Anna D. Tischler

Cyclic diguanylate (c-di-GMP) regulates Vibrio cholerae biofilm formation.

While studying virulence gene regulation in Vibrio cholerae during infection of the host small intestine, we identified VieA as a two‐component response regulator that contributes to activating expression of cholera toxin. Here we report that VieA represses transcription of Vibrio exopolysaccharide synthesis (vps) genes involved in biofilm formation by a mechanism independent of its phosphorelay and DNA‐binding activities. VieA controls the intracellular concentration of the cyclic nucleotide second messenger cyclic diguanylate (c‐di‐GMP) using an EAL domain that functions as a c‐di‐GMP phosphodiesterase. Two‐dimensional thin layer chromatography of nucleotide extracts confirmed that VieA reduces the concentration of c‐di‐GMP, opposing the action of  c‐di‐GMP synthetase proteins. Expression of unrelated V. cholerae c‐di‐GMP synthetase or phosphodiesterae proteins also modulated c‐di‐GMP concentration and vps gene expression. We propose that c‐di‐GMP synthetase and phosphodiesterase domain‐containing proteins contribute to regulating biofilm formation by controlling c‐di‐GMP concentration.

Cyclic Diguanylate Regulates Vibrio cholerae Virulence Gene Expression

ABSTRACT The cyclic dinucleotide second messenger cyclic diguanylate (c-diGMP) has been implicated in regulation of cell surface properties in several bacterial species, including Vibrio cholerae. Expression of genes required for V. cholerae biofilm formation is activated by an increased intracellular c-diGMP concentration. The response regulator VieA, which contains a domain responsible for degradation of c-diGMP, is required to maintain a low concentration of c-diGMP and repress biofilm formation. The VieSAB three-component signal transduction system was, however, originally identified as a regulator of ctxAB, the genes encoding cholera toxin (CT). Here we show that the c-diGMP phosphodiesterase activity of VieA is required to enhance CT production. This regulation occurred at the transcriptional level, and ectopically altering the c-diGMP concentration by expression of diguanylate cyclase or phosphodiesterase enzymes also affected ctxAB transcription. The c-diGMP phosphodiesterase activity of VieA was also required for maximal transcription toxT but did not influence the activity of ToxR or expression of TcpP. Finally, a single amino acid substitution in VieA that increases the intracellular c-diGMP concentration led to attenuation in the infant mouse model of cholera. Since virulence genes including toxT and ctxA are repressed by a high concentration of c-diGMP, while biofilm genes are activated, we suggest that c-diGMP signaling is important for the transition of V. cholerae from the environment to the host.

PilZ domain proteins bind cyclic diguanylate and regulate diverse processes in Vibrio cholerae

Cyclic diguanylate (c-di-GMP) is an allosteric activator and second messenger implicated in the regulation of a variety of biological processes in diverse bacteria. In Vibrio cholerae, c-di-GMP has been shown to inversely regulate biofilm-specific and virulence gene expression, suggesting that c-di-GMP signaling is important for the transition of V. cholerae from the environment to the host. However, the mechanism behind this regulation remains unknown. Recently, it was proposed that the PilZ protein domain represents a c-di-GMP-binding domain. Here we show that V. cholerae PilZ proteins bind c-di-GMP specifically and are involved in the regulation of biofilm formation, motility, and virulence. These findings confirm a role for PilZ proteins as c-di-GMP-sensing proteins within the c-di-GMP signaling network.

The Vibrio cholerae vieSAB Locus Encodes a Pathway Contributing to Cholera Toxin Production

The genes encoding cholera toxin (CT), ctxAB, are coregulated with those for other Vibrio cholerae virulence factors by a cascade of transcriptional activators, including ToxR, TcpP, and ToxT. Additional regulators that modulate expression of ctxAB during infection were recently identified in a genetic selection. A transposon insertion in vieS, the sensor kinase of the VieSAB three-component signal transduction system, resulted in failure to induce expression of a ctxA-recombinase fusion during murine infection. To determine which components of the VieSAB system are essential for CT regulation, ctxAB transcript levels were assessed by RNase protection assay in various vieSAB in-frame deletion mutants after growth in vitro under virulence gene inducing conditions. A threefold reduction in ctxAB transcript levels was observed for the (Delta)vieSAB strain; consistent with this, the (Delta)vieSAB strain produced twofold less CT protein than the wild type, and this defect was complementable in trans. These results suggest that the VieSAB three-component system is required for full activation of the ctxAB operon during in vitro growth as well as during infection. The VieSAB system may regulate ctxAB expression indirectly by affecting production of ToxT, because decreased toxT transcript levels were observed in the (Delta)vieSAB strain.

