Gabriela Kovacikova

Overlapping binding sites for the virulence gene regulators AphA, AphB and cAMP-CRP at the Vibrio cholerae tcpPH promoter

The expression of the Vibrio cholerae virulence factors, toxin‐co‐regulated pilus (TCP) and cholera toxin (CT), are dependent on the ability of the LysR regulator AphB to co‐operate with a second protein, AphA, to activate the expression of the membrane‐bound transcription factors TcpP and TcpH. To gain insights into the mechanism by which AphA and AphB co‐operate to activate the expression of tcpPH, we have purified these two proteins to near homogeneity and show that they are each capable of interacting with the classical tcpPH promoter at distinct binding sites. As shown by tcpP–lacZ promoter deletion experiments, gel shift and DNase I footprinting, AphA binds to and activates from a region of the promoter between −101 and −71 from the start of transcription. AphB binds to and activates from a partially overlapping downstream site between −78 and −43, and these functions are dependent upon a region of partial dyad symmetry that resembles the well‐characterized LysR‐binding motif. A single basepair difference in this region of dyad symmetry has been shown previously to play a critical role in the expression of virulence genes between the two disease‐causing biotypes of V. cholerae, classical and El Tor. We also show here that the tcpPH promoter is negatively influenced by the global regulator cAMP‐CRP. Purified CRP binds to a near‐consensus sequence in the tcpPH promoter in a cAMP‐dependent manner and protects from DNase I digestion a region that is completely within the region protected by AphA and AphB. These findings raise the possibility that the negative effect of cAMP‐CRP on virulence gene expression is the result of its ability to influence AphA‐ and AphB‐dependent transcriptional activation of tcpPH under various conditions.

Dual regulation of genes involved in acetoin biosynthesis and motility/biofilm formation by the virulence activator AphA and the acetate‐responsive LysR‐type regulator AlsR in Vibrio cholerae

AphA is a quorum sensing‐regulated activator that initiates the virulence cascade in Vibrio cholerae by cooperating with the LysR‐type regulator AphB at the tcpPH promoter on the Vibrio pathogenicity island (VPI). To identify the ancestral chromosomal genes in V. cholerae regulated by AphA, we carried out a microarray analysis and show here that AphA influences the expression of 15 genes not associated with the VPI. One set of genes strongly repressed by AphA is involved in the biosynthesis of acetoin, a product synthesized by a variety of bacteria that plays a role in preventing intracellular acidification and which is essential for the viability of V. cholerae in the presence of glucose. Also present in this operon are two putative signal transduction proteins with EAL and GGDEF domains that oppositely influence motility and biofilm formation. Gel mobility shift assays show that AphA binds to a site upstream of the first gene in the acetoin operon. Transcriptional lacZ fusions indicate that at low cell density AphA represses the expression of the acetoin genes up to 15‐fold. Voges Proskauer tests confirm that deletion of AphA increases the production of acetoin under non‐inducing conditions and also that the LysR‐type regulator AlsR divergently transcribed from the operon is required for its production. This is the first report of a specific repressor protein involved in the transcriptional control of acetoin production as well as the co‐regulation of these genes with those that influence motility and biofilm formation. The results here provide a model for the dual regulation of these processes by acetate and quorum sensing through AlsR and AphA.

The Alternative Sigma Factor σE Plays an Important Role in Intestinal Survival and Virulence in Vibrio cholerae

ABSTRACT The alternative sigma factor σΕ (RpoE) is involved in the response to extracytoplasmic stress and plays a role in the virulence of a variety of different bacteria. To assess the role of σΕ in Vibrio cholerae pathogenesis, a ΔrpoE mutant was constructed and analyzed using the infant mouse model. The results here show that σΕ contributes significantly to the virulence of V. cholerae. The ΔrpoE mutant was highly attenuated with a 50% lethal dose more than 3 logs higher than that for the parental strain, and its ability to colonize the intestine was reduced approximately 30-fold. A time course of infection revealed that the number of CFU of the ΔrpoE mutant was approximately 1 log lower than that of the parental strain by 12 h postinoculation and decreased further by 24 h. The defect in virulence in the ΔrpoE mutant thus appears to be a diminished ability to survive within the intestinal environment. The results here also show that σΕ is not required for growth and survival of V. cholerae in vitro at high temperatures but is required under other stressful conditions, such as in the presence of 3% ethanol. As in Escherichia coli, the expression of rpoE in V. cholerae is dependent upon two promoters located upstream of the gene, P1 and P2. P1 appears to be σ70 dependent, whereas the downstream promoter, P2, is positively autoregulated by σΕ.

