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

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

The Mitochondrial Fission Regulator DRP1 Controls Post-Transcriptional Regulation of TNF-α.

The mitochondrial network plays a critical role in the regulation of innate immune signaling and subsequent production of proinflammatory cytokines such as IFN-β and IL-1β. Dynamin-related protein 1 (DRP1) promotes mitochondrial fission and quality control to maintain cellular homeostasis during infection. However, mechanisms by which DRP1 and mitochondrial dynamics control innate immune signaling and the proinflammatory response are incompletely understood. Here we show that macrophage DRP1 is a positive regulator of TNF-α production during sterile inflammation or bacterial infection. Silencing macrophage DRP1 decreased mitochondrial fragmentation and TNF-α production upon stimulation with lipopolysaccharide (LPS) or methicillin-resistant Staphylococcus aureus (MRSA) infection. The defect in TNF-α induction could not be attributed to changes in gene expression. Instead, DRP1 was required for post-transcriptional control of TNF-α. In contrast, silencing DRP1 enhanced IL-6 and IL-1β production, indicating a distinct mechanism for DRP1-dependent TNF-α regulation. Our results highlight DRP1 as a key player in the macrophage pro-inflammatory response and point to its involvement in post-transcriptional control of TNF-α production

Zebrafish as a natural host model for Vibrio cholerae colonization and transmission.

The human diarrheal disease cholera is caused by the aquatic bacterium Vibrio cholerae. V. cholerae in the environment is associated with several varieties of aquatic life, including insect egg masses, shellfish, and vertebrate fish. Here we describe a novel animal model for V. cholerae, the zebrafish. Pandemic V. cholerae strains specifically colonize the zebrafish intestinal tract after exposure in water with no manipulation of the animal required. Colonization occurs in close contact with the intestinal epithelium and mimics colonization observed in mammals. Zebrafish that are colonized by V. cholerae transmit the bacteria to naive fish, which then become colonized. Striking differences in colonization between V. cholerae classical and El Tor biotypes were apparent. The zebrafish natural habitat in Asia heavily overlaps areas where cholera is endemic, suggesting that zebrafish and V. cholerae evolved in close contact with each other. Thus, the zebrafish provides a natural host model for the study of V. cholerae colonization, transmission, and environmental survival

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

Listeria exploits damage and death to spread bad news.

Pathogenic Listeria monocytogenes replicates within the host cytosol; little is known about how it transits from cell to cell, spreading infection. A recent study implicates infection-induced membrane damage as a trigger for efferocytosis, the recognition and uptake of dead cells, thereby tricking neighboring cells into taking up the invader

Genetic screening for bacterial mutants in liquid growth media by fluorescence-activated cell sorting.

Many bacterial pathogens have defined in vitro virulence inducing conditions in liquid media which lead to production of virulence factors important during an infection. Identifying mutants that no longer respond to virulence inducing conditions will increase our understanding of bacterial pathogenesis. However, traditional genetic screens require growth on solid media. Bacteria in a single colony are in every phase of the growth curve, which complicates the analysis and makes screens for growth phase-specific mutants problematic. Here, we utilize fluorescence-activated cell sorting in conjunction with random transposon mutagenesis to isolate bacteria grown in liquid media that are defective in virulence activation. This method permits analysis of an entire bacterial population in real time and selection of individual bacterial mutants with the desired gene expression profile at any time point after induction. We have used this method to identify Vibrio cholerae mutants defective in virulence induction

Mitochondria-Derived Vesicles Deliver Antimicrobial Reactive Oxygen Species to Control Phagosome-Localized Staphylococcus aureus.

Pathogenic bacteria taken up into the macrophage phagosome are the target of many anti-microbial mechanisms. Although mitochondria-derived antimicrobial effectors like reactive oxygen species (mROS) aid in bacterial killing, it is unclear how these effectors reach bacteria within the phagosomal lumen. We show here that endoplasmic reticulum stress triggered upon methicillin-resistant Staphylococcus aureus (MRSA) infection induces mROS that are delivered to bacteria-containing phagosomes via mitochondria-derived vesicles (MDVs). The endoplasmic reticulum stress sensor IRE1α induces mROS, specifically hydrogen peroxide (mH2O2), upon MRSA infection. MRSA infection also stimulates the generation of MDVs, which require the mitochondrial stress response factor Parkin, and contributes to mH2O2 accumulation in bacteria-containing phagosomes. Accumulation of phagosomal H2O2 requires Toll-like receptor signaling and the mitochondrial enzyme superoxide dismutase-2 (Sod2), which is delivered to phagosomes by MDVs. Sod2 depletion compromises mH2O2 production and bacterial killing. Thus, mitochondrial redox capacity enhances macrophage antimicrobial function by delivering mitochondria-derived effector molecules into bacteria-containing phagosomes

Bicarbonate Induces Vibrio cholerae virulence gene expression by enhancing ToxT activity.

Vibrio cholerae is a gram-negative bacterium that is the causative agent of cholera, a severe diarrheal illness. The two biotypes of V. cholerae O1 capable of causing cholera, classical and El Tor, require different in vitro growth conditions for induction of virulence gene expression. Growth under the inducing conditions or infection of a host initiates a complex regulatory cascade that results in production of ToxT, a regulatory protein that directly activates transcription of the genes encoding cholera toxin (CT), toxin-coregulated pilus (TCP), and other virulence genes. Previous studies have shown that sodium bicarbonate induces CT expression in the V. cholerae El Tor biotype. However, the mechanism for bicarbonate-mediated CT induction has not been defined. In this study, we demonstrate that bicarbonate stimulates virulence gene expression by enhancing ToxT activity. Both the classical and El Tor biotypes produce inactive ToxT protein when they are cultured statically in the absence of bicarbonate. Addition of bicarbonate to the culture medium does not affect ToxT production but causes a significant increase in CT and TCP expression in both biotypes. Ethoxyzolamide, a potent carbonic anhydrase inhibitor, inhibits bicarbonate-mediated virulence induction, suggesting that conversion of CO(2) into bicarbonate by carbonic anhydrase plays a role in virulence induction. Thus, bicarbonate is the first positive effector for ToxT activity to be identified. Given that bicarbonate is present at high concentration in the upper small intestine where V. cholerae colonizes, bicarbonate is likely an important chemical stimulus that V. cholerae senses and that induces virulence during the natural course of infection