DksA coordinates bile-mediated regulation of virulence-associated phenotypes in type three secretion system-positive Vibrio cholerae

Abstract

In order to cause disease, pathogenic strains of Vibrio cholerae rely on intricate regulatory networks to orchestrate the transition between their native aquatic environment and the human host. For example, bacteria in a nutrient-starved environment undergo a metabolic shift called the stringent response, which is mediated by the alarmone ppGpp and an RNA-polymerase binding transcriptional factor, DksA. In O1 serogroup strains of V. cholerae, which use the toxin co-regulated pilus (TCP) and cholera toxin (CT) as primary virulence factors, DksA was reported to have additional functions as a mediator of virulence gene expression. However, little is known about the regulatory networks coordinating virulence phenotypes in pathogenic strains that use TCP/CT-independent virulence mechanisms. We therefore investigated whether functions of DksA outside of the stringent response are conserved in type three secretion system (T3SS)-positive V. cholerae . In using the T3SS-positive clinically isolated O39 serogroup strain AM-19226, we observed an increase in dksA expression in the presence of bile at 37 °C. However, DksA was not required for wild-type levels of T3SS structural gene expression, or for colonization in vivo. Rather, data indicate that DksA positively regulates the expression of master regulators in the motility hierarchy. Interestingly, the ΔdksA strain forms a less robust biofilm than the WT parent strain at both 30 and 37 °C. We also found that DksA regulates the expression of hapR, encoding a major regulator of biofilm formation and protease expression. Athough DksA does not appear to modulate T3SS virulence factor expression, its activity is integrated into existing regulatory networks governing virulence-related phenotypes. Strain variations therefore may take advantage of conserved ancestral proteins to expand regulons responding to in vivo signals and thus coordinate multiple phenotypes important for infection.

Introduction

Vibrio cholerae is a genetically and phenotypically diverse species. Both pathogenic and non-pathogenic strains are found in aquatic environments worldwide, and all strains can be classified into serogroups based on the composition of the lipopolysaccharide somatic O-antigen [1]. Pathogenic O1 and O139 serogroup strains cause epidemic outbreaks of the diarrhoeal disease cholera, and invariably encode the toxin co-regulated pilus (TCP) for colonizing the human small intestine, and cholera toxin (CT), which disrupts host cell homeostasis to produce secretory diarrhoea, a hallmark of the disease [2, 3]. Pathogenic strains of other serogroups (non-O1/non-O139) have long been recognized to cause similar disease, although most do not encode TCP or CT and their virulence mechanisms have been less well studied [3–7]. However, a subset of non-O1/non-O139 serogroup strains encode a type three secretion system (T3SS), functionally similar to that found in other Gram-negative pathogens and shown to mediate colonization and disease [8–13].

Regardless of virulence mechanism, all pathogenic strains must have a regulatory network in place that can rapidly and effectively alter gene expression in response to specific external signals. The chemical and physiological parameters of marine reservoirs are dramatically different from conditions encountered within the human small intestine. Bacteria must survive nutrient fluctuations and dynamic changes of signalling molecules during the transition from environment to human host and back again. For example, aquatic environs that harbour V. cholerae provide copepod and zooplankton surfaces for biofilm formation and sources for chitin utilization, whereas the human host presents challenging parameters such as the acidic stomach, competition from resident flora, bile and host immune components [14–18].

A conserved bacterial adaptation to nutrient limitation, known as the stringent response, is characterized by rapid repression of the ribosomal machinery and increased expression of genes associated with stress resistance and survival, resulting in a broad shift in the metabolic profile that facilitates survival [19]. DksA is a ubiquitous regulator of the stringent response in Gram-negative bacteria, but is also recognized as having pleiotropic effects [20, 21]. In Escherichia coli , DksA works in concert with the alarmone ppGpp to induce the stringent response when bacteria sense nutrient-depleted conditions [19, 22]. In most organisms, including V. cholerae , ppGpp alarmone levels are responsible for initiating and implementing the stringent response programme. In E. coli , an auto-feedback regulatory loop is responsible for tightly controlling dksA expression and DksA protein production, although studies on the environmental factors that regulate dksA expression or activity have not been exhaustive [23, 24].

