Abstract
Vibrio cholerae is a motile bacterium responsible for the disease cholera, and motility has been hypothesized to be inversely regulated with virulence. We examined the transcription profiles of V. cholerae strains containing mutations in flagellar regulatory genes (rpoN, flrA, flrC, and fliA) by utilizing whole-genome microarrays. Results revealed that flagellar transcription is organized into a four-tiered hierarchy. Additionally, genes with proven or putative roles in virulence (e.g., ctx, tcp, hemolysin, and type VI secretion genes) were upregulated in flagellar regulatory mutants, which was confirmed by quantitative reverse transcription-PCR. Flagellar regulatory mutants exhibit increased hemolysis of human erythrocytes, which was due to increased transcription of the thermolabile hemolysin (tlh). The flagellar regulatory system positively regulates transcription of a diguanylate cyclase, CdgD, which in turn regulates transcription of a novel hemagglutinin (frhA) that mediates adherence to chitin and epithelial cells and enhances biofilm formation and intestinal colonization in infant mice. Our results demonstrate that the flagellar regulatory system modulates the expression of nonflagellar genes, with induction of an adhesin that facilitates colonization within the intestine and repression of virulence factors maximally induced following colonization. These results suggest that the flagellar regulatory hierarchy facilitates correct spatiotemporal expression patterns for optimal V. cholerae colonization and disease progression.
Vibrio cholerae causes the human diarrheal disease cholera. The bacteria are natural inhabitants of aquatic environments and are introduced into the human population through the ingestion of contaminated food or water. Within the human host, V. cholerae expresses virulence factors that facilitate colonization of the intestine (e.g., toxin-coregulated pilus [TCP]) and that stimulate dramatic fluid loss from host tissues (cholera toxin [CT]) (5, 61). A regulatory cascade consisting of a number of different proteins, including ToxR, TcpP, and ToxT, induces the coordinated expression of CT and TCP maximally within the intestine and under specific in vitro growth conditions (for a review, see reference 7).
V. cholerae is a highly motile organism by virtue of its single polar flagellum. Flagellar genes are transcribed in a four-tiered transcriptional hierarchy (51). The single class I gene product FlrA activates σ54-dependent transcription of class II genes, which encode components of the MS ring-switch-export apparatus as well as the two-component system FlrBC (31). Phosphorylated FlrC activates σ54-dependent transcription of class III genes, which encode the basal body-hook and the flagellin FlaA (10, 11). Finally, the antisigma factor FlgM is secreted through the basal body-hook to allow σ28-dependent transcription of class IV genes, which encode four additional flagellins and some of the motor components (9, 30).
Motility has been linked to the virulence of V. cholerae. Spontaneous nonmotile mutants are defective for fluid accumulation and adherence in the rabbit ileal loop model (53, 58) as well as defective for adherence to isolated rabbit brush borders (15). O1 El Tor biotype nonmotile mutant strains generally show colonization defects in the infant mouse model (35), while nonmotile O1 classical mutants can generally colonize similarly to the motile wild-type strain (16). Nonmotile mutants of live V. cholerae vaccine strains induce reduced disease symptoms, or reactogenicity, in human volunteers while still being able to colonize the intestine (12, 27). Nonmotile mutants have also been shown to express increased levels of virulence factors (CT and TCP), while “hypermotile” mutants (defined by greater swarm diameter in soft agar assays) produce less CT and TCP than wild-type V. cholerae (16). The authors proposed that virulence and motility are inversely regulated in V. cholerae, with motility being downregulated and virulence factor expression simultaneously upregulated when the organisms are colonized on the intestinal cell surface. Since the majority of these studies were performed with undefined motility mutants, the exact connection between flagellar synthesis and cholera pathogenesis has remained unclear.
Recently, Silva et al. provided evidence for this model when they found that transcription of toxT, ctxA, and tcpA is upregulated in a V. cholerae nonmotile (motY) strain (58). Further evidence for this model comes from Ghosh et al., who found that the histone-like nucleoid structuring protein H-NS stimulates motility by stimulating flrA expression while repressing ctxAB and tcpA transcription (18). Though a number of studies have implicated motility as being important for V. cholerae virulence, the contribution of the flagellar regulatory hierarchy to the virulence of V. cholerae has remained obscure. In the current study, we have characterized the transcriptome of flagellar regulatory mutants and found that many nonflagellar genes are also regulated by this hierarchy. Moreover, transcription of a large number of proven and putative virulence genes is upregulated in the flagellar regulatory mutants, revealing a previously unappreciated coordinate regulation of disparate factors that enhance V. cholerae pathogenesis. These studies facilitated the identification of a novel RTX-like hemagglutinin that is positively regulated by the flagellar regulatory hierarchy through a diguanylate cyclase (DGC) and that mediates binding to epithelial cells and enhances intestinal colonization.
MATERIALS AND METHODS
Medium.
Luria broth was used for both liquid growth medium and agar plates, supplemented with ampicillin (50 μg/ml), streptomycin (100 μg/ml), chloramphenicol (2 μg/ml) or kanamycin (35 μg/ml) when appropriate. Medium was supplemented with 2 mM glutamine for growth of rpoN strains. Motility plates consisted of LB with 0.3% agar. LB agar without NaCl was supplemented with 10% sucrose for counterselection when plasmids containing the sacB gene were utilized. LB agar with 2 mg/ml of streptomycin was used for counterselection when plasmids containing the rpsL gene were utilized. Medium was supplemented with 80 μg/ml dl-α,ɛ-diaminopimelic acid (Sigma) for the growth of the Escherichia coli dapA strain.
Plasmid construction.
A complete list of plasmids and oligonucleotide primers used in this study can be found in Table S2 in the supplemental material. Oligonucleotide primer pairs for the promoter regions of the frhA, frhC, cdgD, cdgE, fliK, VC1384, VC1252, and sdhC genes are listed in Table S2 in the supplemental material. Primers were designed to amplify a fragment of approximately 300 bp, based upon the genome sequence of V. cholerae N16961 (24), with restriction sites added to facilitate cloning. Promoter fragments were PCR amplified from 0395 genomic DNA, digested with appropriate restriction enzymes, and ligated into plasmid pRS551 (59), which had been digested similarly to form pKEK790 (fliKp), pKEK791 (cdgEp), pKEK797 (cdgDp), pKEK823 (VC1384p), pKEK837 (VC1252p), pKEK838 (frhCp), pKEK849 (frhAp), and pKEK1105 (sdhCp).
Splicing-by-overlap-extension (SOE) PCR (26) was used to generate the deletion mutations in tlh, cdgD, and frhC. Upstream and downstream gene fragments were PCR amplified in the first reaction by using primers 1 and 2 and primers 3 and 4, respectively, as listed in Table S2 in the supplemental material. The second PCR was performed with primers 1 and 4, with the two fragments generated in the first PCRs used as a template. The resulting SOE fragments were digested with the appropriate restriction enzymes and ligated into pKAS32 (60) to form pKEK844 (Δtlh), pKEK806 (ΔcdgD), and pKEK863 (ΔfrhC). Deletion/insertion mutations of the frhA, cdgE, VC1384, and cdgD genes were constructed by SOE PCR with three fragments: an upstream and a downstream fragment generated with primers 1 and 2 and primers 3 and 4, respectively, as described above, and a fragment containing the chloremphenicol resistance gene (cat). This technique has been described previously (38). The resulting SOE products were digested with the appropriate restriction enzymes and ligated into pKAS32 (60) to form pKEK928 (ΔfrhA::cat), pKEK959 (ΔcdgE::cat), pKEK929 (ΔVC1384::cat), and pKEK836 (ΔcdgD::cat). Complementation plasmids for cdgD and VC1384 were constructed by PCR amplification of these genes by utilizing the primers listed and then cloning into pBAD24 (20) and pWSK30 (67), respectively, to form pKEK822 (cdgD) and pKEK1233 (VC1384).
Bacterial strains.
E. coli strain DH5α (21) was used for cloning manipulations. E. coli strains SM10λpir (42) and WM3046 (dapA) (a gift from William Metcalf, University of Illinois) were used to transfer plasmids to V. cholerae by conjugation. The V. cholerae strains used in this study are listed in Table S2 in the supplemental material. All V. cholerae strains are isogenic with O1 classical strain O395, with the exception of those used in the supplemental biofilm assay, which are isogenic to O1 El Tor strain A1552, and those used in the adult mouse colonization model, which are isogenic to O1 El Tor strain P27459.
V. cholerae mutant strains were constructed by conjugating plasmids containing the gene deletion and/or insertion (described above) into V. cholerae strain KKV598 (O395 ΔlacZ), followed by counterselection on streptomycin as described previously (60). The gbpA V. cholerae strain was constructed using p-28VCA0811 (a kind gift from G. Schoolnik) (40). The correct construction of all strains was verified by PCR and sequencing. The ΔfrhA::cat and ΔcdgD::cat mutations were subsequently moved by CPT1ts transduction (23) into other O1 V. cholerae strains.
Whole-genome transcriptome profiling.
