- Research article
- Open Access
Virulence regulator AphB enhances toxR transcription in Vibrio cholerae
© Xu et al; licensee BioMed Central Ltd. 2010
- Received: 14 August 2009
- Accepted: 6 January 2010
- Published: 6 January 2010
Vibrio cholerae is the causative agent of cholera. Extensive studies reveal that complicated regulatory cascades regulate expression of virulence genes, the products of which are required for V. cholerae to colonize and cause disease. In this study, we investigated the expression of the key virulence regulator ToxR under different conditions.
We found that compared to that of wild type grown to stationary phase, the toxR expression was lower in an aphB mutant strain. AphB has been previously shown to be a key virulence regulator that is required to activate the expression of tcpP. When expressed constitutively, AphB is able to activate the toxR promoter. Furthermore, gel shift analysis indicates that AphB binds toxR promoter region directly. We also characterize the effect of AphB on the levels of the outer membrane porins OmpT and OmpU, which are known to be regulated by ToxR.
Our data indicate that V. cholerae possesses an additional regulatory loop that use AphB to activate the expression of two virulence regulators, ToxR and TcpP, which together control the expression of the master virulence regulator ToxT.
- Minimum Bactericidal Concentration
- Virulence Regulator
- Additional Regulatory Loop
- Porin Expression
- Promoterless lacZ Gene
The primary direct transcriptional activator of V. cholerae virulence genes, including ctxAB and tcpA, is ToxT, a member of the AraC family of proteins . The expression of ToxT is under the control of a complex regulatory pathway. The ToxR protein was identified as the first positive regulator of V. cholerae virulence genes . ToxR activity requires the presence of another protein, ToxS, which is also localized to the inner membrane, but is thought to reside predominantly in the periplasm, where ToxR and ToxS are hypothesized to interact. ToxS serves as a mediator of ToxR function, perhaps by influencing its stability and/or capacity to dimerize . To regulate expression of toxT, ToxR acts in conjunction with a second transcriptional activator, TcpP, which is also membrane-localized with a cytoplasmic DNA-binding and other periplasmic domains . TcpP, like ToxR, requires the presence of a membrane-bound effector protein, TcpH, which interacts with TcpP . Two activators encoded by unlinked genes, AphA and AphB, regulate the transcription of tcpPH. AphA is a dimer with an N-terminal winged-helix DNA binding domain that is structurally similar to those of MarR family transcriptional regulators . AphA cannot activate transcription of tcpP alone, but requires interaction with the LysR-type regulator AphB that binds downstream of the AphA binding site .
The ToxR and ToxS regulatory proteins have long been considered to be at the root of the V. cholerae virulence regulon, called the ToxR regulon. The membrane localization of ToxR suggests that it may directly sense and respond to environmental signals such as temperature, osmolarity, and pH . In addition to regulating the expression toxT, ToxR activates the transcription of ompU and represses the transcription of ompT, outer membrane porins important for V. cholerae virulence [13, 14]. Microarray analysis indicates that ToxR regulates additional genes, including a large number of genes involved in cellular transport, energy metabolism, motility, and iron uptake . It has been reported that levels of ToxR protein appear to remain constant under various in vitro conditions [16, 17] and are modulated by the heat shock response .
To further investigate the relationship between toxR expression and other virulence regulators, we analyzed toxR transcription and ToxR protein levels in various virulence regulator mutants. We found that in addition to activating tcpP, AphB was required for full expression of ToxR in V. cholerae stationary growth phase. AphB regulated toxR directly as purified recombinant AphB binds to the toxR promoter. This study suggests that V. cholerae may use this additional layer of activation to turn on virulence factor production efficiently in optimal conditions.