The stringent response intersects with the virulence factor regulatory network in the O1 serogroup, TCP/CT-positive strain N16961, and the stationary phase sigma factor, RpoS [25, 26]. The stringent response was also reported to alter biofilm formation [27]. Interestingly, Pal and co-workers demonstrated that DksA regulates virulence factor production in strain N16961, and DksA-deficient strains produced less CT in vitro [28, 29]. DksA was also shown to positively regulate additional phenotypes, including motility. Regulation appears indirect, occurring through modulation of the gene encoding FliA, a master regulator of motility that functions as an alternative sigma factor [28, 30–32]. A DksA deletion strain also produced less of the haemagglutinin protease (HAP), which has been hypothesized to play a role in traversing the gut mucosa and in detachment of the intestinal epithelia, although its role in each stage of virulence is not fully understood [26, 33–35]. Regulatory networks governing diverse phenotypes thus overlap in V. cholerae : HAP and biofilm formation are under the control of the HapR transcriptional regulator, which represses genes contributing to biofilm formation but activates the gene responsible for HAP production (hapA), and is itself regulated by FliA [26, 31, 36, 37]. The collective results thus indicate that in TCP/CT-positive strains of V. cholerae , DksA can interact with multiple regulatory networks outside of the stringent response to coordinately regulate pleiotropic phenotypes. Of note, DksA and pppGpp have been shown to play a role outside the stringent response in other pathogens, including regulation of biofilm formation, motility, colonization and T3SS-mediated virulence [38–44].

Most pathogenic V. cholerae strains that are classified as non-O1/non-O139 serogroup strains use alternative virulence mechanisms and lack TCP and CT. In strains that encode a T3SS, we do not currently understand how virulence gene expression is coordinated with other phenotypes. Different strains share core genome-encoded regulatory genes such as dksA and hapR, as well as genes required for phenotypes such as motility and protease production. However, the transcriptional networks that govern virulence gene expression differ among V. cholerae strains [45–49], and it is not fully understood how coordinated responses and phenotypes are wired in different genetic backgrounds and function when bacteria are in the aquatic reservoir vs. the human host. We therefore sought to determine how DksA activity influences virulence gene expression in vitro in a TCP/CT-negative, T3SS-positive, non-O1/non-O139 serogroup strain of V. cholerae , named AM-19226. We evaluated dksA expression under different growth conditions, and paired transcriptional fusion studies with phenotypic assays for motility and biofilm formation to investigate the role of DksA when bacteria are grown under in vitro conditions that include environmental signals present in the human intestine. Finally, we investigated whether DksA influences colonization in vivo using an infant mouse model. Our collective results provide new evidence that DksA functions to regulate gene expression in diverse V. cholerae strains and has an important role in coordinating multiple phenotypes in addition to orchestrating the stringent response.

Methods

Bacterial media and growth conditions

E. coli and V. cholerae strains were maintained at −80 °C in Luria Bertani broth (LB) containing 20 % glycerol, and typically grown in LB unless otherwise indicated. Ampicillin and streptomycin were used at a final concentration of 100 μg ml⁻¹. Bile (Sigma) was prepared as a 10 % (w/v) stock as previously described and used at a final concentration of 0.4 % [45]. M9 minimal medium was supplemented with 0.4 % (v/v) glucose as the carbon source. Overnight liquid cultures were grown at 30 or 37 °C for 16 h on a roller drum.

Strain and plasmid construction

V. cholerae strain MD 992 (AM-19226 R-M+) was used as the parental strain for this study [45]. DksA in-frame deletions were constructed using overlapping PCR and standard allelic exchange methods as previously described [45, 50, 51]. Primer sequences are available on request. Deletions were verified by PCR and Southern blot analyses. DksA-complemented strains were constructed by cloning the dksA coding region downstream of the constitutive tet promoter using a Gateway-compatible pBR322 construct [52]. A lacZ reporter fusion to the T3SS structural operon vcsRTCNS2 was previously described [45]. A strain carrying the dksA promoter–lacZ transcriptional reporter fusion integrated at the V. cholerae lacZ locus was constructed using similar methods, including ~650 bp of sequence upstream of the DksA translational start site. In situ lacZ transcriptional reporter fusions to fliA, flrA, hapR and dksA genes were first constructed as operon fusions using pCVD442-based vectors and allelic exchange to integrate the reporter fusions in single copy into the native gene chromosomal locus of the AM-19226 ΔlacZ strain MD996 [45].

