Post-transcriptional regulation of vibrio cholerae virulence activator toxt

Vibrio cholera, the causative agent of the severe diarreal illness cholera, uses a complex array of gene regulation to induce its virulence determinants. During the early stage of infection, and upon response to unknown signals, virulence genes are turned on. ToxT protein is the primary virulence gene transcription activator. Once ToxT is produced, it amplifies its own expression through an auto-regulatory loop and directly binds and activates expression of various virulence factors including the toxin-coregulated pilus (TCP) and cholera toxin (CT). During the late stage of infection, virulence genes are turned off and the bacteria escape the host to resume their lifestyle in the aquatic environment. While posttranscriptional regulation of ToxT has been observerd, most studies were focused on revealing how toxT expression is initiated. Here, the regulation of ToxT protein at the posttranscriptional level has been explored. In chapter one, experiments are presented that indicate bicarbonate is a positive effector molecule that enhances ToxT activity. Culturing the bacteria in the presence of bicarbonate increases the ToxT activity without inreasing the protein level. Bicarbonate is found in the small intestine where V. cholerae colonize humans and could be the natural signal that the bacteria sense during the course of infection to maximally induce its virulence determinents. In chapter two, the mechanism of virulence down-regulation as V.cholerae terminates its virulence expression during the late stage of infection preparing to enter back into the environment is assessed. The data suggest that virulence expression could terminate through ToxT proteolytic degradation. ToxT proteolysis was observed when culturing the bacteria at high temperature and pH, condition that has been found to repress virulence induction. Further analysis revealed that the unstructured motif which is located between amino acids 100-110 of ToxT is important for this degradation. In the last chapter, a method of utilizing fluorescence-activated cell sorting (FACS) technology in conjuction with transposon mutagenesis is described. This method was used to isolate bacterial mutants that produce different gene expression profiles in response to environmental cues while cultured in vitro in liquid growth media.This technique should be applicable for isolating bacterial mutants that respond differently to chemical and physical inducers or repressors that are present in the liquid growth conditions

A moral dilemma: The use of zinc in patients hospitalized with COVID pneumonia

A clinical decision report using: Yao JS, Paguio JA, Dee EC, Tan HC, Moulick A, Milazzo C, Jurado J, Della Penna N, Celi LA. The minimal effect of zinc on the survival of hospitalized patients with COVID-19: an observational study. Chest. 2021 Jan;159(1):108-111. https://doi.org/10.1016/j.chest.2020.06.082 for a patient hospitalized with COVID pneumonia requiring high flow oxygen

Changes in Quality of Emotional Processing in Trauma Narratives as a Predictor of Outcome in Emotion Focused Therapy for Complex Trauma

The present study tested a model of change in emotional processes over thecourse of Emotion Focused Therapy for Trauma (EFTT). The Classification of AffectiveMeaning States (CAMS; Pascual-Leone & Greenberg, 2005) specifies a sequential shiftfrom maladaptive and unproductive affective processes (i.e. global distress, fear andshame, and rejecting anger), through negative self-evaluation and expressing unmetneeds, to productive affective processes (i.e. hurt and grief, self-compassion, assertiveanger, and acceptance and agency) that aid in adaptive functioning. This study used theCAMS to examine changes in the quality of emotional processes during trauma narrativesin early and late sessions of EFTT. It was expected that a shift from unproductive toproductive affective processes over the course of therapy would be associated withresolution of abuse issues and reduction in trauma symptomology at therapy termination.It was also anticipated that the shift in affective processes would follow the sequence aspresented in the CAMS. Results indicated a greater frequency of productive affectiveprocesses in late narratives; a greater frequency of productive processes was morepredictive of treatment outcome compared to unproductive processes; and greaterincrease in the frequency of productive processes from early to late narrativessignificantly contributed to good treatment outcome. Findings also revealed a significantinteraction between unproductive and productive affective processes in predictingtreatment outcome. These findings are consistent with the proposed hypotheses of thepresent study. Finally, results indicated that clients were significantly more likely tofollow the proposed sequence for lower level processes, which supports the hypothesis.However, results did not produce similarly significant results for higher levels. Rather clients expressed higher levels of productive affective processes in late sessions that werenot preceded by lower levels of processes in the proposed model. The findings haveimplications for guiding the therapeutic process in a productive manner that leads totrauma recovery