Examination of toxR expression under different in vitro conditions using a transcriptional fusion reporter
Influence of virulence regulatory proteins on toxR expression
AphB directly regulates toxR expression
The effects of AphB on ToxR-regulated genes
The ToxR regulon is the classic virulence gene regulation pathway in V. cholerae. In this pathway, AphA and AphB activate tcpP transcriptional expression directly by binding to different promoter regions of tcpP. ToxR and TcpP cooperate in turn by binding different sites of the toxT promoter to activate transcription, leading to the production of the virulence factors TCP and CT. However, the full ToxR regulon is more complex than previously thought. In this paper, we showed that AphA and AphB are also necessary for full ToxR production at the stationary phase. Furthermore, we demonstrated that AphB is sufficient for toxR transcriptional activation in the heterogenic host E. coli through binding of the toxR promoter region. Thus, the effect of AphB on ToxR levels propagates further in the transcription cascade, increasing the transcription of a key gene in V. cholerae pathogenesis, toxT. We have therefore identified another factor responsible for altering end product levels in the V. cholerae virulence axis. Since AphB is at the top of a virulence cascade with multiple end pathways, it appears now that AphB is a central factor in switching the cell from an environmental state to a virulent one. Since it activates ToxR in addition to TcpP, and further influences porin expression, AphB is a divergence point at which nonlinearity is introduced into the V. cholerae virulence pathway. Eukaryotic cells have extremely complex networks of protein and DNA interactions leading to precise control of protein expression levels. Having a more complex network of transcriptional activation and repression in the V. cholerae virulence cascade could enable the bacterial cell to fine-tune its expression levels to optimize its ability to colonize the intestine and spread to other hosts.
Bacterial strains, plasmids and media
All experiments were performed with El Tor Vibrio cholerae C6706  or Escherichia coli DH5α, which were grown in LB with relevant antibiotics at 37°C, except where noted. V. cholerae virulence genes were induced in vitro (the AKI condition) as previously described . Briefly, 3 ml of AKI medium was inoculated with 0.5 μl of overnight culture and incubated for 4 hrs at 37°C without agitation. 1 ml of culture was transferred to a fresh tube and incubated with shaking for a further 4 hrs at 37°C.
P toxR -luxCDABE fusion plasmid was constructed by polymerase chain reaction (PCR) amplifying the toxR promoter regions, ranging from 450 bp, 300 bp, to 130 bp, respectively, and cloning them into the pBBRlux vector . P toxT -luxCDABE plasmid was constructed by cloning toxT promoter regions into the pBBRlux vector. The chromosomal toxR-lacZ transcriptional fusion was constructed by cloning the 5' toxR region into the suicide vector pVIK112, which also contains a promoterless lacZ gene . The resulting plasmid was then integrated into the chromosomes of V. cholerae lacZ- strains by homologous recombination to create a single-copy toxR-lacZ and an intact copy of toxR. P BAD -controlled aphA and aphB plasmids were constructed by cloning aphA and aphB coding sequences into the pBAD24 vector . pBAD-tcpPH plasmid construct was described in . In-frame deletions of toxR, toxS, tcpP, tcpA, toxT, aphA, and aphB were either described previously  or constructed by cloning the regions flanking target genes into the suicide vector pWM91 containing a sacB counter-selectable marker . The resulting plasmids were introduced into V. cholerae by conjugation and deletion mutants were selected for double homologous recombination events.
Lux activity assays
Bacteria were grown at 37°C or 22°C under conditions indicated. At different time points, cultures were withdrawn and luminescence was measured by using a Bio-Tek Synergy HT spectrophotometer. Lux expression is calculated as light units/OD600.
Western blotting and SDS-PAGE electrophoresis
Whole-cell lysates were prepared from bacteria overnight cultures in LB conditions at 37°C and samples were normalized to the amount of total protein as assayed by the Biorad protein assay (Biorad). The isolation of outer membrane (OM) proteins from V. cholerae was performed using the method described by Miller and Mekalanos . Whole-cell lysates or OM preparations were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% polyacrylamide gel and stained with Coomassie brilliant blue for visualization. SDS-PAGE gels were transferred to nitrocellulose membrane for Western blot analysis using polyclonal rabbit anti-ToxR antibody.