V. cholerae cultures of wild-type and isogenic rpoN, flrA, flrC, and fliA mutant strains were grown overnight in LB supplemented with 2 mM glutamine at 37°C with aeration. Cultures were diluted 1:50 in fresh medium and grown to an optical density at 600 nm (OD600) of 0.3 to 0.4. Two-milliliter aliquots of cells were harvested by centrifugation for 2 min at room temperature. The cell pellets were immediately resuspended in 1 ml of TRIzol reagent (Invitrogen) and stored at −80°C. Total cellular RNA was subsequently isolated in accordance with the manufacturer's instructions (Invitrogen) and as described previously (72). The microarrays used in this study are composed of 70-mer oligonucleotides representing the open reading frames present in the V. cholerae genome and were printed at the University of California, Santa Cruz. Whole-genome expression analysis was performed using a common reference RNA, which was isolated from wild-type cells grown in LB supplemented with 2 mM glutamine as described above. Total RNA from test and reference samples was used in cDNA synthesis and microarray hybridization, and scanning was performed as described previously (3). Normalized signal ratios were obtained with LOWESS print-tip normalization using the Bioconductor packages (17) in the R environment. Differentially regulated genes were determined using three biological and two technical replicates with the significance-analysis-of-microarrays program (66), using ≥1.5-fold differences in gene expression and a ≤3% false discovery rate as a cutoff value.
ß-Galactosidase assay.
V. cholerae strains were transformed with the promoter-lacZ fusion plasmids described above, grown in LB plus ampicillin to an OD600 of ∼0.2 to 0.4. Bacterial cells were permeabilized with chloroform and sodium dodecyl sulfate and assayed for ß-galactosidase activity in accordance with the method of Miller (41). All experiments were performed at least three separate times.
Quantitative reverse transcription-PCR.
Total RNA was isolated from V. cholerae strains by using Charge Switch total RNA (Invitrogen), and the isolated RNA was then treated with Turbo DNase (Ambion). Reverse transcription reactions were performed using a high-capacity cDNA reverse transcription kit (Applied Biosystems); reverse transcriptase-negative controls were included for every RNA sample. cDNA was amplified with the Applied Biosystems ABI 7500 real-time PCR system, using SYBR green PCR master mix (Applied Biosystems), and specific primers designed by Primer Express and listed in Table S2 in the supplemental material. The relative expression values (R) were calculated by using the formula R = 2 −(ΔCt target − Ct endogenous control), where Ct is the fractional threshold cycle. The constitutively expressed gyrA cDNA was used as the endogenous control (37).
Hemagglutination and hemolysis assays.
The hemagglutination and hemolysis assays were performed as described by Gardel and Mekalanos (16), with the following alterations. V. cholerae strains were grown to an OD600 of ∼0.6 to 0.8 at 37°C, and then bacterial cells were pelleted, washed twice, and resuspended in KRT buffer at a concentration of 1010 CFU/ml. Bacterial cells were serially diluted in a round-bottomed 96-well microtiter plate. Human type “O” red blood cells were harvested by centrifugation, washed twice, and resuspended in KRT buffer at a 2% concentration. Red blood cells (0.2 ml) were added to 0.1 ml bacteria, the plate was incubated at room temperature for 30 to 60 min, and the hemagglutination titer was recorded. The titer is the reciprocal of the greatest dilution at which hemagglutination occurred. The hemolysis assay was performed in exactly the same manner as the hemagglutination assay; the plates were incubated for 20 h, at which time the OD540 in the supernatants was measured.
HEp-2 cell binding assay.
The HEp-2 cell binding assay was performed as described previously (16), with minor changes. Briefly, HEp-2 cells were grown in a 24-well plate on glass coverslips in Dulbecco's modified Eagle's medium (Gibco) with 10%FBS. V. cholerae strains were grown to an OD600 of ∼0.2 to 0.4 at 37°C. Bacterial cells were then pelleted, washed twice, resuspended in phosphate-buffered saline (PBS), and added to a monolayer of HEp-2 cells at a multiplicity of infection of 50:1. After incubation for 45 min at 30°C, the medium was aspirated, and the wells were washed four times with PBS. The cells were then fixed by methanol for 5 min and stained with Giemsa (1:12.5) for 25 min. Cells were mounted on a glass slide in FluorSave (Calbiochem) and imaged with a Zeiss Axiovert 200 fluorescence microscope. Twenty HEp-2 cells were counted for each bacterial strain, and experiment was performed three separate times, with similar results.
Chitin bead binding assay.
V. cholerae strains were grown to an OD600 of ∼0.6 to 0.8 at 37°C. Bacterial cells were pelleted, washed twice, and resuspended in KRT buffer. A MagnaRack (Bio-Rad) was used for magnetic bead manipulations. Magnetic chitin beads (Bio-Rad) were washed three times with KRT buffer, and then 0.1 ml of chitin bead suspension was mixed with 0.1 ml of bacteria (109 CFU/ml). After incubation for 30 min at room temperature, supernatant was removed and beads were washed three times with KRT buffer. Bound bacterial cells were eluted by vortexing the chitin beads with glass beads (Sigma). Serial dilutions of the input bacteria and the adherent bacteria were plated to quantitate the input and output, respectively. The adhesion factor was determined by dividing the number of output bacteria by the number of input bacteria.
Biofilm assay.
The biofilm assays were performed as described previously (34), with the following modifications. V. cholerae strains were grown overnight at 37°C in LB and then normalized to identical densities based on OD600. Seven microliters of bacterial culture was inoculated into 0.75 ml of LB in 10 ml unscarred borosilicate glass tubes and then incubated statically at 37°C for 21 h. Tubes were rinsed with distilled water and incubated with 1 ml 0.1% crystal violet for 30 min and then rinsed with distilled water and air dried. The stained biofilm was dissolved in 1.5 ml of dimethyl sulfoxide by vortexing and then quantitated by measuring OD570.
Phenotype microarrays.
Phenotype microarrays were performed using PM1 and PM2 carbon source phenotype microarray 96-well plates (Biolog) in accordance with the manufacturer's protocol, with some modifications. V. cholerae strains were grown in LB at 37°C to an OD600 of ∼0.6 to 0.8. Cells were harvested by centrifugation and washed twice with IF-Oa (Biolog) and then resuspended in 20 ml of IF-Oa (105CFU/ml), redox dye (Biolog) was added at 1/10 of the recommended concentration (1 μl/ml), and then the mixture was inoculated into PM plates (0.1 ml/well) and incubated statically at 30°C. Samples were quantitated by measuring OD510.
Mouse intestinal colonization assays.
All animals were handled in strict accordance with good animal practice as defined by the relevant national and/or local animal welfare bodies, and all animal work was approved by the UTSA IACUC.
The competition assay for intestinal colonization in 5-day-old CD-1 suckling mice was performed as described previously (16). The inocula consisted of ∼105 CFU for both wild-type and mutant strains. The competitive indices were calculated by dividing the ratio of mutant to wild-type bacteria in the output by the ratio of mutant to wild-type bacteria in the input.
For adult mouse colonization studies, 5- to 6-week-old female C57BL/6 mice (Harlan, Indianapolis, IN) were anesthetized, and then 50 μl of 8.5% (wt/vol) NaHCO3 was administered intragastrically, followed immediately by treatment with 50 μl of bacterial suspension in PBS, using a 22-gauge feeding needle. At 24 or 48 h postinoculation, mice were euthanized, and small intestines were removed, weighed, and homogenized in PBS. The homogenates were serially diluted in PBS and plated on LB streptomycin agar plates. The detection limit was 100 CFU in the small intestine.
RESULTS
Transcriptome profiling of flagellar regulatory mutants.
To determine which V. cholerae genes are controlled by the flagellar transcription hierarchy, we compared the gene expression profile of the wild-type strain to those of the isogenic rpoN, flrA, flrC, and fliA mutant strains by using a V. cholerae whole-genome microarray. Total RNA was prepared from exponentially grown (OD600, 0.3 to 0.4) V. cholerae rpoN, flrA, flrC, and fliA mutant strains as well as the wild-type parent strain; growth was performed at 37°C in LB supplemented with 2 mM glutamine to overcome the known glutamine requirement of the rpoN strain (31). These RNAs were labeled with Cy3 or Cy5 and applied to a DNA glass-spotted microarray of the V. cholerae genome, and differentially expressed genes were identified by statistical analysis of fluorescence intensity values. Data were analyzed by the significance-analysis-of-microarrays program (66), utilizing a ≤3% false-positive discovery rate and ≥1.5-fold transcript abundance differences. This analysis revealed a surprising number of genes regulated by the flagellar hierarchy.
The alternate sigma factor σ54 is required for V. cholerae flagellar transcription (31), but on the basis of the presence of multiple σ54-dependent transcriptional activators in V. cholerae (32), it controls additional phenotypes as well, including nitrogen assimilation, dicarboxylic acid transport, and quorum sensing (31, 36). The rpoN mutant showed decreased expression of 228 genes and increased expression of 271 genes (Fig. 1; see also Table S1 in the supplemental material). Among the genes positively regulated by σ54 were several expected genes, such as those encoding >50 flagellar/chemotaxis components, glutamine synthetase, phage shock proteins, and dicarboxylic acid transport proteins, as well as genes encoding, e.g., formate dehydrogenase and purine biosynthesis components, along with 77 genes annotated as hypothetical. Among the genes expressed at higher levels in the rpoN mutant than in the wild-type strain were genes involved in biosynthesis of amino acids, cofactors, polysaccharides, the cell wall, fatty acids, protein degradation, and secretion and 116 genes annotated as hypothetical. Of particular note were a number of upregulated genes with proven or putative roles in pathogenesis, including the ctx and tcp genes (discussed below).