Growth curves

Growth curves were plotted using OD measurements to monitor bacterial culture densities in 96-well plates using a BioTek Power Wave XS plate reader. Cultures were grown in LB broth at 30 or 37 °C on a roller drum for 16 h, then sub-cultured 1 : 20 into a 96-well plate containing fresh LB and monitored by OD₆₀₀ for 16 h every 20 min. Growth was measured in LB broth, LB broth supplemented with 0.4 % bile, and M9 minimal medium containing 0.4 % glucose as a carbon source. Growth rate was calculated using the equation: μ (growth rate)=2.303 * log₁₀ ΔOD. Generation time was calculated using the equation: g (generation time in hours)=0.69/μ.

β-Galactosidase assays

β-Galactosidase assays were conducted as previously described and the units reported were micromoles of ONPG hydrolysed per minute per optical density unit at 600 nm (OD₆₀₀) [45]. Briefly, 5 ml cultures from three independent colonies were pelleted and resuspended in Z buffer supplemented with β-mercaptoethanol. Following an OD₆₀₀ reading, 1 % SDS and chloroform were added to lyse the cells. To initiate the reaction, 10 µg ml⁻¹ of ONPG (Sigma) was added to cultures and kinetic readings were taken using a BioTek Power Wave XS plate reader.

Infant mouse colonization assay

Competition assays were conducted as previously described and were in accordance with the University of Rochester Committee on Animal Resources-approved protocols [45, 53, 54]. Briefly, 3- to 5-day-old CD1 mice were intragastrically inoculated with a total of ~10⁵ bacteria at a 1 : 1 ratio of the deletion strain (lacZ ^wtΔdksA) and an isogenic parent strain (ΔlacZ). After 16 h at 30 °C, mice were sacrificed, and the small intestines were surgically removed, homogenized and plated onto LB agar plates containing X-gal. The competitive index (CI) was calculated based on output and input ratios of the parental and the deletion strains, where CI = (mutant output/parental output)/(mutant input/parental input).

HAP assays

HAP secretion was quantified using an azocasein compound that consists of casein conjugated to an azo dye in a standard assay, as previously described [55]. Briefly, three independent cultures were grown for 16 h at 37 °C in LB or LB+0.4 % bile. Samples were pelleted, and supernatants were collected and used for the assay.

Static biofilm assays

Static biofilm assays were performed as previously described [56]. Briefly, 5 ml overnight cultures were grown in LB or LB+0.4 % bile. Cultures were normalized to the same OD₆₀₀ and 10 µl was used to inoculate 1 ml of fresh medium (1 : 100) in borosilicate glass tubes (10×75 mm). Following 30 h of static incubation at 30 and 37 °C for 30 h, the cultures were removed by aspiration, tubes were rinsed with PBS, and incubated with 0.01 % crystal violet stain for 10 min. After washing in PBS, the stained biofilm was resuspended using 100 % DMSO. Total biofilm biomass was measured by absorbance at 570 nm using a BioTek Power Wave XS plate reader. The experiment was conducted using three colonies per strain and repeated three times.

Motility assays

Motility assays were performed as previously described [56]. Briefly, single colonies were used to inoculate LB plates containing 0.3 % agar. The diameters of the bacterial movement zones were measured after incubation for 8 h at 37 °C. The assay was performed three times using five colonies for each strain.