Gel retardation assays
MBP-AphB protein was purified through amylose columns according to the manufacturer's instructions (New England Biolabs). PCR products of the different lengths of toxR promoter regions were digested with EcoRI and end-labeled using [α-32P]dATP and the Klenow fragment of DNA polymerase I. Binding reactions contained 0.1 ng of DNA and MBP-AphB proteins in a buffer consisting of 10 mM Tris-HCl (pH 7.9), 1 mM EDTA, 1 mM dithiothreitol, 60 mM KCl, and 30 mg of calf thymus DNA/ml. After 20 minutes of incubation at 25°C, samples were size-fractionated using 5% polyacrylamide gels in 1× TAE buffer (40 mM Tris-acetate, 2 mM EDTA; pH 8.5). The radioactivity of free DNA and AphB-DNA complexes was visualized by using a Typhoon 9410 PhosphorImager (Molecular Dynamics).
This study was supported by the NIH/NIAID R01 (AI072479) (to J.Z.), and a NSFC key project (30830008) (to B.K.).
- Miller VL, Taylor RK, Mekalanos JJ: Cholera toxin transcriptional activator toxR is a transmembrane DNA binding protein. Cell. 1987, 48 (2): 271-279. 10.1016/0092-8674(87)90430-2.View ArticlePubMedGoogle Scholar
- Herrington DA, Hall RH, Losonsky G, Mekalanos JJ, Taylor RK, Levine MM: Toxin, toxin-coregulated pili, and the toxR regulon are essential for Vibrio cholerae pathogenesis in humans. J Exp Med. 1988, 168 (4): 1487-1492. 10.1084/jem.168.4.1487.View ArticlePubMedGoogle Scholar
- Waldor MK, Mekalanos JJ: Lysogenic conversion by a filamentous phage encoding cholera toxin. Science. 1996, 272 (5270): 1910-1914. 10.1126/science.272.5270.1910.View ArticlePubMedGoogle Scholar
- Kovach ME, Elzer PH, Hill DS, Robertson GT, Farris MA, Roop RM, Peterson KM: Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene. 1995, 166 (1): 175-176. 10.1016/0378-1119(95)00584-1.View ArticlePubMedGoogle Scholar
- DiRita VJ: Co-ordinate expression of virulence genes by ToxR in Vibrio cholerae. Molecular microbiology. 1992, 6 (4): 451-458. 10.1111/j.1365-2958.1992.tb01489.x.View ArticlePubMedGoogle Scholar
- DiRita VJ, Mekalanos JJ: Periplasmic interaction between two membrane regulatory proteins, ToxR and ToxS, results in signal transduction and transcriptional activation. Cell. 1991, 64 (1): 29-37. 10.1016/0092-8674(91)90206-E.View ArticlePubMedGoogle Scholar
- Skorupski K, Taylor RK: Control of the ToxR virulence regulon in Vibrio cholerae by environmental stimuli. Mol Microbiol. 1997, 25 (6): 1003-1009. 10.1046/j.1365-2958.1997.5481909.x.View ArticlePubMedGoogle Scholar
- Hase CC, Mekalanos JJ: TcpP protein is a positive regulator of virulence gene expression in Vibrio cholerae. Proc Natl Acad Sci USA. 1998, 95 (2): 730-734. 10.1073/pnas.95.2.730.PubMed CentralView ArticlePubMedGoogle Scholar
- Beck NA, Krukonis ES, DiRita VJ: TcpH influences virulence gene expression in Vibrio cholerae by inhibiting degradation of the transcription activator TcpP. J Bacteriol. 2004, 186 (24): 8309-8316. 10.1128/JB.186.24.8309-8316.2004.PubMed CentralView ArticlePubMedGoogle Scholar
- De Silva RS, Kovacikova G, Lin W, Taylor RK, Skorupski K, Kull FJ: Crystal structure of the virulence gene activator AphA from Vibrio cholerae reveals it is a novel member of the winged helix transcription factor superfamily. J Biol Chem. 2005, 280 (14): 13779-13783. 10.1074/jbc.M413781200.PubMed CentralView ArticlePubMedGoogle Scholar
