesized by an array of essential glycosyltransferase family C (GT-C) members, which represent potential drug targets. Herein, we have identified and characterized two open reading frames from Corynebacterium glutamicum that encode for putative GT-Cs. Deletion of NCgl2100 and NCgl2097 in C. glutamicum demonstrated their role in the biosynthesis of the branching α(1→2)-Manp residues found in LM and LAM. In addition, utilizing a chemically defined nonasaccharide acceptor, azidoethyl 6-O-benzyl-α-D-mannopyranosyl-(1→6)-[α-D-mannopyranosyl-(1→6)]7-D-mannopyranoside, and the glycosyl donor C50-polyprenol-phosphate-[14C]-mannose with membranes prepared from different C. glutamicum mutant strains, we have shown that both NCgl2100 and NCgl2097 encode for novel α(1→2)-mannopyranosyltransferases, which we have termed MptC and MptD respectively. Complementation studies and in vitro assays also identified Rv2181 as a homologue of Cg-MptC in Mycobacterium tuberculosis. Finally, we investigated the ability of LM and LAM from C. glutamicum, and C. glutamicumΔmptC and C. glutamicumΔmptD mutants, to activate Toll-like receptor 2. Overall, our study enhances our understanding of complex lipoglycan biosynthesis in Corynebacterineae and sheds further light on the structural and functional relationship of these classes of polysaccharides

Maintaining Integrity Under Stress:Envelope Stress Response Regulation of Pathogenesis in Gram-Negative Bacteria

The Gram-negative bacterial envelope is an essential interface between the intracellular and harsh extracellular environment. Envelope stress responses (ESRs) are crucial to the maintenance of this barrier and function to detect and respond to perturbations in the envelope, caused by environmental stresses. Pathogenic bacteria are exposed to an array of challenging and stressful conditions during their lifecycle and, in particular, during infection of a host. As such, maintenance of envelope homeostasis is essential to their ability to successfully cause infection. This review will discuss our current understanding of the σE- and Cpx-regulated ESRs, with a specific focus on their role in the virulence of a number of model pathogens

Enteric YaiW is a surface-exposed outer membrane lipoprotein that affects sensitivity to an antimicrobial peptide

yaiW is a previously uncharacterized gene found in enteric bacteria that is of particular interest because it is located adjacent to the sbmA gene, whose bacA ortholog is required for Sinorhizobium meliloti symbiosis and Brucella abortus pathogenesis. We show that yaiW is cotranscribed with sbmA in Escherichia coli and Salmonella enterica serovar Typhi and Typhimurium strains. We present evidence that the YaiW is a palmitate-modified surface exposed outer membrane lipoprotein. Since BacA function affects the very-long-chain fatty acid (VLCFA) modification of S. meliloti and B. abortus lipid A, we tested whether SbmA function might affect either the fatty acid modification of the YaiW lipoprotein or the fatty acid modification of enteric lipid A but found that it did not. Interestingly, we did observe that E. coli SbmA suppresses deficiencies in the VLCFA modification of the lipopolysaccharide of an S. meliloti bacA mutant despite the absence of VLCFA in E. coli. Finally, we found that both YaiW and SbmA positively affect the uptake of proline-rich Bac7 peptides, suggesting a possible connection between their cellular functions

Alveolar-Capillary Membrane-Related Pulmonary Cells as a Target in Endotoxin-Induced Acute Lung Injury

The main function of the lungs is oxygen transport from the atmosphere into the blood circulation, while it is necessary to keep the pulmonary tissue relatively free of pathogens. This is a difficult task because the respiratory system is constantly exposed to harmful substances entering the lungs by inhalation or via the blood stream. Individual types of lung cells are equipped with the mechanisms that maintain pulmonary homeostasis. Because of the clinical significance of acute respiratory distress syndrome (ARDS) the article refers to the physiological role of alveolar epithelial cells type I and II, endothelial cells, alveolar macrophages, and fibroblasts. However, all these cells can be damaged by lipopolysaccharide (LPS) which can reach the airspaces as the major component of the outer membrane of Gram-negative bacteria, and lead to local and systemic inflammation and toxicity. We also highlight a negative effect of LPS on lung cells related to alveolar-capillary barrier and their response to LPS exposure. Additionally, we describe the molecular mechanism of LPS signal transduction pathway in lung cells

