Karen Skorupski

Parallel quorum sensing systems converge to regulate virulence in Vibrio cholerae.

The marine bacterium Vibrio harveyi possesses two quorum sensing systems (System 1 and System 2) that regulate bioluminescence. Although the Vibrio cholerae genome sequence reveals that a V. harveyi-like System 2 exists, it does not predict the existence of a V. harveyi-like System 1 or any obvious quorum sensing-controlled target genes. In this report we identify and characterize the genes encoding an additional V. cholerae autoinducer synthase and its cognate sensor. Analysis of double mutants indicates that a third as yet unidentified sensory circuit exists in V. cholerae. This quorum sensing apparatus is unusually complex, as it is composed of at least three parallel signaling channels. We show that in V. cholerae these communication systems converge to control virulence.

Positive selection vectors for allelic exchange

We describe here the development and use of two new allelic exchange vectors, pKAS32 and pKAS46. These vectors can be used for allelic exchange in a wide variety of bacterial species because their R6K origin of replication functions only in bacteria engineered to produce the replication protein pi. In addition, these vectors express the Escherichia coli rpsL gene, encoding ribosomal protein S12, which provides a positive selection for bacteria that have exchanged cloned plasmid sequences with the corresponding chromosomal sequences. In this report, we show that these vectors can be used to efficiently introduce point mutations and deletions into the chromosome of Vibrio cholerae.

Control of the ToxR virulence regulon in Vibrio cholerae by environmental stimuli.

Many bacterial pathogens regulate the expression of virulence genes in a co‐ordinate manner in response to changes in the environment. For example, the human pathogen, Vibrio cholerae, possesses a virulence regulon composed of over 20 genes involved in colonization, toxin production and bacterial survival within the host, which are co‐ordinately regulated by external stimuli, such as temperature, pH and osmolarity. Although the expression of the regulon is dependent upon the transcriptional activator ToxR, most of these genes are controlled by a second transcriptional activator, ToxT, which is itself positively regulated by ToxR. The mechanisms by which environmental stimuli influence the ToxR regulon are not yet understood, but ToxR‐mediated control over the expression of toxT clearly plays a role. The recent finding that the global regulator cAMP‐CRP also influences the expression of the ToxR regulon under various environmental conditions raises new issues regarding the pathways and mechanisms by which this regulation is achieved and indicates that multiple overlapping systems are involved.

Vibrio cholerae H-NS silences virulence gene expression at multiple steps in the ToxR regulatory cascade.

H-NS is an abundant nucleoid-associated protein involved in the maintenance of chromosomal architecture in bacteria. H-NS also has a role in silencing the expression of a variety of environmentally regulated genes during growth under nonpermissive conditions. In this study we demonstrate a role for H-NS in the negative modulation of expression of several genes within the ToxR virulence regulon of Vibrio cholerae. Deletion of hns resulted in high, nearly constitutive levels of expression of the genes encoding cholera toxin, toxin-coregulated pilus, and the ToxT virulence gene regulatory protein. For the cholera toxin- and ToxT-encoding genes, elevated expression in an hns mutant was found to occur in the absence of the cognate activator proteins, suggesting that H-NS functions directly at these promoters to decrease gene expression. Deletion analysis of the region upstream of toxT suggests that an extensive region located far upstream of the transcriptional start site is required for complete H-NS-mediated repression of gene expression. These data indicate that H-NS negatively influences multiple levels of gene expression within the V. cholerae virulence cascade and raise the possibility that the transcriptional activator proteins in the ToxR regulon function to counteract the repressive effects of H-NS at the various promoters as well as to recruit RNA polymerase.

A new level in the Vibrio cholerae ToxR virulence cascade: AphA is required for transcriptional activation of the tcpPH operon

The expression of the ToxR virulence regulon is dependent upon the regulatory proteins ToxR/ToxS, TcpP/TcpH and ToxT. We describe here a previously unidentified gene in Vibrio cholerae, aphA (activator of tcpP and tcpH expression), which is required for the transcription of the tcpPH operon. Under conditions normally optimal for virulence gene expression, an in frame aphA deletion decreased the expression of a cholera toxin promoter fusion (ctx–lacZ ) and prevented the production of the toxin co‐regulated pilus (TCP). Plasmids producing ToxT or TcpP/H, but not ToxR, restored ctx–lacZ expression and TCP production in the ΔaphA strain, suggesting that the mutation interferes with toxT expression by influencing the transcription of tcpPH. Indeed, the expression of a chromosomal tcpP–lacZ fusion was reduced in the ΔaphA mutant and increased in both V. cholerae and Escherichia coli by introducing aphA expressed from an inducible promoter. These results support a model in which AphA functions at a previously unknown step in the ToxR virulence cascade to activate the transcription of tcpPH. TcpP/TcpH, together with ToxR/ToxS, then activate the expression of toxTresulting ultimately in the production of virulence factors such as cholera toxin and TCP.

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.