A proteome-wide screen to identify transcription factors interacting with the Vibrio cholerae rpoS promoter.

We describe a proteomic approach to identify transcription factors binding to a target promoter. The method's usefulness was tested by identifying proteins binding to the Vibrio cholerae rpoS promoter in response to cell density. Proteins identified in this screen included the nucleoid-associated protein Fis and the quorum sensing regulator HapR.

Chromatin Immunoprecipitation.

Chromatin immunoprecipitation (ChIP) measures the physical association between a protein and DNA in the cell. In combination with next-generation sequencing, the technique enables the identification of DNA targets for the corresponding protein across an entire genome. Here we describe the immunoprecipitation of Vibrio cholerae DNA bound to the histone-like nucleoid structuring protein (H-NS) tagged with the Flag epitope. The quality of the DNA obtained in this protocol is suitable for next-generation sequencing. The procedure described herein can be readily adapted to other bacteria and DNA-binding proteins.

Deletion of gene encoding the nucleoid-associated protein H-NS unmasks hidden regulatory connections in El Tor biotype Vibrio cholerae.

Hypervirulent atypical El Tor biotype Vibrio cholerae O1 isolates harbour mutations in the DNA-binding domain of the nucleoid-associated protein H-NS and the receiver domain of the response regulator VieA. Here, we provide two examples in which inactivation of H-NS in El Tor biotype vibrios unmasks hidden regulatory connections. First, deletion of the helix-turn-helix domain of VieA in an hns mutant background diminished biofilm formation and exopolysaccharide gene expression, a function that phenotypically opposes its phosphodiesterase activity. Second, deletion of vieA in an hns mutant diminished the expression of σE, a virulence determinant that mediates the envelope stress response. hns mutants were highly sensitive to envelope stressors compared to wild-type. However, deletion of vieA in the hns mutant restored or exceeded wild-type resistance. These findings suggest an evolutionary path for the emergence of hypervirulent strains starting from nucleotide sequence diversification affecting the interaction of H-NS with DNA.

Molecular basis for the differential expression of the global regulator VieA in Vibrio cholerae biotypes directed by H-NS, LeuO and quorum sensing.

VieA is a cyclic diguanylate phosphodiesterase that modulates biofilm development and motility in Vibrio cholerae O1 of the classical biotype. vieA is part of an operon encoding the VieSAB signal transduction pathway that is nearly silent in V. cholerae of the El Tor biotype. A DNA pull-down assay for proteins interacting with the vieSAB promoter identified the LysR-type regulator LeuO. We show that in classical biotype V. cholerae, LeuO cooperates with the nucleoid-associated protein H-NS to repress vieSAB transcription. LeuO and H-NS interacted with the vieSAB promoter of both biotypes with similar affinities and protected overlapping DNA sequences. H-NS was expressed at similar levels in both cholera biotypes. In contrast, El Tor biotype strains expressed negligible LeuO under identical conditions. In El Tor biotype vibrios, transcription of vieSAB is repressed by the quorum sensing regulator HapR, which is absent in classical biotype strains. Restoring HapR expression in classical biotype V. cholerae repressed vieSAB transcription by binding to its promoter. We propose that double locking of the vieSAB promoter by H-NS and HapR in the El Tor biotype prior to the cessation of exponential growth results in a more pronounced decline in VieA specific activity compared to the classical biotype.

Flagellar motility, extracellular proteases and Vibrio cholerae detachment from abiotic and biotic surfaces.