FIG. 1.
Genes showing reduced (A) or increased (B) transcription in V. cholerae flrA, flrC, and fliA mutant strains. Venn diagrams show the number of genes exhibiting transcriptional repression or induction for each respective mutant relative to the wild-type level. Numbers in the overlapping regions represent genes that were coregulated, and the pattern of class II, class III, and class IV flagellar genes is shown in panel A, while the virulence genes discussed in text are shown in panel B.
FlrA is the σ54-dependent activator of class II flagellar genes, and since it lies at the top of the flagellar hierarchy, it is considered the “master regulator” of flagellar gene transcription (51). The flrA mutant showed decreased expression of 152 genes and increased expression of 124 genes (Fig. 1; see also Table S1 in the supplemental material). Among the genes downregulated in the flrA mutant were 50 genes annotated as flagellar/chemotaxis genes, consistent with the known function of FlrA, and 47 genes annotated as hypothetical. FlrC is the σ54-dependent activator of class III flagellar genes, and its activity is regulated by phosphorylation, which presumably occurs at the class II-class III checkpoint (11). The flrC mutant showed decreased expression of 115 genes and increased expression of 92 genes (Fig. 1; see also Table S1 in the supplemental material). Among the genes downregulated in the flrC mutant were 50 genes annotated as flagellar/chemotaxis genes, consistent with the known function of FlrC, and 46 genes annotated as hypothetical. FliA (σ28) is the sigma factor responsible for transcription of class IV flagellar genes, and its activity is regulated by the antisigma factor FlgM, whose secretion presumably occurs at the class III-class IV checkpoint (9). The fliA mutant showed decreased expression of 140 genes and increased expression of 195 genes (Fig. 1; see also Table S1 in the supplemental material). Among the genes downregulated in the fliA mutant were 17 genes annotated as flagellar/chemotaxis genes, consistent with the known function of FliA, and 37 genes annotated as hypothetical. Overlap of expression patterns for both downregulated genes as well as upregulated genes in the flrA, flrC, and fliA mutants can be seen in Fig. 1; the complete list of differentially expressed genes in the four flagellar regulatory mutant strains (including rpoN) is provided in Table S1 in the supplemental material.
Refinement of the flagellar transcription hierarchy through transcriptome profiling.
Cluster analysis of flagellar gene expression in the rpoN, flrA, flrC, and fliA strains revealed that the V. cholerae flagellar transcription hierarchy is organized similarly to the four-tiered hierarchy previously postulated (51). The requirement of flagellar regulatory genes for class II, class III, and class IV flagellar genes is illustrated in Fig. 1A, and selected genes downregulated in the flagellar regulatory mutants are shown in Fig. 2A. For example, in region I, flgBCDEFGHIJ (VC2200 to VC2192), flgKL (VC2191 and VC2190), flaA (VC2189 and VC2188), flgOP (VC2207 and VC2206), and flgT (VC2208) have the RpoN-, FlrA-, and FlrC-dependent expression patterns predicted for class III flagellar genes; flgMN (VC2204 and VC2205) and flaC (VC2187) have the RpoN-, FlrA-, FlrC-, and FliA-dependent expression patterns predicted for class IV flagellar genes; and flgA (VC2203) expression is independent of the flagellar regulatory genes, as previously predicted (51). Previous analysis of the cheV-3 and cheR-2 (VC2202 and VC2201) genes in this region suggested that their expression was also independent of the flagellar regulatory genes, as determined by transcription analysis of the cheV-3 promoter (51). However, the current microarray expression data suggest a class IV expression pattern for cheV-3 and a class III pattern for cheR-2.
FIG. 2.
Cluster analysis of select genes with reduced or increased transcription in flagellar regulatory mutants. (A) Patterns of expression of select genes with reduced expression in flagellar regulatory mutants. The intensity of yellow or blue represents the extent of gene induction or repression, respectively. Shown is the relative expression level of each given gene in the (left to right) flrA, rpoN, flrC, and fliA mutants. The flagellar gene clusters I, II, III, and IV are as described previously (51), and the names/functions of individual genes are denoted. wt, wild type. (B) Cluster analysis of genes with increased expression in all flagellar regulatory mutants. The intensity of yellow represents the extent of gene induction. Shown is the relative expression level of each given gene in the (left to right) flrA, rpoN, flrC, and fliA mutants. The names/functions of individual genes are denoted. (C) Refined flagellar transcription hierarchy (described in the text).
For region II, flrBC (VC2136 and VC2135) and fliEFGHIJ (VC2134 to VC2129) have the RpoN- and FlrA-dependent expression patterns predicted for class II flagellar genes, while flaE (VC2144), flaD (VC2143), and flaB (VC2142) have the RpoN-, FlrA-, FlrC-, and FliA-dependent expression patterns predicted for class IV flagellar genes, and flrA (VC2137) expression was independent of the flagellar regulatory genes (it showed some dependence on RpoN), as predicted previously (51). This microarray analysis also revealed an expression pattern for fliKLMNOPQ (VC2128 to VC2122) predicted for class III flagellar genes (i.e., dependence on FlrC) rather than class II genes as had been predicted previously, suggesting the presence of an FlrC-dependent promoter upstream of fliK. The fliK promoter region was fused to lacZ and assayed for transcription in the flagellar regulatory mutants (Table 1), which confirmed the presence of a class III promoter (RpoN, FlrA, and FlrC dependent) upstream of fliK. Thus, fliKLMNOPQ should be considered class III rather than class II genes. The expression of fliR (VC2121) appeared independent of the flagellar regulatory genes, while the expression of flhB (VC2120) was similar to that of class III genes; further studies of the expression of these genes should reveal their correct placement within the flagellar transcription hierarchy. Finally, flaG-fliD-flaI-fliS (VC2141 to VC2138) were previously classified as class III genes on the basis of the presence of a σ54-dependent promoter upstream of flaG that is dependent on rpoN, flrA, and flrC, but not fliA, for transcription. However, the microarray analysis revealed FliA dependence for flaG expression and FlrC independence for fliD-flaI-fliS expression, suggesting that expression of this region is complicated and that these genes may not in fact be class III genes.
TABLE 1.
Transcription of promoter-lacZ fusions in V. cholerae flagellar regulatory mutant strains
| Promoter fused to lacZ | Mean activity ± SD (Miller units) |
||||
|---|---|---|---|---|---|
| Wild type | rpoN | flrA | flrC | fliA | |
| fliKp | 1,081 ± 2 | 190 ± 1 | 257 ± 2 | 310 ± 3 | 1,264 ± 6 |
| VC1384p | 11,861 ± 810 | 119 ± 2 | 704 ± 21 | 342 ± 20 | 10,051 ± 343 |
| cdgEp | 3,069 ± 92 | 714 ± 9 | 890 ± 5 | 894 ± 36 | 779 ± 3 |
| VC1252p | 15,030 ± 883 | 855 ± 12 | 490 ± 8 | 1,793 ± 122 | 533 ± 28 |
| sdhCp | 14,092 ± 10 | 886 ± 46 | 648 ± 6 | 1,697 ± 77 | 447 ± 7 |
In region III, the flhA operon (VC2069 to VC2058), which contains fliA, along with a number of important chemotaxis genes, has the RpoN- and FlrA-dependent expression patterns predicted for class II flagellar genes, as predicted previously (51). Region IV, which contains only the motAB operon (VC0892 and VC0893), exhibited the RpoN-, FlrA-, FlrC-, and FliA-dependent expression predicted for class IV genes. Microarray analysis of the motY gene (VC1008) revealed the RpoN-, FlrA-, and FlrC-dependent typical of a class III gene, while motX (VC2601) expression was dependent on RpoN, FlrA, FlrC, and FliA, as seen with other class IV genes. Our previous analyses of these genes had characterized motY as a class IV gene and motX as a class III gene on the basis of promoter expression studies. Because the microarray studies here contradicted these earlier studies, we reanalyzed the previous promoter constructs and discovered that the promoter construct annotated as motXp-lacZ actually contained motYp-lacZ and that the promoter construct annotated as motYp-lacZ actually contained motXp-lacZ. Due to this mistake, the motX and motY genes were incorrectly classified, and the current study correctly classifies motY as a class III gene and motX as a class IV gene.
Seven methyl-accepting chemoreceptor protein genes that displayed expression patterns similar to those of class IV genes (i.e., RpoN, FlrA, FlrC, and FliA dependent) were identified: VC1898, VC2439, VCA0176, VCA0658, VCA0773, VCA0923, and VCA0974. Also, an additional cheV gene (VCA0954) that displayed a class IV pattern of expression, similar to cheV-3 (VC2202) in region I, was identified; we suggest that this gene be annotated as cheV-4. Finally, VC1384, which is annotated as a hypothetical gene that has homology to OmpA and thus likely encodes an outer membrane protein, displayed a class III pattern of expression (i.e., dependent on RpoN, FlrA, and FlrC but not FliA). The VC1384 promoter was further analyzed by promoter-lacZ fusion assays with the V. cholerae flagellar regulatory mutants (Table 1), which confirmed this promoter to be a class III promoter. We constructed a ΔVC1384 V. cholerae mutant and analyzed this strain for motility (see Fig. S1 in the supplemental material). The ΔVC1384 strain demonstrated reduced motility compared to that of the wild-type strain, and expression of VC1384 from a plasmid in this strain restored wild-type motility. Moreover, expression of VC1384 from a plasmid in the wild-type strain led to a greater-than-wild-type level of motility, suggesting that this gene plays some role in motility, so we propose reclassifying this gene as a flagellar/motility gene.