Results

DksA is required for optimal growth of strain AM-19226 in minimal media

Similar to E. coli strains, O1 serogroup, epidemic-causing TCP/CT-positive V. cholerae strains require DksA for optimal growth in M9 minimal medium [28]. To determine whether DksA has a role in growth of the TCP/CT-negative, T3SS-positive strain AM-19226, we compared growth of a wild-type strain (dksA ^wt) and a dksA deletion strain. We chose three different media to reflect different conditions: LB as a neutral growth medium, LB supplemented with 0.4 % bile as a physiologically relevant signal during growth in the intestine, and M9 minimal medium supplemented with glucose as a nutrient-limiting condition. We also conducted growth assays at 30 °C (Fig. 1a) and 37 °C (Fig. 1b). At 30 °C, the wild-type and ΔdksA strains produced nearly identical growth curves in both nutrient-rich LB broth and in LB supplemented with bile (Fig. 1a). In minimal medium at 30 °C, however, DksA was required for optimal growth, and the calculated generation time for the deletion strain was 132 min compared to 80 min for the wild-type strain (Fig. 1a). When strains were grown at 37 °C (Fig. 1b), no growth defect was observed in LB. Growth in LB supplemented with bile at 37 °C resulted in an increased generation time for the ΔdksA strain (82 min vs. 57 min, Fig. 1b). However, the mutant strain did reach wild-type levels of growth after ~7 h. There was little effect on growth of the dksA deletion strain in minimal medium at 37 °C, and despite the growth defect observed at 30 °C, the deletion strain achieved a similar level of saturation as the wild-type strain at both temperatures. Providing a constitutively expressed, wild-type copy of the dksA gene in trans restored wild-type growth rates. We therefore conclude that DksA activities are most pronounced when strains are grown in minimal medium at 30 °C and in LB supplemented with bile at 37 °C.

Fig. 1.

DksA is required for optimal growth in minimal media. Growth rates of WT and ΔdksA strains carrying either pBR322 as an empty vector control or expressing dksA constitutively from the tet promoter in pBR322 (pdksA) were assayed at 30 °C (a) and 37 °C (b) in LB broth, LB with 0.4 % bile, and M9 minimal medium supplemented with glucose. Three individual colonies were used for each strain. After overnight growth in LB, strains were subcultured 1 : 200 into LB or M9-glucose minimal media in a 96-well plate and the OD₆₀₀ recorded every 20 min for 12 h. The assay was repeated with similar results.

Bile regulates dksA expression

Experiments conducted in E. coli reported that regulation of dksA expression occurs primarily by negative auto-regulation at the transcriptional level, although expression is modulated by environmental conditions in some organisms such as Pseudomonas [24, 38, 39]. We constructed a dksA–lacZ transcriptional fusion to determine if media and temperature conditions used to generate the growth curves shown in Fig. 1 modulate dksA expression. We engineered a strain carrying a chromosomal lacZ transcriptional fusion in situ at the dksA locus, such that lacZ was transcribed in a bicistronic operon with dksA. The resulting reporter strain was grown overnight under the different conditions described for the growth curves. Fig. 2 shows the results for activities measured at two different temperatures (30 and 37 °C) and in three different media: LB (black bars), LB supplemented with 0.4 % bile (grey bars) and minimal medium supplemented with 0.4 % glucose (hatched bars). At both growth temperatures, cells grown in LB or in minimal medium showed similar levels of LacZ activity. Also at both temperatures, lacZ reporter activity was highest in cells grown in LB supplemented with bile. A three- to four-fold increase in LacZ activity was measured in cells grown in LB with bile at 37 °C, compared to LB alone (compare grey bar to black bar). The same trend was observed for growth at 30 °C, although the difference was less than two-fold. We also assayed the activity of a reporter strain carrying a lacZ transcriptional fusion to only ~650 bp of DksA upstream sequences, which was integrated at the V. cholerae lacZ locus. We obtained similar results to those shown in Fig. 2, suggesting that both reporter fusions were similarly regulated, and that bile-mediated regulation of dksA expression is modulated by sequences within the ~650 bp fragment (data not shown).

Fig. 2.

Bile activates dksA expression. A dksA transcriptional fusion was constructed in situ distal to the dksA coding sequence. Three individual colonies were grown in the indicated media for 16 h at 30 or 37 °C and assayed for β-galactosidase activity. Repeated assays produced similar results. ****P<0.0001 by two-way ANOVA with Tukey's post-hoc test.