Simple inhibitors of histone deacetylase activity that combine features of short-chain fatty acid and hydroxamic acid inhibitors

Butyric acid and trichostatin A (TSA) are anti-cancer compounds that cause the upregulation of genes involved in differentiation and cell cycle regulation by inhibiting histone deacetylase (HDAC) activity. In this study we have synthesized and evaluated compounds that combine the bioavailability of short-chain fatty acids, like butyric acid, with the bidentate binding ability of TSA. A series of analogs were made to examine the effects of chain length, simple aromatic cap groups, and substituted hydroxamates on the compounds\u27 ability to inhibit rat-liver HDAC using a fluorometric assay. In keeping with previous structure-activity relationships, the most effective inhibitors consisted of longer chains and hydroxamic acid groups. It was found that 5-phenylvaleric hydroxamic acid and 4-benzoylbutyric hydroxamic acid were the most potent inhibitors with IC50\u27s of 5 microM and 133 microM respectively

Expression of Colonization Factor CS5 of Enterotoxigenic Escherichia coli (ETEC) Is Enhanced In Vivo and by the Bile Component Na Glycocholate Hydrate

Enterotoxigenic Escherichia coli (ETEC) is an important cause of acute watery diarrhoea in developing countries. Colonization factors (CFs) on the bacterial surface mediate adhesion to the small intestinal epithelium. Two of the most common CFs worldwide are coli surface antigens 5 and 6 (CS5, CS6). In this study we investigated the expression of CS5 and CS6 in vivo, and the effects of bile and sodium bicarbonate, present in the human gut, on the expression of CS5. Five CS5+CS6 ETEC isolates from adult Bangladeshi patients with acute diarrhoea were studied. The level of transcription from the CS5 operon was approximately 100-fold higher than from the CS6 operon in ETEC bacteria recovered directly from diarrhoeal stool without sub-culturing (in vivo). The glyco-conjugated primary bile salt sodium glycocholate hydrate (NaGCH) induced phenotypic expression of CS5 in a dose-dependent manner and caused a 100-fold up-regulation of CS5 mRNA levels; this is the first description of NaGCH as an enteropathogenic virulence inducer. The relative transcription levels from the CS5 and CS6 operons in the presence of bile or NaGCH in vitro were similar to those in vivo. Another bile salt, sodium deoxycholate (NaDC), previously reported to induce enteropathogenic virulence, also induced expression of CS5, whereas sodium bicarbonate did not

A Genome-Wide Approach to Discovery of Small RNAs Involved in Regulation of Virulence in Vibrio cholerae

Small RNAs (sRNAs) are becoming increasingly recognized as important regulators in bacteria. To investigate the contribution of sRNA mediated regulation to virulence in Vibrio cholerae, we performed high throughput sequencing of cDNA generated from sRNA transcripts isolated from a strain ectopically expressing ToxT, the major transcriptional regulator within the virulence gene regulon. We compared this data set with ToxT binding sites determined by pulldown and deep sequencing to identify sRNA promoters directly controlled by ToxT. Analysis of the resulting transcripts with ToxT binding sites in cis revealed two sRNAs within the Vibrio Pathogenicity Island. When deletions of these sRNAs were made and the resulting strains were competed against the parental strain in the infant mouse model of V. cholerae colonization, one, TarB, displayed a variable colonization phenotype dependent on its physiological state at the time of inoculation. We identified a target of TarB as the mRNA for the secreted colonization factor, TcpF. We verified negative regulation of TcpF expression by TarB and, using point mutations that disrupted interaction between TarB and tpcF mRNA, showed that loss of this negative regulation was primarily responsible for the colonization phenotype observed in the TarB deletion mutant