Differences in Gene Expression between the Classical and El Tor Biotypes of Vibrio cholerae O1

ABSTRACT Differences in whole-genome expression patterns between the classical and El Tor biotypes of Vibrio cholerae O1 were determined under conditions that induce virulence gene expression in the classical biotype. A total of 524 genes (13.5% of the genome) were found to be differentially expressed in the two biotypes. The expression of genes encoding proteins required for biofilm formation, chemotaxis, and transport of amino acids, peptides, and iron was higher in the El Tor biotype. These gene expression differences may contribute to the enhanced survival capacity of the El Tor biotype in environmental reservoirs. The expression of genes encoding virulence factors was higher in the classical than in the El Tor biotype. In addition, the vieSAB genes, which were originally identified as regulators of ctxA transcription, were expressed at a fivefold higher level in the classical biotype. We determined the VieA regulon in both biotypes by transcriptome comparison of wild-type and vieA deletion mutant strains. VieA predominantly regulates gene expression in the classical biotype; 401 genes (10.3% of the genome), including those encoding proteins required for virulence, exopolysaccharide biosynthesis, and flagellum production as well as those regulated by σE, are differentially expressed in the classical vieA deletion mutant. In contrast, only five genes were regulated by VieA in the El Tor biotype. A large fraction (20.8%) of the genes that are differentially expressed in the classical versus the El Tor biotype are controlled by VieA in the classical biotype. Thus, VieA is a major regulator of genes in the classical biotype under virulence gene-inducing conditions.

Vibrio cholerae Requires rpoS for Efficient Intestinal Colonization

ABSTRACT Vibrio cholerae is a facultative intestinal pathogen that lives in aquatic environments, often in association with planktonic species. In the suckling mouse, oral inoculation withV. cholerae leads to intestinal colonization and symptoms of diarrheal disease. Results reported here indicate a role for the alternative sigma factor, RpoS, in intestinal colonization in this model of cholera. We constructed within rpoS multiple independent mutations which consistently resulted in a fivefold decrease in colonization ability as assessed by competition assays. These mutations had no detectable effect on the in vitro growth ofV. cholerae in a rich medium. The occurrence of spontaneous suppressor mutations potentially required for viability ofrpoS strains was ruled out by determination of the frequency of insertional inactivation of rpoS in comparison to two other nonessential loci. Finally, both the in vitro and in vivo mutant phenotypes of rpoS strains were fully complemented by providing rpoS in trans or by allelic reversion, indicating that the observed decrease in colonization fitness was indeed due to the loss of functional RpoS.

Spontaneous phthiocerol dimycocerosate-deficient variants of Mycobacterium tuberculosis are susceptible to gamma interferon-mediated immunity

ABSTRACT Onset of the adaptive immune response in mice infected with Mycobacterium tuberculosis is accompanied by slowing of bacterial replication and establishment of a chronic infection. Stabilization of bacterial numbers during the chronic phase of infection is dependent on the activity of the gamma interferon (IFN-γ)-inducible nitric oxide synthase (NOS2). Previously, we described a differential signature-tagged mutagenesis screen designed to identify M. tuberculosis “counterimmune” mechanisms and reported the isolation of three mutants in the H37Rv strain background containing transposon insertions in the rv0072, rv0405, and rv2958c genes. These mutants were impaired for replication and virulence in NOS2−/− mice but were growth-proficient and virulent in IFN-γ−/− mice, suggesting that the disrupted genes were required for bacterial resistance to an IFN-γ-dependent immune mechanism other than NOS2. Here, we report that the attenuation of these strains is attributable to an underlying transposon-independent deficiency in biosynthesis of phthiocerol dimycocerosate (PDIM), a cell wall lipid that is required for full virulence in mice. We performed whole-genome resequencing of a PDIM-deficient clone and identified a spontaneous point mutation in the putative polyketide synthase PpsD that results in a G44C amino acid substitution. We demonstrate by complementation with the wild-type ppsD gene and reversion of the ppsD gene to the wild-type sequence that the ppsD(G44C) point mutation is responsible for PDIM deficiency, virulence attenuation in NOS2−/− and wild-type C57BL/6 mice, and a growth advantage in vitro in liquid culture. We conclude that PDIM biosynthesis is required for M. tuberculosis resistance to an IFN-γ-mediated immune response that is independent of NOS2.