Vibrio cholerae of serogroups O1 and O139, the causative agent of Asiatic cholera, continues to be a major global health threat. This pathogen utilizes substratum-specific pili to attach to distinct surfaces in the aquatic environment and the human small intestine and detaches when conditions become unfavorable. Both attachment and detachment are critical to bacterial environmental survival, pathogenesis and disease transmission. However, the factors that promote detachment are less understood. In this study, we examine the role of flagellar motility and hemagglutinin/protease (HapA) in vibrio detachment from a non-degradable abiotic surface and from the suckling mouse intestine. Flagellar motility facilitated V. cholerae detachment from abiotic surfaces. HapA had no effect on the stability of biofilms formed on abiotic surfaces despite representing >50% of the proteolytic activity present in the extracellular matrix. We developed a balanced lethal plasmid system to increase the bacterial cyclic diguanylate (c-di-GMP) pool late in infection, a condition that represses motility and HapA expression. Increasing the c-di-GMP pool enhanced V. cholerae colonization of the suckling mouse intestine. The c-di-GMP effect was fully abolished in hapA isogenic mutants. These results suggest that motility facilitates detachment in a substratum-independent manner. Instead, HapA appears to function as a substratum-specific detachment factor.

3-Amino 1,8-naphthalimide, a structural analog of the anti-cholera drug virstatin inhibits chemically-biased swimming and swarming motility in vibrios.

A screen for inhibitors of Vibrio cholerae motility identified the compound 3-amino 1,8-naphthalimide (3-A18NI), a structural analog of the cholera drug virstatin. Similar to virstatin, 3-A18NI diminished cholera toxin production. In contrast, 3-A18NI impeded swimming and/or swarming motility of V. cholerae and V. parahemolyticus suggesting that it could target the chemotaxis pathway shared by the polar and lateral flagellar system of vibrios. 3-A18NI did not inhibit the expression of V. cholerae major flagellin FlaA or the assembly of its polar flagellum. Finally, 3-A18NI enhanced V. cholerae colonization mimicking the phenotype of chemotaxis mutants that exhibit counterclockwise-biased flagellum rotation.

H-NS: an overarching regulator of the Vibrio cholerae life cycle.

Vibrio cholerae has become a model organism for studies connecting virulence, pathogen evolution and infectious disease ecology. The coordinate expression of motility, virulence and biofilm enhances its pathogenicity, environmental fitness and fecal-oral transmission. The histone-like nucleoid structuring protein negatively regulates gene expression at multiple phases of the V. cholerae life cycle. Here we discuss: (i) the regulatory and structural implications of H-NS chromatin-binding in the two-chromosome cholera bacterium; (ii) the factors that counteract H-NS repression; and (iii) a model for the regulation of the V. cholerae life cycle that integrates H-NS repression, cyclic diguanylic acid signaling and the general stress response.

Vibrio cholerae hemagglutinin(HA)/protease: An extracellular metalloprotease with multiple pathogenic activities.

Vibrio cholerae of serogroup O1 and O139, the etiological agent of the diarrheal disease cholera, expresses the extracellular Zn-dependent metalloprotease hemagglutinin (HA)/protease also reported as vibriolysin. This enzyme is also produced by non-O1/O139 (non-cholera) strains that cause mild, sporadic illness (i.e. gastroenteritis, wound or ear infections). Orthologs of HA/protease are present in other members of the Vibrionaceae family pathogenic to humans and fish. HA/protease belongs to the M4 neutral peptidase family and displays significant amino acid sequence homology to Pseudomonas aeruginosa elastase (LasB) and Bacillus thermoproteolyticus thermolysin. It exhibits a broad range of potentially pathogenic activities in cell culture and animal models. These activities range from the covalent modification of other toxins, the degradation of the protective mucus barrier and disruption of intestinal tight junctions. Here we review (i) the structure and regulation of HA/protease expression, (ii) its interaction with other toxins and the intestinal mucosa and (iii) discuss the possible role(s) of HA/protease in the pathogenesis of cholera.

Vibrio cholerae Biofilms and Cholera Pathogenesis.

Vibrio cholerae can switch between motile and biofilm lifestyles. The last decades have been marked by a remarkable increase in our knowledge of the structure, regulation, and function of biofilms formed under laboratory conditions. Evidence has grown suggesting that V. cholerae can form biofilm-like aggregates during infection that could play a critical role in pathogenesis and disease transmission. However, the structure and regulation of biofilms formed during infection, as well as their role in intestinal colonization and virulence, remains poorly understood. Here, we review (i) the evidence for biofilm formation during infection, (ii) the coordinate regulation of biofilm and virulence gene expression, and (iii) the host signals that favor V. cholerae transitions between alternative lifestyles during intestinal colonization, and (iv) we discuss a model for the role of V. cholerae biofilms in pathogenicity.

