Rukhsana Chowdhury

Effect of Fatty Acids and Cholesterol Present in Bile on Expression of Virulence Factors and Motility of Vibrio cholerae

ABSTRACT Bile induces pleiotropic responses that affect production of virulence factors, motility, and other phenotypes in the enteric pathogen Vibrio cholerae. Since bile is a heterogeneous mixture, crude bile was fractionated, and the components that mediate virulence gene repression and enhancement of motility were identified by nuclear magnetic resonance, gas chromatography (GC), and GC-mass spectrometry analyses. The unsaturated fatty acids detected in bile, arachidonic, linoleic, and oleic acids, drastically repressed expression of the ctxAB and tcpA genes, which encode cholera toxin and the major subunit of the toxin-coregulated pilus, respectively. The unsaturated fatty acid-dependent repression was due to silencing of ctxAB and tcpA expression by the histone-like nucleoid-structuring protein H-NS, even in the presence of the transcriptional activator ToxT. Unsaturated fatty acids also enhanced motility of V. cholerae due to increased expression of flrA, the first gene of a regulatory cascade that controls motility. H-NS had no role in the fatty acid-mediated enhancement of motility. It is likely that the ToxR/ToxT system that negatively regulates motility is rendered nonfunctional in the presence of unsaturated fatty acids, leading to an increase in motility. Motility and flrA expression were also increased in the presence of cholesterol, another component of bile, in an H-NS- and ToxR/ToxT-independent manner.

Stress response in pathogenic bacteria

Bacterial pathogens survive under two entirely different conditions, namely, their natural environment and in their hosts. Response of these pathogens to stresses encountered during transition from the natural environment to human hosts has been described. The virulence determinants of pathogenic bacteria are under the control of transcriptional activators which respond to fluctuations in growth temperature, osmolarity, metal ion concentration and oxygen tension of the environment. The regulation of stress induced genes may occur at the level of transcription or translation or by post-translational modifications. Under certain stress conditions local changes in the superhelicity of DNA induce or repress genes. In addition to their role in survival of bacteria under stressful situations, the stress induced proteins are also implicated in the manifestation of pathogenicity of bacterial pathogensin vivo.

Effect of Anaerobiosis on Expression of Virulence Factors in Vibrio cholerae

ABSTRACT In Vibrio cholerae, the transmembrane DNA binding proteins, ToxR and TcpP, activate expression of the regulatory gene toxT in response to specific environmental signals. The resulting enhanced level of ToxT leads to a coordinated increase in the production of a subset of virulence factors, including cholera toxin (CT) and toxin-coregulated pilus (TCP). The effect of anaerobiosis on expression of the V. cholerae virulence regulatory cascade was examined. The expression of the major regulatory genes, tcpP, toxR, and toxT, in anaerobically grown V. cholerae was comparable to that in cells grown under aerobic conditions, and no significant difference in the ToxT-dependent expression of tcpA was detected when aerobic and anaerobic cultures were compared. However, in spite of the presence of functional ToxT, ctxAB expression was drastically reduced, and practically no CT was detected in cells grown under anaerobic conditions. In a V. cholerae hns mutant, however, high levels of ctxAB expression occurred even under anaerobic conditions. Also, deletion of the H-NS binding site from the ctxAB promoter eliminated anaerobic repression of ctxAB expression. These results suggest that H-NS directly represses ctxAB expression under anaerobic growth conditions. It has been reported that in the first stage of infection of infant mice by V. cholerae, tcpA is expressed but ctxAB expression is shut off (S. H. Lee, D. L. Hava, M. K. Waldor, and A. Camilli, Cell 99: 625-634, 1999). This pattern is similar to the pattern in anaerobic cultures of V. cholerae. Under all other in vitro conditions, ctxAB and tcpA are known to be coordinately expressed.