We examined the expression patterns of several additional genes downregulated in the flagellar mutants by constructing promoter-lacZ fusions and measuring β-galactosidase activity in the V. cholerae flagellar regulatory mutants (Table 1). The promoter-lacZ results indicated a class IV pattern of expression (i.e., dependent on RpoN, FlrA, FlrC, and FliA) for VC1252, a homologue of competence/damage-inducible CinA protein (PF02464), which contains a probable molybdopterin binding domain (PF00994). Promoter-lacZ transcription measurements also indicated a class IV pattern of expression for the sdhC promoter, which drives transcription of the succinate dehydrogenase operon (sdhCDAB). These results suggest that the flagellar regulatory hierarchy controls some aspects of competence and the tricarboxylic acid cycle/electron transport chain. Promoter-lacZ fusion analysis also indicated that the flagellar hierarchy controls transcription of the frhA, frhC, cdgD, and cdgE promoters (discussed below).
The microarray data have allowed us to refine the flagellar transcription hierarchy of V. cholerae by categorizing individual genes to specific flagellar expression classes based on the expression profiles described above (Fig. 2C).
Flagellar regulatory mutants have increased transcription of diverse virulence genes.
As mentioned above, the rpoN, flrA, flrC, and fliA mutant strains showed increased transcription of a large number of genes, including a number of genes with proven or putative roles in pathogenesis (Fig. 1). Cluster analysis of selected upregulated genes is shown in Fig. 2B. Within the Vibrio pathogenicity island, tagD (VC0824), tcpQCRDSTEF (VC0830 to VC0837), and toxT (VC0838) were upregulated in the flagellar mutants. The tcpA gene, which encodes the TCP structural subunit, lies at the beginning of the tcp operon but differs sufficiently from the probe on the microarray such that no data were available for this gene; upregulated transcription of the tcpA gene in flagellar mutants was subsequently confirmed by quantitative PCR (see below). Within the CTX prophage, ctxA and ctxB (VC1456 and VC1457) were also upregulated in all flagellar mutants. Among the additional putative virulence genes that were upregulated in the flagellar mutants were vieA and vieB (VC1651 and VC1652), response regulators involved in modulation of cyclic di-GMP (c-di-GMP) levels and regulation of CT expression (64, 65); epsL (VC2725), required for CT secretion (1); hlyA and hlyB (VCA0219 and VCA0220), encoding the hemolysins that cause vacuolation and pore formation in many cell types (2); a gene encoding a thermolabile hemolysin (tlh; VCA0218) that has phospholipase activity (14); an additional hemolysin gene (hlx; VCA0594) (45); the gene encoding the chitin-binding protein GbpA (VCA0811), which mediates binding to epithelial cells as well as chitinous surfaces (28); a putative lipase gene (VC1418); and three genes within the type VI secretion system (T6SS) (VCA0107, VCA0108, and VCA0124) involved in translocating proteins associated with virulence across the gram-negative membranes (52).
Additional virulence genes were identified by microarray analysis as upregulated in some but not all of the flagellar mutants, including CT secretion genes (epsF in rpoN, flrA, and flrC; epsG in rpoN and flrA; epsM in flrC and fliA; and epsN in flrC), additional genes in the Vibrio pathogenicity island (orf3 in fliA and rpoN, tcpJ in rpoN and fliA, acfC in fliA, acfA in rpoN and fliA, and acfD in rpoN and flrC); additional genes in the T6SS cluster (VCA0109 in rpoN, fliA, and flrA; vasA in fliA and flrA; VCA0112 in rpoN, fliA, and flrA; VCA0113 in rpoN and flrC; and vasH in rpoN); and genes encoding the RTX toxin subunit RtxA (VC1451 in fliA), a hemagglutinin (VCA0446 in flrA and flrC), and VieS (VC1653 in rpoN, flrA, and flrC).
The results revealed a general trend of widespread upregulation of proven and putative virulence factors. The upregulation of several virulence factors in nonmotile V. cholerae had previously been noted by utilizing phenotypic assays (16), and the current study reveals that the extent of this upregulation occurs at the transcriptional level and includes many genes that have not previously been shown to be linked by common signaling pathways. To confirm the upregulation of selected virulence genes, we performed quantitative real-time PCR on RNA isolated from wild-type and fliA V. cholerae strains, quantitating the message for ctxA, tcpA, hlyA, tagD, gbpA, tlh, and the T6SS gene VCA0108 (Fig. 3A). The results for three biological replicates, with three technical replicates each, revealed increased transcription of all seven genes in the fliA mutant versus those in the wild-type strain, ranging from approximately twofold for tcpA to approximately eightfold for hlyA. These results confirmed those obtained with the microarray analysis and revealed the upregulated transcription of multiple virulence genes in flagellar regulatory mutants.
FIG. 3.
(A) Increased transcription of select virulence genes in the fliA V. cholerae strain. Strains O395 (WT) and KKV1113 (fliA) were grown in LB, and mRNA abundance for the specific genes noted was determined by quantitative reverse transcription-PCR (see Materials and Methods). Results shown represent three biological replicates consisting of three technical replicates for each strain. (B) Utilization of select carbon sources by the rpoN and fliA V. cholerae strains. Strains O395 (WT), KKV56 (rpoN), and KKV1113 (fliA) were assayed for growth on various carbon sources by Biolog PM1 and PM2 phenotype microarrays (see Materials and Methods). Results shown represent two separate experiments for each strain.
Phenotype microarrays indicate that several metabolic pathways are regulated by the flagellar hierarchy.
Phenotype microarrays (Biolog) assaying for growth on 190 different carbon sources were performed with the wild-type and rpoN and fliA mutant V. cholerae strains to determine if the metabolic capabilities of flagellar regulatory mutants differed from those of the wild-type strain. Although phenotype microarray results are often reported as +/−, we have reported them here by OD reading (OD510), which correlates with metabolic activity on the specific carbon sources. RpoN (σ54) is a global regulator that controls the expression of a large number of genes outside the flagellar hierarchy (Fig. 1; see also Table S1 in the supplemental material) (31), and it has been shown previously that a V. cholerae rpoN mutant is defective for growth on succinate (32). This is likely due to σ54-dependent activation of genes involved in dicarboxylic acid transport (54), and thus, it was not surprising that the rpoN mutant exhibited defects in growth on various forms of succinate, malate, and fumarate and that the fliA mutant grew similarly to the wild type (Fig. 3B). The rpoN mutant also grew better on propionate than either the wild type or the fliA mutant, indicating that this metabolic capability is regulated by σ54 but not associated with the flagellar hierarchy. In contrast, both the rpoN and the fliA mutants grew better than the wild type on l-glutamine, β-methyl-d-glucoside, l-histidine, gelatin, laminarin, and mannan as carbon sources (Fig. 3B), indicating that the flagellar regulatory hierarchy normally represses these metabolic capabilities.
Growth of flagellar regulatory mutants on gelatin agar revealed enhanced expression of a gelatinase activity in these mutants in comparison to that in the wild-type strain (see Fig. S2 in the supplemental material), which may explain the enhanced growth of these mutants with gelatin utilized as a carbon source. Phenotype microarrays also demonstrated that the rpoN and fliA mutants grew worse than the wild type with Tween 20 utilized as a carbon source. These results confirm that the flagellar regulatory hierarchy controls metabolic capabilities of the cell in addition to flagellar synthesis.
Increased hemolysis by flagellar regulatory mutants is due to increased expression of the thermolabile hemolysin.
It had been documented previously that spontaneous nonmotile V. cholerae mutants exhibit increased hemolysis of human type O erythrocytes (16), but the exact reason for this behavior has not been characterized. The rpoN, flrA, flrC, and fliA flagellar regulatory mutants also demonstrate increased hemolysis of human type O erythrocytes (11) (Fig. 4 and data not shown). We reasoned that transcription of the hemolysin responsible for this phenotype was likely upregulated in the flagellar regulatory mutants. Three genes annotated as hemolysins are upregulated in the flagellar mutants: VCA0218, VCA0219, and VCA0594 (Fig. 2B; see also Table S1 in the supplemental material). VCA0219 encodes the “El Tor” hemolysin HlyA, and the O395 strain used in these studies contains a deletion in hlyA that renders this strain HlyA− (19); thus, HlyA cannot be the flagellum-regulated hemolysin. VCA0594 encodes a hemolysin, Hlx, which was determined to lyse sheep but not human erythrocytes (45), suggesting that this was also not the flagellum-regulated hemolysin. VCA0218 encodes a thermolabile hemolysin, Tlh, which has phospholipase and lecithinase activity (14).
FIG. 4.
Thermolabile hemolysin is the flagellum-regulated hemolysin. V. cholerae strains O395 (WT), KKV1113 (fliA), KKV2077 (tlh), and KKV2078 (fliA tlh) were assayed for hemolytic activity with human type O erythrocytes as described in Materials and Methods.