DNA Damage and Reactive Nitrogen Species are Barriers to Vibrio cholerae Colonization of the Infant Mouse Intestine

Ingested Vibrio cholerae pass through the stomach and colonize the small intestines of its host. Here, we show that V. cholerae requires at least two types of DNA repair systems to efficiently compete for colonization of the infant mouse intestine. These results show that V. cholerae experiences increased DNA damage in the murine gastrointestinal tract. Agreeing with this, we show that passage through the murine gut increases the mutation frequency of V. cholerae compared to liquid culture passage. Our genetic analysis identifies known and novel defense enzymes required for detoxifying reactive nitrogen species (but not reactive oxygen species) that are also required for V. cholerae to efficiently colonize the infant mouse intestine, pointing to reactive nitrogen species as the potential cause of DNA damage. We demonstrate that potential reactive nitrogen species deleterious for V. cholerae are not generated by host inducible nitric oxide synthase (iNOS) activity and instead may be derived from acidified nitrite in the stomach. Agreeing with this hypothesis, we show that strains deficient in DNA repair or reactive nitrogen species defense that are defective in intestinal colonization have decreased growth or increased mutation frequency in acidified nitrite containing media. Moreover, we demonstrate that neutralizing stomach acid rescues the colonization defect of the DNA repair and reactive nitrogen species defense defective mutants suggesting a common defense pathway for these mutants

The inositol requiring enzyme 1 (IRE1α) RNAse inhibitor, 4µ8C, is also a potent cellular antioxidant

Inositol Requiring Enzyme 1 alpha (IRE1α) is an ER-transmembrane endonuclease that is activated in response to ER stress as part of the unfolded protein response (UPR). Chronic activation of the UPR has been implicated in the pathogenesis of many common disease including diabetes, cancer and neurological pathologies such as Huntington's and Alzheimer's disease. 7-hydroxy-4-methyl-2-oxo-2H-chromene-8-carbaldehyde (4µ8C) is widely used as a specific inhibitor of IRE1α ribonuclease activity in mechanistic studies (IC50 of 6.89 µM in cultured cells). However, in this paper we showed that 4µ8C acts as a potent reactive oxygen species (ROS) scavenger both in a cell free assay and in cultured cells at concentrations lower than that widely used to inhibit IRE1α activity. We demonstrate that i 4µ8C effectively decreases xanthine/xanthine oxidase catalysed superoxide production with an IC50 of 0.2 µM. In cultured endothelial and clonal pancreatic beta-cells, 4µ8C inhibits angiotensin II-induced ROS production with IC50s of 1.92 and 0.29 µM respectively. In light of this discovery, conclusions reached using 4µ8C as an inhibitor of IRE1α should be carefully evaluated. However, this unexpected off-target effect of 4µ8C may prove therapeutically advantageous for the treatment of pathologies that are thought to be caused by, or exacerbated by, both oxidative and ER stress such as endothelial dysfunction and/or diabetes. [Abstract copyright: ©2018 The Author(s).

Transcriptional Regulator PerA Influences Biofilm-Associated, Platelet Binding, and Metabolic Gene Expression in Enterococcus faecalis

Enterococcus faecalis is an opportunistic pathogen and a leading cause of nosocomial infections, traits facilitated by the ability to quickly acquire and transfer virulence determinants. A 150 kb pathogenicity island (PAI) comprised of genes contributing to virulence is found in many enterococcal isolates and is known to undergo horizontal transfer. We have shown that the PAI-encoded transcriptional regulator PerA contributes to pathogenicity in the mouse peritonitis infection model. In this study, we used whole-genome microarrays to determine the PerA regulon. The PerA regulon is extensive, as transcriptional analysis showed 151 differentially regulated genes. Our findings reveal that PerA coordinately regulates genes important for metabolism, amino acid degradation, and pathogenicity. Further transcriptional analysis revealed that PerA is influenced by bicarbonate. Additionally, PerA influences the ability of E. faecalis to bind to human platelets. Our results suggest that PerA is a global transcriptional regulator that coordinately regulates genes responsible for enterococcal pathogenicity