The Global Regulator ArcA Modulates Expression of Virulence Factors in Vibrio cholerae

ABSTRACT A Vibrio cholerae arcA mutant was constructed and used to examine the role of the global anaerobiosis response regulator ArcA in the expression of virulence factors in this important human pathogen. In V. cholerae, expression of the major virulence factors cholera toxin (CT) and toxin-coregulated pilus (TCP) is regulated by the transcriptional activator ToxT. toxT expression, in turn, is controlled by the transmembrane DNA binding proteins ToxR and TcpP. In the V. cholerae arcA mutant, although ToxR and TcpP were unaffected, Northern blot and reverse transcription-PCR analyses indicated that the expression of toxT was significantly decreased with concomitant reduction in the expression of CT and TCP. CT and TCP expression was completely restored in the V. cholerae arcA mutant strain by expressing a cloned toxT gene in the mutant. These results suggest that ArcA functions as a positive regulator of toxT expression under both aerobic and anaerobic conditions, although as expected, the effect was more pronounced during anaerobic growth. This was reflected in a reduction of virulence of the V. cholerae arcA mutant strain in the infant mouse cholera model.

Role of the Histone-Like Nucleoid Structuring Protein in Colonization, Motility, and Bile-Dependent Repression of Virulence Gene Expression in Vibrio cholerae

ABSTRACT Bile-mediated repression of virulence gene expression is relieved in a Vibrio cholerae hns mutant. The mutant also exhibited reduced motility due to lower flrA expression, higher in vivo production of the virulence factors, and lower colonization efficiency. The colonization defect of the mutant was due to low FlrA production.

The Molecular Basis of Inactivation of Metronidazole-Resistant Helicobacter pylori Using Polyethyleneimine Functionalized Zinc Oxide Nanoparticles

In view of the world wide prevalence of Helicobacter pylori infection, its potentially serious consequences, and the increasing emergence of antibiotic resistant H. pylori strains there is an urgent need for the development of alternative strategies to combat the infection. In this study it has been demonstrated that polyethyleneimine (PEI) functionalized zinc oxide (ZnO) nanoparticles (NPs) inhibit the growth of a metronidazole-resistant strain of H. pylori and the molecular basis of the anti-bacterial activity of ZnO-PEI NP has been investigated. The ZnO-PEI NP was synthesized using a wet chemical method with a core size of approximately 3–7 nm. Internalization and distribution of ZnO-PEI NP without agglomeration was observed in H. pylori cytosol by electron microscopy. Several lines of evidence including scanning electron microscopy, propidium iodide uptake and ATP assay indicate severe membrane damage in ZnO-PEI NP treated H. pylori. Intracellular ROS generation increased rapidly following the treatment of H. pylori with ZnO-PEI NP and extensive degradation of 16S and 23S rRNA was observed by quantitative reverse-transcriptase PCR. Finally, considerable synergy between ZnO-PEI NP and antibiotics was observed and it has been demonstrated that the concentration of ZnO-PEI NP (20 µg/ml) that is non-toxic to human cells could be used in combination with sub-inhibitory concentrations of antibiotics for the inhibition of H. pylori growth.

Cigarette smoke induces p-benzoquinone–albumin adduct in blood serum: Implications on structure and ligand binding properties

Earlier we had reported that irrespective of the source cigarette smoke (CS) contains substantial amounts of p-benzosemiquinone, which is readily converted to p-benzoquinone (p-BQ) by disproportionation and oxidation by transition metal containing proteins. Here we show that after CS-exposure, p-BQ-protein adducts are formed in the lungs as well as serum albumin of guinea pigs. We also show that serum of human smokers contains p-BQ-albumin adduct. It is known that human serum albumin (HSA) plays a very important role in binding and transport of a variety of ligands, including fatty acids and drugs. We show in vitro that p-BQ forms covalent adducts with free amino groups of all twenty amino acids as well as ɛ-amino groups of lysine residues of HSA in a concentration dependent manner. When HSA is incubated with p-BQ in the molar ratio of 1:1, the number of p-BQ incorporated is 1. At the molar ratio of 1:60, the number of p-BQ incorporated is 40. The formation of HSA-p-BQ adduct has been demonstrated by absorption spectroscopy, MALDI-MS and MALDI-TOF-TOF-MS analyses. Upon complexation with p-BQ, the secondary structure and conformation of HSA are altered, as evidenced by steady state and time-resolved fluorescence, circular dichroism, 8-anilino-1-napthalenesulfonic acid binding and differential scanning calorimetry. Alteration of the structure and conformation of HSA results in impairment of its ligand binding properties with respect to myristic acid, quercitin and paracetamol. This might be one of the reasons why transport and distribution of lipids and drugs are impaired in smokers.