- Kovacikova G, Lin W, Skorupski K: Vibrio cholerae AphA uses a novel mechanism for virulence gene activation that involves interaction with the LysR-type regulator AphB at the tcpPH promoter. Mol Microbiol. 2004, 53 (1): 129-142. 10.1111/j.1365-2958.2004.04121.x.View ArticlePubMedGoogle Scholar
- Wong SM, Carroll PA, Rahme LG, Ausubel FM, Calderwood SB: Modulation of expression of the ToxR regulon in Vibrio cholerae by a member of the two-component family of response regulators. Infect Immun. 1998, 66 (12): 5854-5861.PubMed CentralPubMedGoogle Scholar
- Li CC, Crawford JA, DiRita VJ, Kaper JB: Molecular cloning and transcriptional regulation of ompT, a ToxR-repressed gene in Vibrio cholerae. Mol Microbiol. 2000, 35 (1): 189-203. 10.1046/j.1365-2958.2000.01699.x.View ArticlePubMedGoogle Scholar
- Sperandio V, Bailey C, Giron JA, DiRita VJ, Silveira WD, Vettore AL, Kaper JB: Cloning and characterization of the gene encoding the OmpU outer membrane protein of Vibrio cholerae. Infect Immun. 1996, 64 (12): 5406-5409.PubMed CentralPubMedGoogle Scholar
- Bina J, Zhu J, Dziejman M, Faruque S, Calderwood S, Mekalanos J: ToxR regulon of Vibrio cholerae and its expression in vibrios shed by cholera patients. Proc Natl Acad Sci USA. 2003, 100 (5): 2801-2806. 10.1073/pnas.2628026100.PubMed CentralView ArticlePubMedGoogle Scholar
- DiRita VJ, Neely M, Taylor RK, Bruss PM: Differential expression of the ToxR regulon in classical and E1 Tor biotypes of Vibrio cholerae is due to biotype-specific control over toxT expression. Proc Natl Acad Sci USA. 1996, 93 (15): 7991-7995. 10.1073/pnas.93.15.7991.PubMed CentralView ArticlePubMedGoogle Scholar
- Reidl J, Klose KE: Vibrio cholerae and cholera: out of the water and into the host. FEMS microbiology reviews. 2002, 26 (2): 125-139. 10.1111/j.1574-6976.2002.tb00605.x.View ArticlePubMedGoogle Scholar
- Parsot C, Mekalanos JJ: Expression of ToxR, the transcriptional activator of the virulence factors in Vibrio cholerae, is modulated by the heat shock response. Proc Natl Acad Sci USA. 1990, 87 (24): 9898-9902. 10.1073/pnas.87.24.9898.PubMed CentralView ArticlePubMedGoogle Scholar
- Kovacikova G, Skorupski K: A Vibrio cholerae LysR homolog, AphB, cooperates with AphA at the tcpPH promoter to activate expression of the ToxR virulence cascade. J Bacteriol. 1999, 181 (14): 4250-4256.PubMed CentralPubMedGoogle Scholar
- Hammer BK, Bassler BL: Regulatory small RNAs circumvent the conventional quorum sensing pathway in pandemic Vibrio cholerae. Proc Natl Acad Sci USA. 2007, 104 (27): 11145-11149. 10.1073/pnas.0703860104.PubMed CentralView ArticlePubMedGoogle Scholar
- Lee SH, Hava DL, Waldor MK, Camilli A: Regulation and temporal expression patterns of Vibrio cholerae virulence genes during infection. Cell. 1999, 99 (6): 625-634. 10.1016/S0092-8674(00)81551-2.View ArticlePubMedGoogle Scholar
- Iwanaga M, Yamamoto K, Higa N, Ichinose Y, Nakasone N, Tanabe M: Culture conditions for stimulating cholera toxin production by Vibrio cholerae O1 El Tor. Microbiol Immunol. 1986, 30 (11): 1075-1083.View ArticlePubMedGoogle Scholar