The Vibrio cholerae Hybrid Sensor Kinase VieS Contributes to Motility and Biofilm Regulation by Altering the Cyclic Diguanylate Level

Phosphorelay systems are important mediators of signal transduction during bacterial adaptation to new environments. Previously we described the vieSAB operon, encoding a putative three-protein component phosphorelay involved in regulating Vibrio cholerae virulence gene expression. At least part of the regulatory activity of VieSAB is exerted through the cyclic diguanylate (c-di-GMP)-degrading activity of the putative response regulator VieA. So far no direct evidence that VieSAB encodes a phosphorelay system exists. In addition, the role VieS plays in modulating VieA activity remains unclear. To address these questions, we expressed and purified VieA and a soluble cytoplasmic portion of VieS and used them in autophosphorylation and phosphotransfer assays. These assays showed that VieS has kinase activity in vitro and is able to selectively phosphorylate VieA. A phenotypic comparison revealed that deletion of vieS results in increased biofilm production comparable to that seen for deletion of vieA, whereas motility was decreased only slightly in the DeltavieS mutant compared to the profound defect observed in a DeltavieA mutant. We also found that the DeltavieS strain has a lower level of vieA transcript and, similar to a DeltavieA mutant, an increased intracellular level of c-di-GMP. Further analysis using site-directed vieA mutants showed that some of the phenotypes observed were due to the phosphorylation status of VieA. The evidence presented in this report is the first to link VieS and VieA biochemically and genetically, lending support to the hypothesis that these proteins function together in a signaling system.

Mycobacterium tuberculosis Requires Phosphate-Responsive Gene Regulation To Resist Host Immunity

ABSTRACT Mycobacterium tuberculosis persists in the tissues of mammalian hosts despite inducing a robust immune response dominated by the macrophage-activating cytokine gamma interferon (IFN-γ). We identified the M. tuberculosis phosphate-specific transport (Pst) system component PstA1 as a factor required to resist IFN-γ-dependent immunity. A ΔpstA1 mutant was fully virulent in IFN-γ−/− mice but attenuated in wild-type (WT) mice and mice lacking specific IFN-γ-inducible immune mechanisms: nitric oxide synthase (NOS2), phagosome-associated p47 GTPase (Irgm1), or phagocyte oxidase (phox). These phenotypes suggest that ΔpstA1 bacteria are sensitized to an IFN-γ-dependent immune mechanism(s) other than NOS2, Irgm1, or phox. In other species, the Pst system has a secondary role as a negative regulator of phosphate starvation-responsive gene expression through an interaction with a two-component signal transduction system. In M. tuberculosis, we found that ΔpstA1 bacteria exhibited dysregulated gene expression during growth in phosphate-rich medium that was mediated by the two-component sensor kinase/response regulator system SenX3-RegX3. Remarkably, deletion of the regX3 gene suppressed the replication and virulence defects of ΔpstA1 bacteria in NOS2−/− mice, suggesting that M. tuberculosis requires the Pst system to negatively regulate activity of RegX3 in response to available phosphate in vivo. We therefore speculate that inorganic phosphate is readily available during replication in the lung and is an important signal controlling M. tuberculosis gene expression via the Pst-SenX3-RegX3 signal transduction system. Inability to sense this environmental signal, due to Pst deficiency, results in dysregulation of gene expression and sensitization of the bacteria to the host immune response.

Contrasting persistence strategies in Salmonella and Mycobacterium.

Long-term survival of persistent bacterial pathogens in mammalian hosts critically depends on their ability to avoid elimination by innate and adaptive immune responses. The persistent human pathogens that cause typhoid fever and tuberculosis exemplify alternative strategies for survival in the host: immune evasion and immune adaptation, respectively. Salmonella enterica serotype Typhi evades host innate immune responses and inflammation by expressing factors that interfere with its detection as a Gram-negative bacterium, enabling persistent colonization of an immunologically privileged niche, the gallbladder. In contrast, Mycobacterium tuberculosis has adapted to survive within phagocytic cells, which typically eliminate invading microbes, by deploying stress resistance mechanisms that counteract the harsh environment of the phagolysosome.