Vibrio cholerae AphA uses a novel mechanism for virulence gene activation that involves interaction with the LysR‐type regulator AphB at the tcpPH promoter

AphA is required for expression of the Vibrio cholerae virulence cascade and for its regulation by quorum sensing. In order to activate transcription, AphA functions together with a second protein, the LysR‐type regulator AphB, at the tcpPH promoter. As AphA is a member of a new and largely uncharacterized regulator family, random mutagenesis was used to gain insights into how this protein activates transcription. As shown here, 17 amino acid substitutions were identified in AphA that reduced expression of the tcpPH promoter and prevented the protein from binding DNA. The amino acids involved in DNA recognition inferred from a dominant‐negative analysis were located throughout the N‐terminal domain from amino acids 18 to 67. This region of AphA has a conserved domain architecture similar to that of MarR, a multiple antibiotic resistance repressor. The analogous positions of the dominant‐negative mutations in AphA and MarR confirm that the DNA‐binding domains of these proteins are similar and indicate that AphA is a new member of the winged helix family of transcription factors. We also show that AphB is capable of rescuing two of the DNA binding‐defective AphA mutants, suggesting that the proteins interact directly on the DNA. Disruption of this interaction by insertion of half a helical turn between the two binding sites prevented AphB from rescuing the mutants and prevented the expression of the virulence cascade in a wild‐type background. These results provide a novel mechanism for the initiation of virulence gene expression at tcpPH.

The LysR-Type Virulence Activator AphB Regulates the Expression of Genes in Vibrio cholerae in Response to Low pH and Anaerobiosis

AphB is a LysR-type activator that initiates the expression of the virulence cascade in Vibrio cholerae by cooperating with the quorum-sensing-regulated activator AphA at the tcpPH promoter on the Vibrio pathogenicity island (VPI). To identify the ancestral chromosomal genes in V. cholerae regulated by AphB, we carried out a microarray analysis and show here that AphB influences the expression of a number of genes that are not associated with the VPI. One gene strongly activated by AphB is cadC, which encodes the ToxR-like transcriptional activator responsible for activating the expression of lysine decarboxylase, which plays an important role in survival at low pH. Other genes activated by AphB encode a Na(+)/H(+) antiporter, a carbonic anhydrase, a member of the ClC family of chloride channels, and a member of the Gpr1/Fun34/YaaH family. AphB influences each of these genes directly by recognizing a conserved binding site within their promoters, as determined by gel mobility shift assays. Transcriptional lacZ fusions indicate that AphB activates the expression of these genes under aerobic conditions in response to low pH and also under anaerobic conditions at neutral pH. Further experiments show that the regulation of cadC by AphB in response to low pH and anaerobiosis is mirrored in the heterologous organism Escherichia coli, is independent of the global regulators Fnr and ArcAB, and depends upon the region of the promoter that contains the AphB binding site. These results raise the possibility that the activity of AphB is influenced by the pH and oxygen tension of the environment.

Integration Host Factor Positively Regulates Virulence Gene Expression in Vibrio cholerae

Virulence gene expression in Vibrio cholerae is dependent upon a complex transcriptional cascade that is influenced by both specific and global regulators in response to environmental stimuli. Here, we report that the global regulator integration host factor (IHF) positively affects virulence gene expression in V. cholerae. Inactivation of ihfA and ihfB, the genes encoding the IHF subunits, decreased the expression levels of the two main virulence factors tcpA and ctx and prevented toxin-coregulated pilus and cholera toxin production. IHF was found to directly bind to and bend the tcpA promoter region at an IHF consensus site centered at position -162 by using gel mobility shift assays and DNase I footprinting experiments. Deletion or mutation of the tcpA IHF consensus site resulted in the loss of IHF binding and additionally disrupted the binding of the repressor H-NS. DNase I footprinting revealed that H-NS protection overlaps with both the IHF and the ToxT binding sites at the tcpA promoter. In addition, disruption of ihfA in an hns or toxT mutant background had no effect on tcpA expression. These results suggest that IHF may function at the tcpA promoter to alleviate H-NS repression.

The Virulence Activator AphA Links Quorum Sensing to Pathogenesis and Physiology in Vibrio cholerae by Repressing the Expression of a Penicillin Amidase Gene on the Small Chromosome

Activation of the tcpPH promoter on the Vibrio pathogenicity island by AphA and AphB initiates the Vibrio cholerae virulence cascade and is regulated by quorum sensing through the repressive action of HapR on aphA expression. To further understand how the chromosomally encoded AphA protein activates tcpPH expression, site-directed mutagenesis was used to identify the base pairs critical for AphA binding and transcriptional activation. This analysis revealed a region of partial dyad symmetry, TATGCA-N6-TNCNNA, that is important for both of these activities. Searching the V. cholerae genome for this binding site permitted the identification of a second one upstream of a penicillin V amidase (PVA) gene on the small chromosome. AphA binds to and footprints this site, which overlaps the pva transcriptional start, consistent with its role as a repressor at this promoter. Since aphA expression is under quorum-sensing control, the response regulators LuxO and HapR also influence pva expression. Thus, pva is repressed at low cell density when AphA levels are high, and it is derepressed at high cell density when AphA levels are reduced. Penicillin amidases are thought to function as scavengers for phenylacetylated compounds in the nonparasitic environment. That AphA oppositely regulates the expression of pva from that of virulence, together with the observation that PVA does not play a role in virulence, suggests that these activities are coordinated to serve V. cholerae in different biological niches.

