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.

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.

A comparison between electrospinning and rotary-jet spinning to produce PCL fibers with low bacteria colonization.

One of the important components in tissue engineering is material structure, providing a model for fixing and the development of cells and tissues, which allows for the transport of nutrients and regulatory molecules to and from cells. The community claims the need for new materials with better properties for use in the clinic. Poly (ε-caprolactone) (PCL) is a biodegradable polymer, semi crystalline, with superior mechanical properties and has attracted an increasing interest due to its usefulness in various biomedical applications. Herein, two different methods (electrospinning versus rotary jet spinning) with different concentrations of PCL produced ultra thin-fibers each with particular characteristics, verified and analyzed by morphology, wettability, thermal and cytotoxicity features and for bacteria colonization. Different PCL scaffold morphologies were found to be dependent on the fabrication method used. All PCL scaffolds showed greater mammalian cell interactions. Most impressively, rotary-jet spun fibers showed that a special rough surface decreased bacteria colonization, emphasizing that no nanoparticle or antibiotic was used; maybe this effect is related with physical (scaffold) and/or biological mechanisms. Thus, this study showed that rotary jet spun fibers possess a special topography compared to electrospun fibers to reduce bacteria colonization and present no cytotoxicity when in contact with mammalian cells.

Lose to win: marT pseudogenization in Salmonella enterica serovar Typhi contributed to the surV-dependent survival to H2O2, and inside human macrophage-like cells.

The difference in host range between Salmonella enterica serovar Typhimurium (S. Typhimurium) and Salmonella enterica serovar Typhi (S. Typhi) can be partially attributed to the gain of functions, to the loss of functions (i.e. pseudogenization), or to a combination of both processes. As previously reported, the loss of functions by pseudogenization may play a role in bacterial evolution, especially in host-restricted pathogens such as S. Typhi. The marT-fidL operon, located at the SPI-3, encodes the MarT transcriptional regulator and a hypothetical protein (i.e. FidL) with no significant similarities to known proteins, respectively. Even though predicted S. Typhimurium FidL exhibit 99.4% identity with S. Typhi FidL, marT has been annotated as a pseudogene in S. Typhi. In this work, we found that S. Typhi expressing S. Typhimurium marT-fidL exhibited an increased accumulation of reactive oxygen species (ROS), leading to a decreased survival in presence of H2O2. Moreover, we found that that the presence of a functional copy of S. Typhimurium marT-fidL in S. Typhi resulted in a repression of surV (STY4039), an ORF found in the S. Typhi SPI-3 but absent from S. Typhimurium SPI-3, that contribute to the resistance to H2O2 by decreasing the accumulation of ROS. Finally, we observed that the presence of S. Typhimurium marT-fidL in S. Typhi negatively affected the survival inside macrophage-like cells, but not in epithelial cells, after 24h post infection. Therefore, this work provides evidence arguing that marT pseudogenization in Salmonella Typhi contributed to the surV-dependent survival against H2O2, and inside human macrophage-like cells. This is a good example of how the loss of functions (marT pseudogenization) and the gain of functions (presence of surV) might contribute to phenotypic changes improving virulence.

Pseudogenization of sopA and sopE2 is functionally linked and contributes to virulence of Salmonella enterica serovar Typhi.

The difference in host range between Salmonella enterica serovar Typhimurium (S. Typhimurium) and S. enterica serovar Typhi (S. Typhi) can be partially attributed to pseudogenes. Pseudogenes are genomic segments homologous to functional genes that do not encode functional products due to the presence of genetic defects. S. Typhi lacks several protein effectors implicated in invasion or other important processes necessary for full virulence of S. Typhimurium. SopA and SopE2, effectors that have been lost by pseudogenization in S. Typhi, correspond to an ubiquitin ligase involved in cytokine production by infected cells, and to a guanine exchange factor necessary for invasion of epithelial cells, respectively. We hypothesized that sopA and/or sopE pseudogenization contributed to the virulence of S. Typhi. In this work, we found that S. Typhi expressing S. Typhimurium sopE2 exhibited a decreased invasion in different epithelial cell lines compared with S. Typhi WT. S. Typhimurium sopA completely abolished the hypo-invasive phenotype observed in S. Typhi expressing S. Typhimurium sopE2, suggesting that functional SopA and SopE2 participate concertedly in the invasion process. Finally, the expression of S. Typhimurium sopA and/or sopE2 in S. Typhi, determined changes in the secretion of IL-8 and IL-18 in infected epithelial cells.

Salmonella Typhi shdA: pseudogene or allelic variant?

ShdA from Salmonella Typhimurium (ShdASTm) is a large outer membrane protein that specifically recognizes and binds to fibronectin. ShdASTm is involved in the colonization of the cecum and the Peyer's patches of terminal ileum in mice. On the other hand, shdA gene from Salmonella Typhi (shdASTy) has been considered a pseudogene (i.e. a nonfunctional sequence of genomic DNA) due to the presence of deletions and mutations that gave rise to premature stop codons. In this work we show that, despite the deletions and mutations, shdASTy is fully functional. S. Typhi ΔshdA mutants presented an impaired adherence and invasion of HEp-2 pre-treated with TGF-ß1, an inducer of fibronectin production. Moreover, shdA from S. Typhi and S. Typhimurium seem to be equivalent since shdASTm restored the adherence and invasion of S. Typhi ΔshdA mutant to wild type levels. In addition, anti-FLAG mAbs interfered with the adherence and invasion of the S. Typhi shdA-3xFLAG strain. Finally, shdASTy encodes a detectable protein when heterologously expressed in Escherichia coli DH5α. The data presented here show that shdASTy is not a pseudogene, but a different functional allele compared with shdASTm.