We constructed a V. cholerae strain with a deletion in the tlh gene (Δtlh) (see Materials and Methods) and measured human O erythrocyte hemolysis by V. cholerae wild-type and fliA, tlh, and fliA tlh mutant strains (Fig. 4). The fliA mutant exhibited high levels of hemolysis in comparison to the wild-type strain, confirming that the hemolysin is negatively regulated by the flagellar regulatory hierarchy. Importantly, the hemolytic activity of the fliA strain is abrogated when tlh is inactivated, demonstrating that the flagellum-regulated hemolysin is the thermolabile hemolysin Tlh. Because the transcription of tlh is increased in the flagellar regulatory mutants (Fig. 3A), we can conclude that the flagellar regulatory hierarchy represses the transcription of tlh. In an infant mouse small intestine colonization assay (see Fig. 6), the tlh V. cholerae strain colonized only slightly worse than the wild-type parent (competitive index, 0.67; P = 0.029). This indicates that thermolabile hemolysin does not play a critical role in colonization of the intestine, at least in this particular animal model.
FIG. 6.
V. cholerae frhA and cdgD strains are defective for colonization of the infant mouse intestine. V. cholerae strains KKV2093 (frhA), KKV1966 (cdgD), and KKV2077 (tlh) were coinoculated with the wild-type strain O395 perorally into infant mice at a ratio of ∼1:1, intestinal homogenates were recovered at 24 h postinoculation, and numbers of CFU of wild-type and mutant strains were determined. The competitive index is given as the ratio of mutant/wild-type bacteria in the output divided by the ratio of mutant/wild-type bacteria in the input; each value shown is from an individual mouse.
The decrease in hemagglutination associated with flagellar regulatory mutants is due to decreased expression of a novel RTX-like hemagglutinin, flagellum-regulated hemagglutinin.
It had been documented previously that spontaneous nonmotile V. cholerae mutants also exhibit decreased hemagglutination of human type O erythrocytes (16), but the exact reason for this behavior has not been characterized. We have observed agglutinated erythrocytes under the microscope, and V. cholerae bacteria can be seen bound to the surfaces of the erythrocytes, indicating that an adhesin on the bacterial surface acts as the hemagglutinin (data not shown). We reasoned that transcription of the hemagglutinin responsible for this phenotype was likely downregulated in the flagellar regulatory mutants. A gene annotated as encoding an “agglutination protein,” VC1621, is downregulated in the flagellar mutants, as is a hypothetical gene, VC1620, immediately adjacent and divergently transcribed (Fig. 5A; see also Table S1 in the supplemental material). The VC1620 and VC1621 promoters were measured for transcription by fusing the VC1620-VC1621 intergenic region in both orientations into a lacZ transcriptional fusion vector and then assaying for beta-galactosidase activity in the flagellar regulatory mutants (Fig. 5B). Transcription of both the VC1620 and the VC1621 promoters is downregulated in the flagellar mutants, confirming that the flagellar regulatory hierarchy controls transcription of these genes.
FIG. 5.
The flagellum-regulated hemagglutinin FrhA mediates binding to erythrocytes, epithelial cells, and chitin and enhances biofilm formation. (A) V. cholerae frhA locus. (B) Flagellar regulatory proteins and CdgD positively regulate transcription of the frhA and frhC promoters. V. cholerae strains O395 (WT), KKV56 (rpoN), KKV98 (flrC), KKV1113 (fliA), and KKV1966 (cdgD) carrying plasmids pKEK849 (frhAp-lacZ) and pKEK838 (frhCp-lacZ) were measured for β-galactosidase activity. (C) The cdgD promoter requires the flagellar regulatory proteins for transcription. V. cholerae strains O395 (WT), KKV56 (rpoN), KKV59 (flrA), KKV98 (flrC), and KKV1113 (fliA) carrying plasmid pKEK797 (cdgDp-lacZ) were measured for β-galactosidase activity. (D) FrhA and CdgD enhance biofilm formation. (E) FrhA and CdgD are required for flagellum-regulated hemagglutination of human O erythrocytes. (F) FrhA and CdgD enhance adhesion to Hep-2 cells. (G) FrhA and CdgD enhance V. cholerae chitin binding. For panels D to G, biofilm formation, hemagglutination, Hep-2 binding, and chitin binding, respectively, for the V. cholerae strains O395 (WT), KKV1113 (fliA), KKV2092 (cdgD), and KKV2093 (frhA) carrying plasmid pBAD24 (“−”) or pKEK822 (“pcdgD”) were measured as described in Materials and Methods. All assays were performed in triplicate, and error bars are shown for panels B, C, D, F, and G; panel E shows representative results for three separate experiments. For panels B, C, F, and G, P was <0.001, and for panel D, P was <0.01 for comparison of the mutant to the wild type or for comparison of the mutant strain complemented with cdgDp to the parent mutant strain by Student's two-tailed t test, with the exception of the frhA strain complemented with cdgDp, which was not significantly different from the frhA strain without cdgDp.
VC1620 is predicted to encode a large, 2,251-amino-acid (aa) protein that contains an RTX-like repeat region at the C terminus with a signature type I secretion motif (Fig. 5A). Due to the functional properties of this protein (see below), we have renamed it the flagellum-regulated hemagglutinin A (frhA) protein. The region between aa 484 and 995 shows homology to cadherin domains (cd00031), including two identical 114-aa sequences from aa 768 to 881 and aa 882 to 995. This 114-aa sequence is repeated as many as six times in the homologous protein in other clinical O1/O139 V. cholerae strains (e.g., 2740-80, MAK757, and MO10) (see alignment in Fig. S3 in the supplemental material), suggesting an important function for this motif within the human host. The 114-aa sequence shares 36% identity and 46% similarity with human cadherin 18 and similar levels of identity/similarity with other eukaryotic cadherins (e.g., insect, fish, and rodent cadherins). Cadherins are normally glycoproteins involved in calcium-dependent cell-cell adhesion of eukaryotic cells; thus, a role for this protein in adhesion to eukaryotic cells might be anticipated. The cadherin domains are typically repeated multiple times within the protein, providing a “zipper” mechanism for strong interactions between cadherin proteins on adjoining cells (57). FrhA also contains a region at the C terminus (aa 1514 to 2251) that shares homology with the C-terminal portions of RTX toxins and related Ca2+-binding proteins (COG2931); this includes the type I secretion signal that directs the protein through this secretion pathway.
Type I secretion substrates require additional proteins for secretion (for a review, see reference 25). The best-studied type I secretion substrate, HlyA of E. coli, requires the inner membrane protein HlyB, the periplasm-spanning protein HlyD, and the outer membrane protein TolC for secretion across the inner and outer membranes (62). Type I secretion proteins are typically encoded by the same operon as the secretion substrate. FrhA is encoded by a single gene operon, but the divergently transcribed operon, carrying VC1621 and VC1622, is also regulated by the flagellar regulatory hierarchy (Table 1), as mentioned above. VC1621 is predicted to encode a type I secretion component that shares homology with TolC and facilitates movement of the substrate across the outer membrane. Because we have shown that VC1621 also contributes to flagellum-regulated hemagglutination (see below), we have renamed this gene frhC. VC1622 shares homology with OmpA family proteins (pfam00691) and is predicted to encode an outer membrane porin. Downstream of frhA lies an operon containing VC1618 and VC1619 (Fig. 5A). VC1618 is predicted to encode a major facilitator superfamily transmembrane protein that facilitates the movement of a substrate(s) across the cytoplasmic membrane; it shares some homology to HlyB (see Fig. S4 in the supplemental material). We suspect that this gene may represent the type I secretion component involved in translocating FrhA across the inner membrane but have not yet analyzed its transcription or its function. However, the E. coli cytoplasmic membrane component HlyB also contains a nucleotide binding domain at its C terminus with ATPase activity important for energizing the secretion process (56), but the homology between VC1618 and HlyB is weak in this region, and VC1618 lacks the Walker A and B motifs and C and D loops important for nucleotide binding and ATPase activity (see Fig. S4 in the supplemental material). Thus, VC1618 likely has no ATPase activity, and no other obvious candidates with homology to nucleotide binding proteins/ATPases could be found near frhA. VC1619 does not show obvious homology to any known type I secretion components but encodes a member of pfam07209, a family of proteins of unknown function.
We constructed V. cholerae strains containing mutations in frhA and frhC and analyzed these strains for their ability to agglutinate human type O erythrocytes. The frhA V. cholerae strain failed to agglutinate erythrocytes, similar to a fliA strain (Fig. 5E). Attempts to clone frhA into a plasmid for complementation purposes were unsuccessful, due to the large and repetitive nature of this gene; however, polar effects in this single gene operon are not anticipated, and we have reconstructed this strain several times with an identical resultant phenotype; thus, we are confident that frhA encodes the flagellum-regulated hemagglutinin. The frhC V. cholerae strain also failed to agglutinate erythrocytes, similar to a fliA strain (see Fig. S5 in the supplemental material), demonstrating the involvement of FrhC in flagellum-regulated hemagglutination as well. The frhA and frhC strains exhibited motility patterns similar to those of the wild-type strain in soft agar, suggesting that these genes are not involved in motility/chemotaxis (data not shown).
Flagellum-regulated hemagglutinin mediates binding to epithelial cells and chitin and increased biofilm formation.