DksA regulates the expression of motility and biofilm genes, but not T3SS structural genes

We previously reported that the expression of genes encoding the AM-19226 T3SS structural apparatus is increased ~4–5-fold when bacteria are grown at 37°C in LB supplemented with bile [45]. We were therefore interested in investigating whether DksA influences T3SS gene expression, using a previously described lacZ reporter fusion to a T3SS structural apparatus encoding operon, vcsRTCNS2-lacZ [45]. Simultaneously, we wanted to assess whether DksA modulates expression of virulence-related phenotypes, such as motility, biofilm formation and protease production in the T3SS-positive background. We measured expression from a lacZ transcriptional reporter fusion to the hapR gene, encoding a regulator of protease production and biofilm formation, and genes encoding motility regulatory proteins FlrA and FliA [30, 31, 34, 36]. All strains were grown in LB or LB supplemented with 0.4 % bile at 30 and 37 °C. Reporter expression in the dksA deletion background was calculated relative to the isogenic, wild-type parent strain background, with expression in the wild-type strain set to 100 % (Fig. 3). vcsRTCNS-lacZ expression was decreased ~50 % in the ΔdksA strain grown at 30 °C in LB compared to the wild-type strain, although wild-type levels of expression were observed in the presence of bile. The data are consistent with our previously published results showing that bile increases T3SS gene expression, and may indicate that bile suppresses the effect of the dksA deletion seen at 30 °C [45]. At 37 °C, vcsRTCNS-lacZ expression in LB and LB containing bile was similar to wild-type levels (Fig. 3b). We observed ~40–50 % decreased hapR-, flrA- and fliA-lacZ reporter fusion expression during growth in both LB and LB with bile at 30 °C (Fig. 3a). Interestingly, growth in LB medium alone at 37 °C resulted in only an ~25 % reduction of hapR, flrA and fliA reporter gene expression compared to the isogenic parent strain (Fig. 3b). However, the decrease in expression was more pronounced (~50 %) in the presence of bile at 37 °C. We therefore conclude that in the presence of bile, DksA can promote the expression of genes encoding products that regulate biofilm formation and motility.

Fig. 3.

DksA activates expression of genes encoding transcriptional regulators of biofilm and motility. Expression of fliA, flrA, hapR and the T3SS operon vcsRTCNS2 was measured in a dksA deletion background using lacZ transcriptional fusions and three independent colonies per experiment. Strains were grown in LB or LB+0.4 % bile for 16 h at 30 or 37 °C. The results are presented as activities in the dksA deletion strain relative to the WT strain, set at 100 %. The percentage activity reported is the average of three independent experiments.

A dksA deletion strain is not attenuated for colonization in vivo

Although we did not observe a role for DksA in modulating T3SS gene expression under conditions similar to those expected during infection (37 °C and bile), we tested the ability of a ΔdksA strain to colonize in vivo, using a standard infant mouse model where results are reported as a CI relative to the wild-type strain [45, 53, 54]. As shown in Fig. 4, the ΔdksA strain colonizes as well or ~2-fold better than the isogenic parent strain, indicating that the loss of DksA does not negatively impact colonization in the mouse model. In fact, lack of DksA may result in a small increase in colonization or bacteria retained in the small intestine due to decreased HA protease and/or biofilm dispersal (see data below and Discussion).

Fig. 4.

Deletion of dksA does not negatively affect in vivo colonization. A competition assay with WT AM-19226 and ΔdksA strains was conducted. Three- to 5-day-old mice were gavaged with WT and ΔdksA strains of V. cholerae . CIs were calculated using the ratio of output and input c.f.u. values for the WT and deletion strain, and the horizontal bar indicates the mean CI. The experiment was repeated and produced similar results.