The quorum sensing regulator HapR downregulates the expression of the virulence gene transcription factor AphA in Vibrio cholerae by antagonizing Lrp- and VpsR-mediated activation

HapR is a quorum sensing‐regulated transcription factor that represses the virulence cascade in Vibrio cholerae by binding to a specific site centred at −71 in the aphA promoter, ultimately preventing activation of the tcpPH promoter on the Vibrio pathogenicity island. In an effort to elucidate the mechanism by which HapR represses aphA expression, we identified two transcriptional regulators, Lrp and VpsR, both of which activate the aphA promoter. Lrp, the leucine‐responsive regulatory protein, binds to a region between −136 and −123 in the promoter to initiate aphA expression. VpsR, the response regulator that controls biofilm formation, binds to a region between −123 and −73 to activate aphA expression. HapR represses aphA expression by antagonizing the functions of both of these activators. The HapR binding site at −71 lies downstream of the Lrp binding site and overlaps the VpsR binding site. HapR binding thus directly blocks access of VpsR to the promoter. A naturally occurring point mutation in the aphA promoter (G‐77T), which has previously been shown to prevent HapR binding, also prevents VpsR binding. In the absence of HapR, either Lrp or VpsR is capable of achieving nearly full expression of the aphA promoter, but when present together their effects are to some degree additive. The aphA promoter is also negatively autoregulated and an AphA binding site is centred at −20. The results here provide a model for the dual activation of the aphA promoter by Lrp and VpsR as well as its dual repression by HapR and AphA.

Crystal structure of the virulence gene activator AphA from Vibrio cholerae reveals it is a novel member of the winged helix transcription factor superfamily.

AphA is a member of a new and largely uncharacterized family of transcriptional activators that is required for initiating virulence gene expression in Vibrio cholerae, the causative agent of the frequently fatal epidemic diarrheal disease cholera. AphA activates transcription by an unusual mechanism that appears to involve a direct interaction with the LysR-type regulator AphB at the tcpPH promoter. As a first step toward understanding the molecular basis for tcpPH activation by AphA and AphB, we have determined the crystal structure of AphA to 2.2 Å resolution. AphA is a dimer with an N-terminal winged helix DNA binding domain that is architecturally similar to that of the MarR family of transcriptional regulators. Unlike this family, however, AphA has a unique C-terminal antiparallel coiled coil domain that serves as its primary dimerization interface. AphA monomers are highly unstable by themselves and form a linked topology, requiring the protein to partially unfold to form the dimer. The structure of AphA also provides insights into how it cooperates with AphB to activate transcription, most likely by forming a heterotetrameric complex at the tcpPH promoter.

Characterization of Vibrio cholerae O1 El Tor Biotype Variant Clinical Isolates from Bangladesh and Haiti, Including a Molecular Genetic Analysis of Virulence Genes

ABSTRACT Vibrio cholerae serogroup O1, the causative agent of the diarrheal disease cholera, is divided into two biotypes: classical and El Tor. Both biotypes produce the major virulence factors toxin-coregulated pilus (TCP) and cholera toxin (CT). Although possessing genotypic and phenotypic differences, El Tor biotype strains displaying classical biotype traits have been reported and subsequently were dubbed El Tor variants. Of particular interest are reports of El Tor variants that produce various levels of CT, including levels typical of classical biotype strains. Here, we report the characterization of 10 clinical isolates from the International Centre for Diarrhoeal Disease Research, Bangladesh, and a representative strain from the 2010 Haiti cholera outbreak. We observed that all 11 strains produced increased CT (2- to 10-fold) compared to that of wild-type El Tor strains under in vitro inducing conditions, but they possessed various TcpA and ToxT expression profiles. Particularly, El Tor variant MQ1795, which produced the highest level of CT and very high levels of TcpA and ToxT, demonstrated hypervirulence compared to the virulence of El Tor wild-type strains in the infant mouse cholera model. Additional genotypic and phenotypic tests were conducted to characterize the variants, including an assessment of biotype-distinguishing characteristics. Notably, the sequencing of ctxB in some El Tor variants revealed two copies of classical ctxB, one per chromosome, contrary to previous reports that located ctxAB only on the large chromosome of El Tor biotype strains.