RNA-Seq analysis and whole genome DNA-binding profile of the Vibrio cholerae histone-like nucleoid structuring protein (H-NS).

The data described in this article pertain to the genome-wide transcription profiling of a Vibrio cholerae mutant lacking the histone-like nucleoid structuring protein (H-NS) and the mapping of the H-NS chromosome binding sites [1, 2]. H-NS is a nucleoid-associated protein with two interrelated functions: organization of the bacterial nucleoid and transcriptional silencing [3]. Both functions require DNA binding and protein oligomerization [4, 5]. H-NS commonly silences the expression of virulence factors acquired by lateral gene transfer [6]. The highly pleiotropic nature of hns mutants in V. cholerae indicates that H-NS impacts a broad range of cellular processes such as virulence, stress response, surface attachment, biofilm development, motility and chemotaxis. We used a V. cholerae strain harboring a deletion of hns and a strain expressing H-NS tagged at the C-terminus with the FLAG epitope to generate datasets representing the hns transcriptome and DNA binding profile under laboratory conditions (LB medium, 37°C). The datasets are publicly available at the Gene Expression Omnibus (GEO) repository (http://www.ncbi.nlm.nih.gov/geo/) with accession numbers GSE62785 and GSE64249.

Repression by H-NS of genes required for the biosynthesis of the Vibrio cholerae biofilm matrix is modulated by the second messenger cyclic diguanylic acid.

Expression of Vibrio cholerae genes required for the biosynthesis of exopolysacchide (vps) and protein (rbm) components of the biofilm matrix is enhanced by cyclic diguanylate (c-di-GMP). In a previous study, we reported that the histone-like nucleoid structuring (H-NS) protein represses the transcription of vpsA, vpsL and vpsT. Here we demonstrate that the regulator VpsT can disrupt repressive H-NS nucleoprotein complexes at the vpsA and vpsL promoters in the presence of c-di-GMP, while H-NS could disrupt the VpsT-promoter complexes in the absence of c-di-GMP. Chromatin immunoprecipitation-Seq showed a remarkable trend for H-NS to cluster at loci involved in biofilm development such as the rbmABCDEF genes. We show that the antagonistic relationship between VpsT and H-NS regulates the expression of the rbmABCDEF cluster. Epistasis analysis demonstrated that VpsT functions as an antirepressor at the rbmA/F, vpsU and vpsA/L promoters. Deletion of vpsT increased H-NS occupancy at these promoters while increasing the c-di-GMP pool had the opposite effect and included the vpsT promoter. The negative effect of c-di-GMP on H-NS occupancy at the vpsT promoter required the regulator VpsR. These results demonstrate that c-di-GMP activates the transcription of genes required for the biosynthesis of the biofilm matrix by triggering a coordinated VpsR- and VpsT-dependent H-NS antirepression cascade.

Role of methylthioadenosine/S-adenosylhomocysteine nucleosidase in Vibrio cholerae cellular communication and biofilm development.

In Vibrio cholerae, the genes required for biofilm development are repressed by quorum sensing at high cell density due to the accumulation in the medium of two signaling molecules, cholera autoinducer 1 (CAI-1) and autoinducer 2 (AI-2). A significant fraction of toxigenic V. cholerae isolates, however, exhibit dysfunctional quorum sensing pathways. It was reported that transition state analogs of the enzyme methylthioadenosine/S-adenosylhomocysteine nucleosidase (MtnN) required to make AI-2 inhibited biofilm formation in the prototype quorum sensing-deficient strain N16961. This finding prompted us to examine the role of both autoinducers and MtnN in biofilm development and virulence gene expression in a quorum sensing-deficient genetic background. Here we show that deletion of mtnN encoding methylthioadenosine/S-adenosylhomocysteine nucleosidase, cqsA (CAI-1), and/or luxS (AI-2) do not prevent biofilm development. However, two independent mtnN mutants exhibited diminished growth rate and motility in swarm agar plates suggesting that, under certain conditions, MtnN could influence biofilm formation indirectly. Nevertheless, MtnN is not required for the development of a mature biofilm.