DksA contributes to HAP production in strain AM-19226

HAP, encoded by the hapA gene, is a secreted protein postulated to function in colonization and/or detachment from the small intestine during infection [34, 57]. In TCP/CT-positive O1 serogroup strain C6709, HAP production was decreased in a ΔdksA strain [26, 28]. To determine the role of DksA in HAP production in T3SS-positive strain AM-19226, we grew isogenic WT and ΔdksA strains carrying an empty vector control plasmid, and a ΔdksA-complemented strain (pDksA) for 16 h at 37 °C in LB or LB+0.4 % bile, and then quantified HAP activity using a standard azocasein protease assay [28]. As shown in Fig. 5, the ΔdksA strain produced less protease compared to the wild-type strain (38 %) when cells were grown in LB. We observed a more dramatic decrease, to 11 % of WT levels, when ΔdksA cells were grown in LB+0.4 % bile. Protease production was partially restored in a strain containing the pdksA complementing plasmid (Fig. 5). The results of the azocasein assay combined with results shown in Fig. 3 are consistent with indirect DksA regulation of hapA expression via HapR [26].

Fig. 5.

A dksA deletion reduces HAP in AM19226. In order to assay protease activity, WT and ΔdksA strains carrying pBR322 and a dksA deletion strain carrying the complementing plasmid (Δdks+pdksA) were assayed for their ability to degrade azocasein dye. Three colonies from each strain were grown in LB or LB+0.4 % bile at 37 °C for 16 h. Cell suspensions were normalized to an OD₆₀₀ of 1.0, and supernatants were collected and incubated with azocasein substrate. Degradation of the azocasein substrate was measured by recording changes in absorbance. The results are representative of three independent experiments. ****P<0.0001, two-way ANOVA with Dunnett's post-hoc test.

DksA influences motility in V. cholerae AM-19226

As described earlier, a complex hierarchy of transcriptional regulatory proteins govern the regulation of motility in V. cholerae [32]. Reports of motility defects in an N16961 dksA-deletion strain [28], combined with the bile-mediated regulation of dksA expression, led us to test the hypothesis that DksA activity influences motility in strain AM-19226. We measured the motility of the dksA deletion strain and the isogenic parent strain when grown on soft agar LB plates using a standard assay, and included strain N16961 for comparison. In general, strain AM-19226 was less motile than strain N16961 at all time points, and the effect of a dksA deletion on N16961 motility was marked, as previously reported [28] (Table 1). The AM-19226 ΔdksA strain consistently displayed a smaller diameter zone of growth compared to the isogenic wild-type strain. The difference was overall less pronounced than that observed for strain N16961, but did reach statistical significance (P<0.001) at later time points. We also conducted a preliminary motility assay using plates containing 0.4 % bile. Although bile stimulated motility overall in strain AM-19226, there was no difference in the diameter of growth between the WT and the AM-19226 ΔdksA strain, suggesting that bile suppresses any DksA effect under the conditions tested (data not shown).

Table 1.

DksA activates motility in both epidemic O1 serogroup strain N16961 and O39 serogroup strain AM19226

Motility assays were conducted using soft agar LB plates (0.3 % agar). Eight to 10 individual colonies of either AM-19226 or N16961 WT and corresponding dksA deletion strains were inoculated into the centre of a plate and incubated for 8, 10 or 14 h at 37 °C before measuring the growth diameter (mm). The experiment was repeated with similar results. Average growth diameter and standard deviation are reported, with P-value calculated to compare diameters of each deletion strain to the respective WT strain at the indicated time points.

	Strain	Average growth diameter (mm)	sd	P-value
	AM-19226	11.6	1.35
	AM-19226 ΔdksA	9.2	0.37	0.0026
	N16961	20.0	0.96
	N16961 ΔdksA	8.3	0.46	2.55E-14
LB 10 h
	AM-19226	24.0	1.49
	AM-19226 ΔdksA	21.5	0.53	0.0005
	N16961	42.9	1.73
	N16961 ΔdksA	21.2	0.78	3.04E-18
LB 14 h
	AM-19226	34.2	2.27
	AM-19226 ΔdksA	28.8	1.00	0
	N16961	59.0	2.31
	N16961 ΔdksA	29.2	1.40	5.83E-18