Since FrhA mediates binding to and agglutination of erythrocytes, we wished to determine if FrhA could also mediate binding to epithelial cells. Gardel and Mekalanos (16) observed that spontaneous nonmotile V. cholerae bacteria were defective at binding to the human epithelial Hep-2 cell line, but the exact reason for this behavior has not been characterized. The wild-type V. cholerae strain binds to Hep-2 cells, but the frhA mutant strain shows a >10-fold defect in Hep-2 cell binding, similar to a fliA strain (Fig. 5F). The frhC mutant strain was also defective for binding to Hep-2 cells (data not shown), consistent with the idea that flagellum-regulated hemagglutinin mediates adhesion to human epithelial cells. To ensure that the involvement of FrhA in epithelial cell binding is not restricted to the parent V. cholerae strain used in these studies, we also introduced the frhA mutation into an O1 El Tor V. cholerae strain, P27459, and found that this frhA mutant also showed a greater-than-sevenfold defect for epithelial cell binding in comparison to its parent wild-type strain (see Fig. S6 in the supplemental material).
GlcNAc residues on the surfaces of epithelial cells and chitinous surfaces are bound by a V. cholerae chitin-binding protein, GbpA (28). We wondered whether FrhA could also mediate binding to chitin. Utilizing chitin beads, we found that the frhA mutant showed a greater-than-sixfold defect in binding to chitin in comparison to the wild-type strain, and a similar binding defect was seen in the fliA mutant (Fig. 5G). The previously characterized chitin-binding protein GbpA is encoded by VCA0811, which is upregulated in the flagellar regulatory mutants, including the fliA mutant (Fig. 2B and 3A; see also Table S1 in the supplemental material). The fliA mutant was defective for binding chitin despite upregulated GbpA expression, which suggested that FrhA plays a more important role in chitin binding in this assay. To confirm this, we constructed gbpA and gbpA fliA V. cholerae strains and measured these for chitin binding in the same assay (see Fig. S7 in the supplemental material). In our hands, the gbpA and gbpA fliA mutants did not behave differently from their respective isogenic wild-type and fliA parent strains in this assay, suggesting that, at least under the conditions tested, FrhA plays a more dominant role in chitin binding than GbpA.
Biofilm development by V. cholerae is a complex process involving multiple genes, and flagellar synthesis and motility contribute to the ability of V. cholerae to form biofilms (68, 72). Nonmotile V. cholerae strains are known to be defective during the early stages of biofilm formation involving attachment to abiotic surfaces (69). We determined whether FrhA contributes to V. cholerae biofilm formation by measuring biofilms formed at the air-liquid interface in tubes of liquid cultures. The parent wild-type strain, O395, is known to form only weak biofilms under these conditions, yet the frhA mutant showed a greater-than-twofold defect in biofilm formation in comparison to the wild-type strain, and this defect was similar to that seen in the fliA mutant (Fig. 5D). We also introduced the frhA mutation into an O1 El Tor V. cholerae strain, A1552, which is known to make robust biofilms (72), and in this background, the frhA mutant also showed an approximate threefold defect in biofilm formation in comparison to the A1552 parent strain (see Fig. S8 in the supplemental material). In both strain backgrounds, prolonged incubation led to similar levels of biofilm formation, suggesting that the defect was in the initial stages of biofilm formation. These results demonstrate that FrhA contributes to biofilm formation of V. cholerae.
The flagellar regulatory hierarchy controls expression of a cyclic diguanylate synthase, CdgD, which regulates transcription of flagellum-regulated hemagglutinin.
The second messenger c-di-GMP modulates complex behaviors of V. cholerae, and its presence positively regulates biofilm formation and negatively regulates virulence (37, 63, 64). A large number of proteins with DGC (also known as GGDEF) and c-di-GMP phosphodiesterase (also known as EAL) domains are present in V. cholerae; thus, the roles of specific c-di-GMP-modulatory proteins are often obscured by functional redundancy. The transcriptome profiling results (Fig. 2A) indicated that transcription of two DGC genes is downregulated in the flagellar regulatory mutants VCA0697 (cdgD) and VC1367 (cdgE). Transcription of both of these DGC genes has previously been observed to be downregulated in rugose variants of V. cholerae (37). In this study, Lim et al. found that a cdgD mutant exhibited enhanced motility and diminished early biofilm formation but that a cdgE mutant had no obvious phenotype.
We measured transcription of cdgDp-lacZ and cdgEp-lacZ fusions in the wild-type and rpoN, flrA, flrC, and fliA mutant strains (Fig. 5C and Table 1). These results confirmed that cdgD and cdgE transcription is downregulated in all the flagellar regulatory mutants. We constructed cdgD and cdgE mutant V. cholerae strains and found that, unlike the fliA mutant strain (Fig. 3A), the cdgD and cdgE strains were unaffected for transcription of ctx, tcp, gbpA, hlyA, tlh, or T6SS genes (data not shown). Consistent with the unaffected tlh transcription, the cdgD and cdgE mutants showed low hemolytic activity against human erythrocytes, similar to the wild-type strain (data not shown). Moreover, the cdgD strain did not have the enhanced gelatinase activity characteristic of the flagellar regulatory mutants (see Fig. S2 in the supplemental material). These data indicate that suppression of the transcription of these various virulence genes by the flagellar regulatory hierarchy is not mediated by flagellar regulation of these DGC proteins.
However, the cdgD mutant was defective for hemagglutination of human erythrocytes, similar to the fliA and frhA mutants (Fig. 5E). When CdgD was expressed from a plasmid in the cdgD strain, hemagglutination returned to wild-type levels. Importantly, when CdgD was expressed from a plasmid in the fliA strain, hemagglutination also returned to wild-type levels, indicating that the failure of the flagellar regulatory mutant to agglutinate red blood cells is due to its reduced expression of CdgD. Finally, when CdgD was expressed from a plasmid in the frhA strain, there was no restoration of hemagglutination, confirming that FrhA is the hemagglutinin responsible for the FliA- and CdgD-dependent hemagglutination. Likewise, when CdgD was expressed from a plasmid in the frhC strain, there was no restoration of hemagglutination (see Fig. S5 in the supplemental material). Interestingly, when CdgD was expressed from a plasmid in the wild-type strain, it increased hemagglutination activity almost threefold. In contrast, the cdgE mutant showed no alteration in hemagglutination of human erythrocytes, and expression of CdgE from a plasmid had no effect on hemagglutination by any of these strains (data not shown). These results suggested that the flagellar regulatory hierarchy controlled FrhA expression by controlling CdgD expression. In order to confirm this, we measured frhAp-lacZ and frhCp-lacZ transcription in the cdgD V. cholerae strain (Fig. 5B). As suspected, the cdgD mutant strain showed the low level of frhA and frhC transcription present in the flagellar regulatory mutant strains, demonstrating that the flagellar transcription hierarchy controls flagellum-regulated hemagglutinin expression via CdgD regulation of frh transcription.
We measured the cdgD mutant for the other phenotypes associated with FrhA, including epithelial cell binding, chitin binding, and biofilm formation. The cdgD mutant was defective for epithelial cell binding (Fig. 5F) and chitin binding (Fig. 5G), similar to the fliA and frhA mutant strains. Expression of CdgD in the cdgD and fliA mutant strains restored Hep-2 cell binding and chitin binding, demonstrating that the flagellar regulatory hierarchy controls these activities by controlling expression of CdgD. As expected, expression of CdgD in the frhA mutant was unable to restore epithelial cell binding and chitin binding, consistent with FrhA mediating these functions. The cdgD mutant was also defective for biofilm formation (Fig. 5D), although it formed biofilms more poorly than the fliA and frhA mutants. Expression of CdgD in the cdgD and fliA mutant strains restored wild-type levels of biofilm formation, but expression of CdgD in the frhA mutant did not enhance biofilm formation. These results are consistent with the flagellar regulatory hierarchy controlling flagellum-regulated hemagglutinin-dependent enhanced biofilm formation via the expression of CdgD.
FrhA enhances V. cholerae intestinal colonization.
Since FrhA enhances binding of V. cholerae to epithelial cells, we determined whether FrhA also enhances colonization of the intestine. We examined the ability of the frhA mutant to colonize the infant mouse intestine, the most widely used animal model for V. cholerae (29). In this model, the ability of the mutant to colonize the intestine in the presence of the isogenic wild-type strain is examined at 24 h postinoculation. We found a significant colonization defect of the frhA mutant under these conditions, approximately twofold (competitive index, 0.44; P = 0.003) (Fig. 6). The cdgD mutant showed a slightly greater colonization defect in this assay, approximately threefold (competitive index, 0.31; P = 0.0001) (Fig. 6). These results demonstrate that FrhA and CdgD contribute to intestinal colonization of the infant mouse by V. cholerae.