Thermodynamic and infrared analyses of the interaction of chlorpromazine with phospholipid monolayers.

An investigation has been made of the interaction between chlorpromazine (CPZ) and monolayers of 1,2-dipalmitoyl-sn-3-glycerophosphatidylcholine (DPPC) and 1,2-dipalmitoyl-sn-3-glycero[phospho-rac-(1-glycerol)] (DPPG), both at the air/water interface and in transferred Langmuir-Blodgett films. The Gibbs free energy, DeltaG, and the compressibility modulus (C(S)(-1)), obtained from the surface pressure isotherms, indicated changes in the in-plane interactions of CPZ/DPPG mixed monolayers, with positive values of DeltaG. The arrangement of CPZ in the zwitterionic DPPC monolayers causes a weaker interaction in CPZ/DPPC mixed monolayers, with the DeltaG fluctuating around zero. IR measurements in transferred monolayers showed that CPZ did not affect the conformational order of the acyl chains, its effects being limited to the bands corresponding to the headgroups. Furthermore, since no shift was observed for the acyl chain bands, the phase transition induced by CPZ is not a liquid expanded (LE) to liquid condensed (LC) transition, as the latter is associated with chain ordering. Taken together, the IR and compressibility results demonstrate that the effect from CPZ cannot be correlated with temperature changes in the subphase for pure monolayers, in contrast to models proposed by other authors.

The interaction of meso-tetraphenylporphyrin with phospholipid monolayers.

In this article, we investigate the interaction of meso-tetraphenylporphyrin (TPP) with phospholipid monolayers. Pure TPP molecules form films at the air-water interface with large extension of aggregation, which is confirmed by UV-vis spectra of transferred monolayers. For mixed films of TPP with dipalmitoyl phosphatidyl choline (DPPC) or dipalmitoyl phosphatidyl glycerol (DPPG), on the other hand, aggregation is only significant at high surface pressures or high concentrations of TPP (above 0.1 molar ratio). This was observed via Brewster angle microscopy (BAM) for the Langmuir films and UV-vis spectroscopy for transferred layers onto solid substrates. TPP indeed causes the DPPC and DPPG monolayers to expand, especially at the liquid-expanded to liquid-condensed phase transition for DPPC. The effects from TPP cannot be explained using purely geometrical considerations, as the area per TPP molecule obtained from the isotherms is at least twice the expected value from the literature. Therefore, interaction between TPP and DPPC or DPPG should be cooperative, so that more phospholipid molecules are affected than just the first neighbors to a TPP molecule.

Interaction of two phenothiazine derivatives with phospholipid monolayers.

This paper addresses the cooperative interaction of two phenothiazine drugs, viz. trifluoperazine (TFP) and chlorpromazine (CPZ), with phospholipid monolayers as the model membrane system. Surface pressure and surface potential isotherms were obtained for mixed Langmuir monolayers of either dipalmitoyl-phosphatidyl-choline (DPPC) or dipalmitoyl-phosphatidyl-glycerol (DPPG) co-spread with TFP or CPZ. The changes in monolayer behavior caused by incorporation of a few molar ratio of drug molecules were practically within the experimental dispersion for the zwitterionic DPPC, and therefore a more refined analysis will be required to probe the interactions in an unequivocal way. For the charged DPPG, on the other hand, the surface pressure and the dipole moment were significantly affected even for TFP or CPZ concentrations as low as 0.002 molar ratio. Overall, the effects from CPZ and TFP are similar, but small differences exist which are probably due to the different protonation properties of the two drugs. For both drugs, changes are more prominent at the liftoff of the surface pressure, i.e. at the gas-condensed phase transition, with the surface pressure and surface potential isotherms becoming more expanded with the drug incorporation. With DPPG/CPZ monolayers, in particular, an additional phase transition appears at higher CPZ concentrations, which resembles the effects from increasing the subphase temperature for a pure DPPG monolayer. The dipole moment for DPPG/CPZ and DPPG/TFP monolayers decreases with the drug concentration, which means that the effects from the charged drugs are not associated with changes in the double-layer potential. Otherwise, the effective dipole moment should increase with the drug concentration. The changes caused in surface pressure and dipole moment by small concentrations of TFP or CPZ can only be explained by some cooperative effect through which the contribution from DPPG molecules changes considerably, i.e. even DPPG molecules that are not neighbor to a CPZ or TFP molecule are also affected. Such changes may occur either through a significant reorientation of the DPPG molecules or to a change in their hydration state. We discuss the cooperativity semi-quantitatively by estimating the number of lipid molecules affected by the drug interaction. CPZ and TFP also affect the morphology of DPPG monolayers, which was confirmed with Brewster angle microscopy. The biological implications from the cooperative, non-specific interaction of CPZ and TFP with membranes are also commented upon.