DksA modulates biofilm formation in strain AM-19226

HapR is a pleiotropic regulator that acts as a repressor of biofilm formation in addition to its role as an activator of protease production [58–60]. To determine if DksA plays a role in biofilm formation, we conducted standard biofilm assays at both 30 and 37 °C. When bacteria were grown in LB, biofilm formation was reduced ~2-fold in a ΔdksA strain at both 30 and 37 °C (Fig. 6, black bars). Surprisingly, when LB was supplemented with 0.4 % bile, we observed reduced biofilm formation in ΔdksA strains only at 30 °C, but not at 37 °C (Fig. 6, grey bars), again suggesting that bile suppresses the effects of a dksA deletion. Interestingly, the effect occurs at the higher temperature for biofilm formation. Biofilm production was partially or fully restored in strains containing the pdksA complementing plasmid.

Fig. 6.

DksA activity contributes to biofilm formation. WT, ΔdksA and ΔdksA strains carrying either pBR322 (empty vector) or the pdksA complementing plasmid were assayed for their ability to form static biofilms during growth in LB and LB+0.4 % bile at 30 (panel a) and 37 °C (panel b)using six individual colonies for each strain. Biofilm formation was assayed using standard methods for static biofilms and biofilm mass was measured by crystal violet staining and absorbance at OD₅₇₀. The assay was repeated twice and produced similar results. *P<0.05 and **P<0.005 by two-way ANOVA with Dunnett's post-hoc test.

Discussion

During the course of infection, pathogenic strains of V. cholerae transition from their native aquatic environment to a human host. Bacteria are therefore exposed to diverse environmental parameters, such as temperature, bile and nutrient availability, which must be sensed and translated into an appropriate phenotypic response. To cope with a nutrient-depleted environment, cells undergo a ‘stringent response’, a rapid metabolic shift that employs the DksA protein to modulate gene expression. Although historically associated solely with the stringent response, studies using an O1 serogroup, TCP⁺/CT⁺ strain of V. cholerae concluded that DksA also functions to regulate motility, protease secretion, biofilm formation and CT production [27–29]. V. cholerae strains are genetically and phenotypically diverse, and the mechanisms of gene regulation frequently differ among strains despite the presence of common factors. We were therefore interested to understand the role of DksA in modulating gene expression in a TCP/CT-negative, T3SS-positive strain of V. cholerae, AM-19226.

Consistent with a role in modulating the stringent response, DksA is important in V. cholerae strain N16961 for growth under nutrient-limited conditions [28]. We showed that DksA is also important for growth in nutrient-limited conditions in strain AM-19226, suggesting that in this capacity, DksA has a well-conserved role across strains (Fig. 1). Interestingly, however, the growth defect was less pronounced when cells were grown at 37 °C compared to 30 °C. It is possible that at 37 °C, an additional factor(s) can partially alleviate the growth defect caused by the absence of DksA, or alternatively, that temperature affects DksA function.

In E. coli , dksA expression is tightly regulated by an auto-feedback loop that prevents large fluctuations in gene expression and protein production [23]. To examine whether virulence-related environmental signals influence dksA expression in strain AM-19226, we constructed a lacZ transcriptional reporter fusion to dksA and grew the resulting strain in LB, LB+0.4 % bile and minimal media. We included LB with 0.4 % bile in the culture medium to determine if dksA expression might be coordinately regulated with T3SS genes, as DksA was shown to play a role in virulence in other organisms. Interestingly, dksA expression increased >4.5-fold in the presence of bile at 37 °C, suggesting that dksA expression responds to an environmental parameter present during infection. Consistent with that interpretation, the enhanced expression was temperature-dependent, as expression at 30 °C was increased less than 2-fold in the presence of bile compared to growth in LB alone. Similar results were observed using a dksA promoter–lacZ transcriptional reporter fusion integrated at the V. cholerae lacZ locus (data not shown). Preliminary Western blot analysis using a polyclonal antibody against E. coli DksA (kind gift of M. Cashel) resulted in cross-reacting bands with V. cholerae DksA. Thus, we were unable to definitively determine whether bile resulted in increased DksA protein production (data not shown).