RNA-seq analysis identifies new genes regulated by the histone-like nucleoid structuring protein (H-NS) affecting Vibrio cholerae virulence, stress response and chemotaxis.

The histone-like nucleoid structuring protein (H-NS) functions as a transcriptional silencer by binding to AT-rich sequences at bacterial promoters. However, H-NS repression can be counteracted by other transcription factors in response to environmental changes. The identification of potential toxic factors, the expression of which is prevented by H-NS could facilitate the discovery of new regulatory proteins that may contribute to the emergence of new pathogenic variants by anti-silencing. Vibrio cholerae hns mutants of the El Tor biotype exhibit altered virulence, motility and environmental stress response phenotypes compared to wild type. We used an RNA-seq analysis approach to determine the basis of the above hns phenotypes and identify new targets of H-NS transcriptional silencing. H-NS affected the expression of 18% of all predicted genes in a growth phase-dependent manner. Loss of H-NS resulted in diminished expression of numerous genes encoding methyl-accepting chemotaxis proteins as well as chemotaxis toward the attractants glycine and serine. Deletion of hns also induced an endogenous envelope stress response resulting in elevated expression of rpoE encoding the extracytoplamic sigma factor E (σE). The RNA-seq analysis identified new genes directly repressed by H-NS that can affect virulence and biofilm development in the El Tor biotype cholera bacterium. We show that H-NS and the quorum sensing regulator HapR silence the transcription of the vieSAB three-component regulatory system in El Tor biotype V. cholerae. We also demonstrate that H-NS directly represses the transcription of hlyA (hemolysin), rtxCA (the repeat in toxin or RTX), rtxBDE (RTX transport) and the biosynthesis of indole. Of these genes, H-NS occupancy at the hlyA promoter was diminished by overexpression of the transcription activator HlyU. We discuss the role of H-NS transcriptional silencing in phenotypic differences exhibited by V. cholerae biotypes.

The LuxR-type regulator VpsT negatively controls the transcription of rpoS, encoding the general stress response regulator, in Vibrio cholerae biofilms.

Cholera is a waterborne diarrheal disease caused by Vibrio cholerae strains of serogroups O1 and O139. Expression of the general stress response regulator RpoS and formation of biofilm communities enhance the capacity of V. cholerae to persist in aquatic environments. The transition of V. cholerae between free-swimming (planktonic) and biofilm life-styles is regulated by the second messenger cyclic di-GMP (c-di-GMP). We previously reported that increasing the c-di-GMP pool by overexpression of a diguanylate cyclase diminished RpoS expression. Here we show that c-di-GMP repression of RpoS expression is eliminated by deletion of the genes vpsR and vpsT, encoding positive regulators of biofilm development. To determine the mechanism of this regulation, we constructed a strain expressing a vpsT-FLAG allele from native transcription and translation signals. Increasing the c-di-GMP pool induced vpsT-FLAG expression. The interaction between VpsT-FLAG and the rpoS promoter was demonstrated by chromatin immunoprecipitation. Furthermore, purified VpsT interacted with the rpoS promoter in a c-di-GMP-dependent manner. Primer extension analysis identified two rpoS transcription initiation sites located 43 bp (P1) and 63 bp (P2) upstream of the rpoS start codon. DNase I footprinting showed that the VpsT binding site at the rpoS promoter overlaps the primary P1 transcriptional start site. Deletion of vpsT significantly enhanced rpoS expression in V. cholerae biofilms that do not make HapR. This result suggests that VpsT and c-di-GMP contribute to the transcriptional silencing of rpoS in biofilms prior to cells entering the quorum-sensing mode.