We also measured intestinal colonization in the adult mouse intestinal colonization model, a recently developed model for longer-term colonization studies (46, 47). The O395 background V. cholerae strain used for the studies mentioned above does not colonize the intestines of adult mice. We therefore constructed frhA and cdgD mutant strains in the P27459 V. cholerae background; the colonization behavior of this strain in the adult mouse intestine has been extensively characterized (47). Colonization was monitored at 24 and 48 h postinoculation. In this model, the frhA mutant showed a trend toward a defect in colonization, although the differences in colonization between this mutant and the wild-type strain did not reach statistical significance (see Fig. S9 in the supplemental material). For example, at 24 h, 5 of 15 mice inoculated with the frhA strain had no detectable CFU within the intestine, in contrast to the observation that only 1 of 16 mice inoculated with the wild-type strain had no detectable CFU within the intestine at this time point. At 48 h, the average number of intestinal CFU recovered for the frhA strain was more than 10-fold lower than the average number of intestinal CFU recovered for the wild-type strain. These results suggest that FrhA may contribute to intestinal colonization in the adult mouse model, although more animals would be needed to obtain statistical significance. In contrast, the cdgD mutant colonized similarly to the wild-type strain at 24 h and 48 h, suggesting that CdgD does not noticeably affect intestinal colonization in this model and/or this V. cholerae strain background. Our results provide the first demonstration that an adhesin under the control of the flagellar regulatory hierarchy is required for enhanced intestinal colonization.
DISCUSSION
The effect of motility on V. cholerae pathogenesis has been noted by several different laboratories, but the exact connection between flagellar synthesis and virulence has been elusive. This has been partly due to an incomplete understanding of flagellar synthesis, since many of the earlier studies were performed with spontaneous nonmotile V. cholerae strains prior to the elucidation of the flagellar regulatory hierarchy. Using defined flagellar regulatory mutants, we have reexamined the effect of the flagellar regulatory cascade upon V. cholerae virulence, utilizing whole-genome transcriptome analyses. Our results are consistent with the previous report by Gardel and Mekalanos (16) that suggested that motility and the expression of specific virulence factors are inversely regulated.
Transcriptome studies demonstrate that a large number of V. cholerae genes are both positively and negatively regulated by the flagellar regulatory hierarchy. This was not unexpected for the alternate sigma factor σ54, which regulates flagellar synthesis, but which also controls the expression of a number of other genes through the action of multiple σ54-dependent regulatory proteins (31, 32). However, previously, the only known function of the σ54-dependent regulators FlrA and FlrC was in the stimulation of flagellar and chemotaxis gene transcription. The alternate sigma factor σ28, the activator of class IV flagellar genes, was recently shown to modulate virulence factor expression via repression of hapR expression, a component of the quorum-sensing circuit (39); however, this circuit is not operative in the O395 strain used in the present studies, due to inactivation of the hapR gene. Our transcriptome analyses revealed that many genes with no known roles in flagellar synthesis or chemotaxis are downregulated in the absence of the flagellar regulators. Even more striking is the upregulation of a variety of proven and putative virulence genes in the absence of the flagellar regulators, demonstrating a previously unknown coordination in the transcriptional regulation of various unlinked factors that enhance V. cholerae virulence (discussed below).
The flagellar regulatory hierarchy regulates flagellar as well as other genes.
Our laboratory had previously described the four-tiered flagellar transcription hierarchy, based upon analyses of flagellar promoter expression in the regulatory mutants (51). The current transcriptome analysis of the flagellar regulatory mutants confirms the previous hierarchical model and has also allowed us to refine and expand the flagellar hierarchy (Fig. 2C). For example, we had previously designated the fliE to -Q genes as a single operon under the control of the class II fliE promoter. However, the microarray analysis revealed that the fliK to -Q genes showed a class III pattern of expression, suggesting the presence of a class III (FlrC-dependent) promoter upstream of fliK, which was confirmed by lacZ fusion assays (Table 1). The transcriptome analyses also allowed us to identify new flagellar genes, such as the class III flgOP genes. FlgP is a lipoprotein required for motility and enhanced intestinal colonization, which we described previously (44). In the present study, the transcriptome analyses identified VC1384 as a potential class III gene, which was confirmed by a promoter-lacZ assay (Table 1). Inactivation of VC1384 leads to reduced V. cholerae motility, while expression of VC1384 in a wild-type strain leads to increased motility (see Fig. S1 in the supplemental material), indicating that this gene should be considered a motility gene. Finally, there are 43 genes annotated as methyl-accepting chemotaxis protein genes in the V. cholerae genome (24), and the transcriptome analyses indicate that seven methyl-accepting chemotaxis protein genes (VC1898, VC2439, VCA0176, VCA0658, VCA0773, VCA0923, and VCA0974) display the expression pattern of class IV flagellar genes.
Among the additional genes downregulated in the flagellar regulatory mutants were a number of genes involved in amino acid and cofactor biosynthesis, cell division, energy metabolism, protein and nucleic acid synthesis, transport, and regulation. The change in transcription patterns suggests major alterations in cell physiology in nonflagellated cells. For example, we confirmed that the succinate dehydrogenase operon is downregulated in the flagellar regulatory mutants. Because succinate dehydrogenase participates in both the tricarboxylic acid cycle and oxidative phosphorylation, its expression has been shown to be downregulated under anaerobic conditions in other bacteria (48). The cell division gene ftsZ is also downregulated in the flagellar regulatory mutants, suggesting that cell division is decreased in the absence of flagella. Genes encoding a novel adhesin and c-di-GMP synthases are also downregulated in the flagellar regulatory mutants; these are discussed below.
However, in addition to the physiologic capabilities downregulated in the flagellar regulatory mutants observed in the transcriptome studies, phenotype microarrays revealed enhanced catabolic capabilities in these mutants for certain carbon sources. For example, the flagellar mutants showed enhanced growth on polymers of glucose and mannose (laminarin and mannan) and on gelatin, glutamine, and histidine as carbon sources. The flagellar regulatory mutants show enhanced gelatinase activity in comparison with that of the wild-type strain (see Fig. S2 in the supplemental material), which may explain their enhanced growth with gelatin utilized as a carbon source. The results indicate that nonflagellated V. cholerae bacteria are physiologically programmed for enhanced utilization of certain nutrients, as might be expected under conditions where flagellar synthesis would be downregulated, such as during intestinal colonization or biofilm formation.
The flagellar regulatory hierarchy positively regulates FrhA, a novel adhesin that enhances intestinal colonization.
It had previously been shown that spontaneous nonmotile V. cholerae strains were decreased for hemagglutination of human erythrocytes (16), but the identity of the flagellum-regulated hemagglutinin was unknown. The transcriptome analyses facilitated the identification of FrhA, a novel adhesin that facilitates V. cholerae binding not only to human erythrocytes but also to epithelial cells, chitin, and abiotic surfaces during biofilm development. FrhA is in the RTX family of toxin and Ca2+-binding proteins based on homology within the C terminus (COG2931). This large, 234-kDa protein also contains four cadherin repeats typically found in the cadherin family of proteins. Different sequenced pathogenic V. cholerae strains have up to four additional cadherin repeats in FrhA (see Fig. S3 in the supplemental material), suggesting an important function for these repeats within the human host. A cadherin repeat is an independently folding sequence of ∼110 aa with a Ca2+-binding pocket, and most cadherin proteins contain multiple tandem cadherin repeats (49). Eukaryotic cadherins are surface-localized proteins that mediate Ca2+-dependent cell-cell adhesion, and we anticipate that the cadherin repeats in FrhA likely mediate the adherence of V. cholerae to erythrocytes and epithelial cells, perhaps to cadherins on these cells’ surfaces. During the course of these studies, a report by Chatterjee et al. described the presence of two tandem RTX-like genes in V. cholerae (6); upon closer inspection of the genome of the strain utilized in their studies, N16961, we found that a stop codon within the coding sequence of frhA is present in strain N16961, thus making it appear as if two tandem RTX-like genes are present, rather than a single pseudogene. Sequencing of frhA from strain N16961 confirmed the presence of this stop codon (data not shown).
The C terminus of FrhA contains a type I secretion signal motif, suggesting that it is secreted through a type I-dependent mechanism, similar to other RTX family members (4). We showed that the divergently transcribed FrhC protein, which has homology with the type I secretion component TolC, is also regulated by the flagellar hierarchy and is also required for hemagglutination, strongly suggesting that FrhC is required for FrhA secretion. However, the other components normally involved in type I secretion (i.e., HlyB and HlyD homologs) were not obviously encoded nearby, although VC1618 shares some homology with HlyB.
The flagellar regulatory hierarchy controls the expression of a DGC, CdgD, and this protein in turn regulates the transcription of frhA and frhC. CdgD is predicted to contain two transmembrane segments (aa 27 to 41 and aa 327 to 339) by the DAS algorithm (13) as well as a PAS sensory box domain (which normally binds prosthetic groups) and the GGDEF DGC domain at its C terminus. CdgD was previously shown to negatively regulate flagellum-mediated motility (37). Since we have shown here that the flagellar regulatory hierarchy controls CdgD expression, this demonstrates a regulatory loop whereby the flagellum-regulated DGC in turn downregulates flagellum-mediated motility. CdgD was also previously shown to enhance early biofilm development in El Tor V. cholerae (37). We have confirmed this observation here in the classical V. cholerae background and, moreover, demonstrated that CdgD-dependent enhancement of biofilm formation is due to CdgD-dependent transcription of the flagellum-regulated hemagglutinin.
