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.
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.
Results and Discussion
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.).
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