A highly specific cell-based high-throughput screening assay for ligands of cyclic adenosine monophosphate receptor protein in gram-negative bacteria.

Quorum sensing is a cell-cell communication process in bacteria that involves the production, release, and subsequent detection of chemical signal molecules called autoinducers. In Vibrio cholerae, multiple input signals activate the expression of the quorum sensing regulator HapR, which acts to repress the expression of virulence factors. We have shown that CRP, the cyclic adenosine monophosphate (cAMP) receptor protein, enhances quorum sensing by activating the biosynthesis of cholera autoinducer 1, the major signaling molecule that contributes to the activation of HapR. Thus, proquorum sensing CRP agonists could inhibit virulence and lead to new drugs to treat severe cholera. In this study, we show that expression of the quorum sensing-regulated luxCDABE operon can be used as a robust readout for CRP activity. Further, we describe and validate a highly specific cell-based luminescence high-throughput screening assay for proquorum sensing CRP ligands. A pilot screen of 9,425 compounds yielded a hit rate of 0.02%, one hit being cAMP itself. The Z' value for this assay was 0.76 and its coefficient of variance 8% for the positive control compound. To our knowledge, this is the first cell-based assay for ligands of the highly conserved CRP protein of Gram-negative bacteria. The use of this assay to screen large chemical libraries could identify lead compounds to treat cholera, as well as small molecules to probe ligand-receptor interactions in the CRP molecule.

A quinazoline-2,4-diamino analog suppresses Vibrio cholerae flagellar motility by interacting with motor protein PomB and induces envelope stress.

Vibrio cholerae strains of serogroups O1 and O139, the causative agents of the diarrheal illness cholera, express a single polar flagellum powered by sodium motive force and require motility to colonize and spread along the small intestine. In a previous study, we described a high-throughput assay for screening for small molecules that selectively inhibit bacterial motility and identified a family of quinazoline-2,4-diamino analogs (Q24DAs) that (i) paralyzed the sodium-driven polar flagellum of Vibrios and (ii) diminished cholera toxin secreted by El Tor biotype V. cholerae. In this study, we provide evidence that a Q24DA paralyzes the polar flagellum by interacting with the motor protein PomB. Inhibition of motility with the Q24DA enhanced the transcription of the cholera toxin genes in both biotypes. We also show that the Q24DA interacts with outer membrane protein OmpU and other porins to induce envelope stress and expression of the extracellular RNA polymerase sigma factor σ(E). We suggest that Q24DA-induced envelope stress could affect the correct folding, assembly, and secretion of pentameric cholera toxin in El Tor biotype V. cholerae independently of its effect on motility.

The histone-like nucleoid structuring protein (H-NS) is a repressor of Vibrio cholerae exopolysaccharide biosynthesis (vps) genes.

The capacity of Vibrio cholerae to form biofilms has been shown to enhance its survival in the aquatic environment and play important roles in pathogenesis and disease transmission. In this study, we demonstrated that the histone-like nucleoid structuring protein is a repressor of exopolysaccharide (vps) biosynthesis genes and biofilm formation.

Interaction of the histone-like nucleoid structuring protein and the general stress response regulator RpoS at Vibrio cholerae promoters that regulate motility and hemagglutinin/protease expression.

The bacterium Vibrio cholerae colonizes the human small intestine and secretes cholera toxin (CT) to cause the rice-watery diarrhea characteristic of this illness. The ability of this pathogen to colonize the small bowel, express CT, and return to the aquatic environment is controlled by a complex network of regulatory proteins. Two global regulators that participate in this process are the histone-like nucleoid structuring protein (H-NS) and the general stress response regulator RpoS. In this study, we address the role of RpoS and H-NS in the coordinate regulation of motility and hemagglutinin (HA)/protease expression. In addition to initiating transcription of hapA encoding HA/protease, RpoS enhanced flrA and rpoN transcription to increase motility. In contrast, H-NS was found to bind to the flrA, rpoN, and hapA promoters and represses their expression. The strength of H-NS repression at the above-mentioned promoters was weaker for hapA, which exhibited the strongest RpoS dependency, suggesting that transcription initiation by RNA polymerase containing σ(S) could be more resistant to H-NS repression. Occupancy of the flrA and hapA promoters by H-NS was demonstrated by chromatin immunoprecipitation (ChIP). We show that the expression of RpoS in the stationary phase significantly diminished H-NS promoter occupancy. Furthermore, RpoS enhanced the transcription of integration host factor (IHF), which positively affected the expression of flrA and rpoN by diminishing the occupancy of H-NS at these promoters. Altogether, we propose a model for RpoS regulation of motility gene expression that involves (i) attenuation of H-NS repression by IHF and (ii) RpoS-dependent transcription initiation resistant to H-NS.