- Kovacikova G, Lin W, Skorupski K: 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. J Bacteriol. 2003, 185 (16): 4825-4836. 10.1128/JB.185.16.4825-4836.2003.PubMed CentralView ArticlePubMedGoogle Scholar
- Kovacikova G, Lin W, Skorupski K: 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. Mol Microbiol. 2005, 57 (2): 420-433. 10.1111/j.1365-2958.2005.04700.x.View ArticlePubMedGoogle Scholar
- Kovacikova G, Skorupski K: Binding site requirements of the virulence gene regulator AphB: differential affinities for the Vibrio cholerae classical and El Tor tcpPH promoters. Mol Microbiol. 2002, 44 (2): 533-547. 10.1046/j.1365-2958.2002.02914.x.View ArticlePubMedGoogle Scholar
- Provenzano D, Lauriano CM, Klose KE: Characterization of the role of the ToxR-modulated outer membrane porins OmpU and OmpT in Vibrio cholerae virulence. J Bacteriol. 2001, 183 (12): 3652-3662. 10.1128/JB.183.12.3652-3662.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- Provenzano D, Klose KE: Altered expression of the ToxR-regulated porins OmpU and OmpT diminishes Vibrio cholerae bile resistance, virulence factor expression, and intestinal colonization. Proc Natl Acad Sci USA. 2000, 97 (18): 10220-10224. 10.1073/pnas.170219997.PubMed CentralView ArticlePubMedGoogle Scholar
- Provenzano D, Schuhmacher DA, Barker JL, Klose KE: The virulence regulatory protein ToxR mediates enhanced bile resistance in Vibrio cholerae and other pathogenic Vibrio species. Infect Immun. 2000, 68 (3): 1491-1497. 10.1128/IAI.68.3.1491-1497.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- Krukonis ES, Yu RR, Dirita VJ: The Vibrio cholerae ToxR/TcpP/ToxT virulence cascade: distinct roles for two membrane-localized transcriptional activators on a single promoter. Mol Microbiol. 2000, 38 (1): 67-84. 10.1046/j.1365-2958.2000.02111.x.View ArticlePubMedGoogle Scholar
- Dziejman M, Balon E, Boyd D, Fraser CM, Heidelberg JF, Mekalanos JJ: Comparative genomic analysis of Vibrio cholerae: genes that correlate with cholera endemic and pandemic disease. Proc Natl Acad Sci USA. 2002, 99 (3): 1556-1561. 10.1073/pnas.042667999.PubMed CentralView ArticlePubMedGoogle Scholar
- Kalogeraki VS, Winans SC: Suicide plasmids containing promoterless reporter genes can simultaneously disrupt and create fusions to target genes of diverse bacteria. Gene. 1997, 188 (1): 69-75. 10.1016/S0378-1119(96)00778-0.View ArticlePubMedGoogle Scholar
- Guzman LM, Belin D, Carson MJ, Beckwith J: Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol. 1995, 177 (14): 4121-4130.PubMed CentralPubMedGoogle Scholar
- Metcalf WW, Jiang W, Daniels LL, Kim SK, Haldimann A, Wanner BL: Conditionally replicative and conjugative plasmids carrying lacZ alpha for cloning, mutagenesis, and allele replacement in bacteria. Plasmid. 1996, 35 (1): 1-13. 10.1006/plas.1996.0001.View ArticlePubMedGoogle Scholar
- Miller VL, Mekalanos JJ: A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J Bacteriol. 1988, 170 (6): 2575-2583.PubMed CentralPubMedGoogle Scholar
- Miller JH: Experiments in Molecular Genetics. 1972, Cold Spring Harbor, Cold Spring Harbor Laboratory PressGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.