The mechanism(s) by which DGCs modulate gene expression is still not entirely clear, especially in V. cholerae. The expression of high levels of c-di-GMP causes global transcription patterns that generally favor exopolysaccharide expression and biofilm formation, while low levels of c-di-GMP lead to gene expression characteristic of flagellated and virulent V. cholerae (3, 64). Proteins containing PilZ domains bind c-di-GMP and contribute to some of these phenotypes (50). However, a large number of proteins containing DGC (>40) and c-di-GMP phosphodiesterase domains (>20) exist in V. cholerae, a number of these domains reside within the same protein, and many are expressed simultaneously under any given condition. Thus, it is not clear exactly how presumably small fluctuations in the intracellular c-di-GMP pool stimulated by diverse GGDEF- and EAL-containing proteins can lead to specific gene expression changes under a certain set of environmental conditions. For example, both CdgD and CdgE are positively regulated by the flagellar regulatory hierarchy, and both are predicted to be membrane-associated DGCs, yet only CdgD is involved in regulating flagellum-regulated hemagglutinin and mediating binding to epithelial cells and chitin. Previously, CdgD, and not CdgE, was found to influence biofilm formation and motility, despite both being expressed under biofilm-inducing conditions (37). In fact, we have evidence suggesting that both have DGC activity; specifically, overexpression of both CdgD and CdgE in the O395 V. cholerae strain leads to inhibition of CT expression (data not shown), similar to the effect of overexpression of the DGC VCA0956 (64).
Given these observations, it may be that DGCs influence gene expression changes in a more localized manner, rather than nonspecifically contributing to a common intracellular c-di-GMP pool. In other words, a specific Cdg, such as CdgD, may interact with a specific cognate c-di-GMP binding protein and thus influence gene expression in a specific manner. However, when intracellular c-di-GMP levels rise significantly, as has been observed with the overexpression of Cdgs, this would influence multiple c-di-GMP-responsive proteins, which would otherwise only respond to their cognate Cdg. The future identification of the c-di-GMP-responsive protein that directly regulates flagellum-regulated hemagglutinin should help in the dissection of flagellar regulation of this adhesin.
FrhA enhances V. cholerae intestinal colonization, and this was observed in the infant mouse model and suggested to occur in the adult mouse model. In infant mice, the ability of the mutant to compete with the wild-type strain is observed at 24 h postinoculation, and the frhA mutant had an approximately twofold defect in colonization. In the adult mouse, the number of successfully colonized mice at 24 h and the average intestinal burden at 48 h were lower in mice inoculated with the frhA strain than in mice inoculated with the wild-type strain at these same time points. These results demonstrate that FrhA enhances intestinal colonization, and given its role as an adhesin to epithelial cells, we predict that FrhA facilitates binding to the intestinal epithelial surface.
The most important intestinal colonization factor, TCP, facilitates bacterium-bacterium interactions and microcolony formation within the intestine (61), but no direct role in binding to intestinal cells has been identified for TCP. The GlcNAc-binding protein GbpA was identified as a protein that enhances intestinal colonization (28), and we showed here that GbpA expression is upregulated in the flagellar regulatory mutants. However, the nonmotile mutants with enhanced GbpA expression showed reduced binding to erythrocytes, epithelial cells, and even chitin, which was due to reduced FrhA expression, suggesting that FrhA may play a more dominant role in binding to these surfaces, at least in the laboratory. However, it appears likely that multiple adhesins contribute to intestinal colonization as well as biofilm formation, and thus, the individual contributions of select adhesins with redundant functions may seem modest in the absence of a single essential adhesin.
Our results demonstrate that the flagellar regulatory hierarchy controls transcription of CdgD, which in turn regulates flagellum-regulated hemagglutinin transcription, presumably through DGC activity, and FrhA then mediates binding to surfaces, which enhances intestinal colonization as well as biofilm formation. We suggest that this adhesin is utilized to initiate colonization of the intestine or development of a biofilm, when the bacteria are flagellated and motile upon approach to the epithelial cell surface or the environmental surface, respectively (Fig. 7).
FIG. 7.
Proposed influence of the flagellar regulatory hierarchy on V. cholerae behavior within the intestine.
The flagellar regulatory hierarchy coordinately regulates diverse virulence genes.
The ToxR/TcpP/ToxT regulon has been extensively studied, because this regulatory circuit controls the expression of the two most important virulence factors for V. cholerae, TCP and CT (reviewed in reference 7). In the flagellar regulatory mutants, transcription of the ctx, tcp, and acf genes was upregulated, and this is probably due to the upregulation of transcription of toxT, the regulator that directly binds and activates the ctx, tcp, and acf genes. Interestingly, transcription of tcpP or toxR was not upregulated in the flagellar mutants. TcpP and ToxR together activate the transcription of toxT, and expression of TcpP is correlated with CT and TCP expression, because ToxR is constitutively expressed (22, 33). Thus, the upregulation of toxT, ctx, tcp, and acf genes in the absence of tcpP upregulation suggests that the flagellar regulatory cascade influences the transcription of these genes in a TcpP- and ToxR-independent manner. The genes encoding VieSAB were also upregulated in the flagellar regulatory mutants; VieA is a c-di-GMP phosphodiesterase that stimulates CT expression and downregulates biofilm formation (63, 64). VieA stimulates CT expression by decreasing c-di-GMP, and this effect is TcpP and ToxR independent (64), suggesting that the upregulation of ctx transcription in the flagellar regulatory mutants may be mediated by the upregulation of vieA transcription.
Among the additional virulence genes upregulated in the flagellar regulatory mutants was the gene encoding the “El Tor” hemolysin HlyA (also called cytolysin). This is a pore-forming exotoxin that causes cell vacuolation (8, 43), and although classical V. cholerae strains (such as that used in this study) contain the hlyA gene, an 11-bp deletion within the coding sequence renders the resulting product nonhemolytic (2). HlyA expression is under the control of HlyU (70), but transcription of the hlyU gene was not upregulated in the flagellar regulatory mutants.
Spontaneous nonmotile V. cholerae strains were previously shown to have increased hemolytic activity toward human type O erythrocytes (16), but the identity of the flagellum-regulated hemolysin was unknown. We have shown here that transcription of thermolabile hemolysin (tlh) is increased in the flagellar regulatory mutants and that inactivation of tlh in these mutants renders them nonhemolytic, thus identifying thermolabile hemolysin as the flagellum-regulated hemolysin. Thermolabile hemolysin has phospholipase and lecithinase activity (14), and the tlh gene is adjacent to and divergently transcribed from the hlyA gene. Because hlyA and tlh are similarly upregulated in the flagellar regulatory mutants, the divergent tlh and hlyA promoters are likely coordinately regulated by the same inducing factor(s), although this regulation does not involve increased expression of HlyU. The tlh mutant colonized infant mice similarly to the wild-type strain (see Fig. S9 in the supplemental material), consistent with a previous study with rabbits (14) indicating the lack of a dominant role for this protein in virulence. However, a number of virulence factors with possibly redundant roles are expressed simultaneously within the intestine, which may obscure the individual contributions of these various factors, as has been suggested by the studies of Olivier et al. (47).
Interestingly, HlyU also controls expression of Hcp (hemolysin-coregulated protein), one of the secreted factors found in the type VI secretion cluster (52, 71). Although transcription of the hcp gene was not altered in the flagellar regulatory mutants, there was an upregulation of several type VI secretion genes that lie in a cluster (VCA0107, VCA0108, VCA0109, VCA0112, VCA0113, VCA0117, and VCA0124). The T6SS is required for virulence of V. cholerae in Dictyostelium discoideum, and previous studies suggested that this cluster of genes was positively regulated by σ54 and the σ54-dependent activator encoded by VCA0117 (52). We observed upregulation of these genes in the flagellar regulatory mutants, including the rpoN strain, which indicates an additional σ54-independent, flagellum-dependent mechanism responsible for increased transcription.
The eps genes were also upregulated in the flagellar regulatory mutants; these genes encode the type II secretion system responsible for CT secretion as well as secretion of CTXφ, hemagglutinin/protease, chitinase, neuraminidase, and lipase (55). The eps genes have previously been shown to be induced in response to increased intracellular c-di-GMP (3), but in the case of the flagellar regulatory mutants, intracellular c-di-GMP levels would likely be lower than those in the wild-type strain, suggesting an alternate (flagellum-dependent) mechanism for regulating extracellular protein secretion. The chitin-binding protein GbpA was also upregulated in the flagellar regulatory mutants, and GbpA expression has previously been shown to be stimulated by the presence of GlcNAc (40). The upregulation of gbpA in the flagellar regulatory mutants suggests an alternative GlcNAc-independent, flagellum-dependent mechanism of control over its expression.
Our results demonstrate coordination of the transcriptions of diverse virulence genes via the flagellar regulatory hierarchy (Fig. 7). The upregulation of CT, TCP, extracellular protein secretion, HlyA, thermolabile hemolysin, GbpA, and the T6SS as a result of the downregulation of flagellar synthesis would be predicted to enhance prolonged colonization and pathogenesis at the epithelial cell surface. The flagellar regulatory system thus acts as an important signaling component of the pathogenic process, positively regulating factors that assist in arrival at the colonization site (motility and chemotaxis) and initial adherence upon arrival (flagellum-regulated hemagglutinin), while negatively regulating diverse virulence genes that facilitate persistence of colonized, less (non)motile bacteria; after colonization we predict that the flagellar regulatory system downregulates flagellar, chemotaxis, and adherence genes, and thus the repression of the TCP, CT, and other virulence factors is relieved, which facilitates a productive colonization phase of the bacteria.
Supplementary Material
Acknowledgments
This study was supported by NIH grant AI43486 to K.E.K.
Footnotes
Published ahead of print on 28 August 2009.
Supplemental material for this article may be found at http://jb.asm.org/.
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