Interplay among cyclic diguanylate, HapR, and the general stress response regulator (RpoS) in the regulation of Vibrio cholerae hemagglutinin/protease.

Vibrio cholerae secretes the Zn-dependent metalloprotease hemagglutinin (HA)/protease (mucinase), which is encoded by hapA and displays a broad range of potential pathogenic activities. Expression of HA/protease has a stringent requirement for the quorum-sensing regulator HapR and the general stress response regulator RpoS. Here we report that the second messenger cyclic diguanylic acid (c-di-GMP) regulates the production of HA/protease in a negative manner. Overexpression of a diguanylate cyclase to increase the cellular c-di-GMP pool resulted in diminished expression of HA/protease and its positive regulator, HapR. The effect of c-di-GMP on HapR was independent of LuxO but was abolished by deletion of the c-di-GMP binding protein VpsT, the LuxR-type regulator VqmA, or a single-base mutation in the hapR promoter that prevents autorepression. Though expression of HapR had a positive effect on RpoS biosynthesis, direct manipulation of the c-di-GMP pool at a high cell density did not significantly impact RpoS expression in the wild-type genetic background. In contrast, increasing the c-di-GMP pool severely inhibited RpoS expression in a ΔhapR mutant that is locked in a regulatory state mimicking low cell density. Based on the above findings, we propose a model for the interplay between HapR, RpoS, and c-di-GMP in the regulation of HA/protease expression.

A high-throughput screening assay for inhibitors of bacterial motility identifies a novel inhibitor of the Na+-driven flagellar motor and virulence gene expression in Vibrio cholerae.

Numerous bacterial pathogens, particularly those that colonize fast-flow areas in the bladder and gastrointestinal tract, require motility to establish infection and spread beyond the initially colonized tissue. Vibrio cholerae strains of serogroups O1 and O139, the causative agents of the diarrheal illness cholera, express a single polar flagellum powered by sodium motive force and require motility to colonize and spread along the small intestine. Therefore, motility may be an attractive target for small molecules that can prevent and/or block the infective process. In this study, we describe a high-throughput screening (HTS) assay to identify small molecules that selectively inhibit bacterial motility. The HTS assay was used to screen an ∼8,000-compound structurally diverse chemical library for inhibitors of V. cholerae motility. The screen identified a group of quinazoline-2,4-diamino analogs that completely suppressed motility without affecting the growth rate in broth. A further study on the effects of one analog, designated Q24DA, showed that it induces a flagellated but nonmotile (Mot(-)) phenotype and is specific for the Na(+)-driven flagellar motor of pathogenic Vibrio species. A mutation conferring phenamil-resistant motility did not eliminate inhibition of motility by Q24DA. Q24DA diminished the expression of cholera toxin and toxin-coregulated pilus as well as biofilm formation and fluid secretion in the rabbit ileal loop model. Furthermore, treatment of V. cholerae with Q24DA impacted additional phenotypes linked to Na(+) bioenergetics, such as the function of the primary Na(+) pump, Nqr, and susceptibility to fluoroquinolones. The above results clearly show that the described HTS assay is capable of identifying small molecules that specifically block bacterial motility. New inhibitors such as Q24DA may be instrumental in probing the molecular architecture of the Na(+)-driven polar flagellar motor and in studying the role of motility in the expression of other virulence factors.