The El Tor Biotype of Vibrio cholerae Exhibits a Growth Advantage in the Stationary Phase in Mixed Cultures with the Classical Biotype

Vibrio cholerae strains of the O1 serogroup that typically cause epidemic cholera can be classified into two biotypes, classical and El Tor. The El Tor biotype emerged in 1961 and subsequently displaced the classical biotype as a cause of cholera throughout the world. In this study we demonstrate that when strains of the El Tor and classical biotypes were cocultured in standard LB medium, the El Tor strains clearly had a competitive growth advantage over the classical biotype starting from the late stationary phase and could eventually take over the population. The classical biotype produces extracellular protease(s) in the stationary phase, and the amounts of amino acids and small peptides in the late stationary and death phase culture filtrates of the classical biotype were higher than those in the corresponding culture filtrates of the El Tor biotype. The El Tor biotype cells could utilize the amino acids more efficiently than the classical biotype under the alkaline pH of the stationary phase cultures but not in medium buffered to neutral pH. The growth advantage of the El Tor biotype was also observed in vivo using the ligated rabbit ileal loop and infant mouse animal models.

Intracellular Development of Choleraphage Φ149 under Permissive and Nonpermissive Conditions: An Electron Microscopic Study

Intracellular development of choleraphage phi 149 following infection of Vibrio cholerae and Vibrio eltor cells under different conditions was examined by thin-section electron microscopy. Degradation of the host DNA following infection and formation of mature phage particles inside the infected cells were demonstrated. The concatemeric DNA intermediate formed during intracellular replication of phi 149 DNA in permissive hosts was resolved. In confirmation of biochemical evidence, no concatemeric DNA intermediate was observed for infection in high-phosphate medium.

Bile and Unsaturated Fatty Acids Inhibit the Binding of Cholera Toxin and Escherichia coli Heat-Labile Enterotoxin to GM1 Receptor

ABSTRACT Cholera toxin (CT) is an archetypal bacterial toxin that binds with a high affinity to the receptor ganglioside GM1 on the intestinal epithelial surface and that causes the severe watery diarrhea characteristic of the disease cholera. Blockage of the interaction of CT with the GM1 receptor is an attractive approach for therapeutic intervention. We report here that crude bile prevents the interaction of CT with GM1 and reduces CT-mediated fluid accumulation in the rabbit intestine. The unsaturated fatty acids detected in crude bile, arachidonic, linoleic, and oleic acids, were found to be the most effective. Crude bile and the unsaturated fatty acids interacted with CT but not GM1 to prevent CT-GM1 binding. Neither crude bile nor the unsaturated fatty acids had any effect on the subunit structure of CT. The binding of CT to unsaturated fatty acids resulted in a shift of the apparent pI of CT from 6.8 to 8.2 and a marked decrease in intrinsic fluorescence. The Kd was calculated from fluorescence quenching assays. It was demonstrated by the rabbit ileal loop model that practically no fluid accumulated in the intestinal loops when CT was administered together with inhibitory concentrations of linoleic acid. The bile present in the intestine was sufficient to inhibit the activity of up to 300 ng CT. Bile and unsaturated fatty acids also inhibited the binding of Escherichia coli heat-labile enterotoxin (LT) to GM1, and no fluid accumulation was observed in rabbit ileal loops when LT was administered together with linoleic acid.