Genetic regulation of the ompX porin of Salmonella Typhimurium in response to hydrogen peroxide stress

BACKGROUND: Salmonella Typhimurium is a Gram negative pathogen that causes a systemic disease in mice resembling typhoid fever. During its infective cycle, S. Typhimurium is phagocytized by macrophages and proliferates inside a Salmonella containing vacuole where Salmonella is exposed and survives oxidative stress induced by H2O2 through modulation of gene expression. After exposure of Salmonella to H2O2, the expression of the porin encoding gene ompX increases, as previously shown by microarray analysis. Expression of ompX mRNA is regulated at a post transcriptional level by MicA and CyaR sRNAs in aerobiosis. In addition, sequence analysis predicts a site for OxyS sRNA in ompX mRNA. RESULTS: In this work we sought to evaluate the transcriptional and post transcriptional regulation of ompX under H2O2 stress. We demonstrate that ompX expression is induced at the transcriptional level in S . Typhimurium under such conditions. Unexpectedly, an increase in ompX gene transcript and promoter activity after challenges with H2O2 does not translate into increased protein levels in the wild type strain, suggesting that ompX mRNA is also regulated at a post transcriptional level, at least under oxidative stress. In silico gene sequence analysis predicted that sRNAs CyaR, MicA, and OxyS could regulate ompX mRNA levels. Using rifampicin to inhibit mRNA expression, we show that the sRNAs (MicA, CyaR and OxyS) and the sRNA:mRNA chaperone Hfq positively modulate ompX mRNA levels under H2O2 induced stress in Salmonella during the exponential growth phase in Lennox broth. CONCLUSIONS: Our results demonstrate that ompX mRNA is regulated in response to H2O2 by the sRNAs CyaR, MicA and OxyS is Salmonella Typhimurium.

Carbapenem-resistant and cephalosporin-susceptible: a worrisome phenotype among Pseudomonas aeruginosa clinical isolates in Brazil

Abstract The mechanisms involved in the uncommon resistance phenotype, carbapenem resistance and broad-spectrum cephalosporin susceptibility, were investigated in 25 Pseudomonas aeruginosa clinical isolates that exhibited this phenotype, which were recovered from three different hospitals located in São Paulo, Brazil. The antimicrobial susceptibility profile was determined by CLSI broth microdilution. β-lactamase-encoding genes were investigated by PCR followed by DNA sequencing. Carbapenem hydrolysis activity was investigated by spectrophotometer and MALDI-TOF assays. The mRNA transcription level of oprD was assessed by qRT-PCR and the outer membrane proteins profile was evaluated by SDS-PAGE. Genetic relationship among P. aeruginosa isolates was assessed by PFGE. Carbapenems hydrolysis was not detected by carbapenemase assay in the carbapenem-resistant and cephalosporin-susceptible P. aueruginosa clinical isolates. OprD decreased expression was observed in all P. aeruginosa isolates by qRT-PCR. The outer membrane protein profile by SDS-PAGE suggested a change in the expression of the 46 kDa porin that could correspond to OprD porin. The isolates were clustered into 17 genotypes without predominance of a specific PFGE pattern. These results emphasize the involvement of multiple chromosomal mechanisms in carbapenem-resistance among clinical isolates of P. aeruginosa, alert for adaptation of P. aeruginosa clinical isolates under antimicrobial selective pressure and make aware of the emergence of an uncommon phenotype among P. aeruginosa clinical isolates.

Mutational and acquired carbapenem resistance mechanisms in multidrug resistant Pseudomonas aeruginosa clinical isolates from Recife, Brazil

An investigation was carried out into the genetic mechanisms responsible for multidrug resistance in nine carbapenem-resistant Pseudomonas aeruginosaisolates from different hospitals in Recife, Brazil. Susceptibility to antimicrobial agents was determined by broth microdilution. Polymerase chain reaction (PCR) was employed to detect the presence of genes encoding β-lactamases, aminoglycoside-modifying enzymes (AMEs), 16S rRNA methylases, integron-related genes and OprD. Expression of genes coding for efflux pumps and AmpC cephalosporinase were assessed by quantitative PCR. The outer membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The blaSPM-1, blaKPC-2 and blaGES-1 genes were detected in P. aeruginosaisolates in addition to different AME genes. The loss of OprD in nine isolates was mainly due to frameshift mutations, premature stop codons and point mutations. An association of loss of OprD with the overexpression of MexAB-OprM and MexXY-OprM was observed in most isolates. Hyper-production of AmpC was also observed in three isolates. Clonal relationship of the isolates was determined by repetitive element palindromic-PCR and multilocus sequence typing. Our results show that the loss of OprD along with overexpression of efflux pumps and β-lactamase production were responsible for the multidrug resistance in the isolates analysed.

Active immunization against riboflavin carrier protein results in peri-implantation embryonic loss leading to pregnancy termination in rats: use of alternate adjuvants.

To investigate the mechanism of pregnancy termination following immuno-neutralization of riboflavin carrier protein (RCP) and to use acceptable adjuvants, we actively immunized female rats with reduced and carboxymethylated RCP (RCM-RCP) using various adjuvants (during primary immunization) such as sodium phthalylated lipopolysaccharide (SPLPS), purified S. typhi outer membrane proteins (porins) and a combination of them. Rats (5-14 per group) were immunized with alugel adsorbed RCM-RCP (100 microg/dose) either alone or with SPLPS or porins or SPLPS+porins. Control animals received RCM-RCP emulsified with Fruend's completelincomplete adjuvants (FCA/FIA). All animals received five boosters at intervals of 21 days. The lowest (4 X 10(-3)) and the highest (> 70 X 10(-3)) anti-RCM-RCP antibody titers were observed in alugel adsorbed-RCM-RCP group and control groups, respectively. Immunized animals showed reduced fertility following 3rd, 4th and 5th boosters. Reduction in fertility was 30-60% in alugel adsorbed RCM-RCP group, 90-100% in FCA-RCM-RCP group and 80-90% in SPLPS+porins group. Fertility reduction was not strictly correlatable with the serum antibody titers. RCP-specific IgG could be localized in the uterine endometrial glands and luminal epithelial cells in the immunized animals. Animals in the FCA/FIA group showed abnormal implantation/resorption sites and their histological sections showed degenerated embryos. But, day 5 preimplantation embryos were normal. These results show that (a) SPLPS+porins can be used as adjuvants in place of FCA/FIA for active immunization against RCM-RCP and (b) early termination of pregnancy in the immunized animals is due largely to the failure of normal embryo implantation.

Aquaporins and water transfer across epithelial barriers

The cloning and molecular characterization of water channels, generically called aquaporins have marked a pivotal point in our understanding of water movements across epithelial barriers. Nevertheless, the mechanisms underlying water transfer across these barriers at the molecular and cellular level are not yet clarified. We analyze here the role of the different driving forces moving water across epithelia and the biophysical properties of the water channels. We will also review the recently cloned epithelial members of the aquaporin family, including their expression and distribution in different tissues.