coli showed that an ompC knockout mutant had increased levels of OmpA [40], however, changes in permeability were not evaluated. Furthermore, this has not been evaluated in a S. Typhimurium or E. coli ∆ompW strain. Figure 2 Bacterial concentration of S . Typhimurium 14028s and Δ ompW exposed to H 2 O 2 or NaOCl. Cultures of 14028s and ΔompW were grown to OD ~ 0.4 and treated with H2O2 4 mM or NaOCl 5 CX-6258 mw mM in LB medium. Time of treatment is indicated. Bacterial concentrations were determined by plating. The values are the concentrations of surviving
SYN-117 mw bacteria after exposure to H2O2 or NaOCl. Experiments were performed in triplicate. Error bars indicate SD. Our data supports the proposed model where OmpW allows the influx of small polar molecules, like H2O2 and HOCl. The crystal structure of OmpW from E. coli mTOR activity revealed that the cross-section of the barrel has approximate dimensions of 17 × 12 Å along the length of the barrel and although the interior of the channel has a hydrophobic character, the observed single channel activities shows that polar molecules traverse the barrel [17]. Taken together, these
results provide biochemical and genetic evidence indicating that both toxic compounds are channeled through OmpW. From our knowledge, this is the first direct evidence of HOCl diffusion through porins. Furthermore, preliminary analyses indicate that H2O2 and HOCl channeling is common for S. Typhimurium OmpD, OmpC and OmpF porins (unpublished data). Hydrogen peroxide and hypochlorous acid exposure results in ompW negative regulation Since the OmpW porin channels H2O2 and HOCl through the OM and exposure to these molecules is detrimental to bacteria, we hypothesized that ompW should be negatively regulated when S. Typhimurium is exposed to H2O2 and HOCl. To study ADP ribosylation factor this effect, wild type S. Typhimurium cells were grown to mid-log
phase, exposed to H2O2 or HOCl and ompW mRNA levels were measured by qRT-PCR. As seen in Figure 3, exposure to H2O2 and HOCl resulted in lower levels of ompW transcripts (0.27 ± 0.04 and 0.156 ± 0.079, respectively) relative to control untreated cells. In agreement with our results of ompW negative regulation, similar results were observed by Wang et al. (2010) who showed that S. Enteritidis and Typhimurium cells exposed to HOCl results in modulation of ompD, ompC, ompF (negatively) and ompA (positively) expression. Furthermore, Calderón et al. (2011) demonstrated that the S. Typhimurium ompD gene is negatively regulated in response to H2O2. Therefore, our and all the published data suggest that in the presence of OCl- or H2O2 there might be a general lowering in the concentration of porins in the outer membrane, in order to diminish the permeability. To assess the specificity of our assay, we evaluated ompD, ompC and arcB transcript levels as positive (ompD and ompC) and negative controls (arcB).