A glycomic approach reveals a new mycobacterial polysaccharide

Mycobacterium tuberculosis lipoarabinomannan (LAM) and biosynthetically related lipoglycans and glycans play an important role in host–pathogen interactions. Therefore, the elucidation of the complete biosynthetic pathways of these important molecules is expected to afford novel therapeutic targets. The characterization of biosynthetic enzymes and transporters involved in the formation and localization of these complex macromolecules in the bacterial cell envelope largely relies on genetic manipulation of mycobacteria and subsequent analyses of lipoglycan structural alterations. However, lipoglycans are present in relatively low amounts. Their purification to homogeneity remains tedious and time-consuming. To overcome these issues and to reduce the biomass and time required for lipoglycan purification, we report here the development of a methodology to efficiently purify lipoglycans by sodium deoxycholate–polyacrylamide gel electrophoresis. This faster purification method can be applied on a small amount of mycobacterial cells biomass (10–50 mg), resulting in tens of micrograms of purified lipoglycans. This amount of purified products was found to be sufficient to undertake structural analyses of lipoglycans and glycans carbohydrate domains by a combination of highly sensitive analytical procedures, involving cryoprobe NMR analysis of intact macromolecules and chemical degradations monitored by gas chromatography and capillary electrophoresis. This glycomic approach was successfully applied to the purification and structural characterization of a newly identified polysaccharide, structurally related to LAM, in the model fast-growing species Mycobacterium smegmatis

Characterization of the σ^E-dependent <i>rpoEp3</i> promoter of <i>Salmonella enteric</i> serovar Typhimurium

Using a two-plasmid system, we recently identified σ&lt;sup&gt;E&lt;/sup&gt;-dependent promoters directing expression of the σ&lt;sup&gt;E&lt;/sup&gt; regulon genes in &lt;i&gt;Salmonella enteric&lt;/i&gt; serovar Typhimurium (&lt;i&gt;S&lt;/i&gt;. Typhimurium). Comparison of the promoters revealed a consensus sequence almost identical to the σ&lt;sup&gt;E&lt;/sup&gt;-dependent &lt;i&gt;rpoEp3&lt;/i&gt; promoter directing expression of &lt;i&gt;rpoE&lt;/i&gt;. This two-plasmid system was previously optimized to identify nucleotides critical for the &lt;i&gt;rpoEp3&lt;/i&gt; promoter activity. However, two highly conserved nucleotides in the σ&lt;sup&gt;E&lt;/sup&gt; consensus sequence were not identified by this screening. In the present study, we have improved the two-plasmid screening system using a new optimized error-prone PCR mutagenesis. Together with site-directed mutagenesis, we further identified nucleotides critical for activity of the &lt;i&gt;rpoEp3&lt;/i&gt; promoter and quantified the effect of the particular mutation upon promoter activity. All the identified critical nucleotides of the &lt;i&gt;rpoEp3&lt;/i&gt; promoter (in capital) were located in the −35 (ggAACtt) and −10 (gTCtaA) regions and corresponded to the most conserved nucleotides in the σ&lt;sup&gt;E&lt;/sup&gt; consensus sequence. The expression of the wild-type and mutated &lt;i&gt;rpoEp3&lt;/i&gt; promoters was confirmed in &lt;i&gt;S&lt;/i&gt;. Typhimurium and was found to exhibit a different pattern of σ&lt;sup&gt;E&lt;/sup&gt; activation compared with &lt;i&gt;Escherichia coli&lt;/i&gt;, with a peak &lt;i&gt;rpoEp3&lt;/i&gt; promoter activity in early stationary phase followed by a decrease in late stationary phase

A mutant of Salmonella enterica serovar Typhimurium RNA polymerase extracytoplasmic stress response sigma factor σE with altered promoter specificity

The alternative sigma factor σE is critical for envelope stress response and plays a role in pathogenicity of a variety of different bacteria. We previously identified several critical nucleotides in the Salmonella enterica serovar Typhimurium (S. Typhimurium) σE-dependent rpoEp3 promoter that corresponded to the most conserved nucleotides in the σE consensus sequence of the −10 and −35 promoter elements. In the present study, we exploited a previously established Escherichia coli (E. coli) two-plasmid system with an error-prone PCR mutagenesis to identify mutants in the rpoE gene that suppress the mutation of the most conserved residue A-30G of the rpoEp3 promoter. This analysis identified amino-acid changes in the conserved arginine residue (R171G, R171C) located in the conserved region 4.2 of σE that enabled efficient recognition of the mutated rpoEp3 promoter. However, the change of this conserved arginine to alanine (R171A) resulted in an almost complete loss of σE activity. The activity of the mutant σE factors in directing transcription of the wild-type (WT) and the A-30G mutated rpoEp3 promoters was investigated by S1-nuclease mapping using RNA isolated from the E. coli two-plasmid system. In addition to suppression of the A-30G mutated rpoEp3 promoter, both mutant sigma factors (R171G, R171C) also efficiently directed transcription from the WT rpoEp3 promoter and from the rpoEp3 promoter with other mutations in the −35 element, indicating relaxed recognition of the σE-dependent promoters by both mutants. The activity of both mutant σE factors was confirmed in vivo in S. Typhimurium. In conclusion, replacement of the conserved R171 residue in σE by different amino-acid residues exhibited intriguingly different phenotypes; R171A almost completely abolished sigma factor activity, whereas R171G and R171C impart a relaxed recognition phenotype to σE