To investigate how DksA was integrated into existing motility and biofilm regulatory networks, we first measured the activity of strains carrying lacZ transcriptional reporter fusions to operons encoding Class I and Class II motility regulators flrA and fliA and the gene encoding the main regulator of biofilm formation, hapR. Because we were also interested in understanding whether DksA functioned in virulence gene regulation, we included a reporter fusion to a T3SS structural operon, vcsRTCNS2-lacZ, and tested the ΔdksA strain for the ability to colonize intestinal tissues using the infant mouse model. Interestingly, the ΔdksA strain was not attenuated for colonization and did not show decreased T3SS gene expression at 37 °C in the presence of bile. Instead, we found that DksA influenced T3SS gene expression only during growth conditions at 30 °C in LB alone, which are not conditions typically associated with human infection. The results raise the intriguing possibility that the T3SS may have a function outside the human host or at later stages of infection, although further experiments are required to investigate such possibilities.

Notably, our results suggest that DksA activates expression of genes encoding important regulators of biofilm and motility gene expression at both 30 and 37 °C, and the effect was most pronounced when strains were grown at 37 °C in the presence of bile (Fig. 3). Collectively, the results of our transcriptional reporter fusion studies suggest a role for DksA in temperature-dependent gene regulation that is influenced by the presence of bile. We therefore assayed biofilm formation at both temperatures and in LB with and without bile, to better understand the breadth of the DksA influence. The results shown in Fig. 6 suggest that bile influences biofilm formation in part via DksA, suggesting that bile either influences DksA activity, perhaps via alterations in DNA structure [61], or alternatively, may induce the expression of another factor that functions in the complex regulatory and signalling network governing biofilm formation [62, 63]. Reports demonstrating that DksA plays a role in Salmonella biofilm formation and pleiotropic phenotypes in Pseudomonas , combined with our results, suggest a more widespread and common role for DksA in regulating previously unrecognized phenotypes outside of the stringent response in pathogenic bacteria [40, 42]. Thus, we favour the interpretation that although DksA may not be acting directly on the T3SS virulence genes in V. cholerae strain AM-19226, DksA activity responds to the same signals as the T3SS (bile), thus coordinating virulence-related phenotypes.

Consistent with a proposed role in modulating virulence-related phenotypes, DksA was shown to play a role in the production of HAP in V. cholerae TCP⁺/CT⁺ strain C6709 [26, 28]. HAP is encoded by hapA, which is directly regulated by HapR [36]. Thus, we predicted a role for DksA in protease production via HapR, based on our results in the biofilm assays and with hapR expression studies. We therefore assayed HAP production in WT and ΔdksA strains, and found that DksA does indeed modulate protease production in cells grown in LB and LB+0.4 % bile at 37 °C (Fig. 5). The results are consistent with observations in a CT-positive strain, suggesting that the network response is conserved between diverse strains [26]. As indicated earlier, HAP production may be important for the V. cholerae exit strategy from an infected intestine. Interestingly, DksA was shown to be important for biofilm dispersal in Pseudomonas , and bile acids can influence biofilm formation and detachment [57, 63–65]. The role of bile in modulating the intestinal microbiome and in the ability of V. cholerae to establish an infection provides additional evidence that bile has widespread effects on bacterial physiologies and behaviours [66]. It is therefore interesting to speculate that the small but measurable increased competitive index observed for the ΔdksA strain (Fig. 4) may be biologically relevant.

DksA has been shown to play a role in motility in V. cholerae strain N16961, E. coli and Salmonella [21, 28, 42]. Although we observed hypomotility in a ΔdksA N16961 strain, consistent with the results reported by others, the magnitude of the difference was much smaller for strain AM-19226. One interpretation is that motility does not appear to respond as dramatically to the presence of bile as seen in strain N16961, and thus the bile signal may be moderated by other signals or transcriptional regulatory components that are part of the motility regulon and cascade. However, our results are consistent with the transcriptional reporter fusion data shown in Fig. 2, indicating that DksA contributes to the positive regulation of flrA and fliA expression. FliA functions as an alternative sigma factor in the regulatory hierarchy governing production of flagella and motility, further suggesting that DksA-mediated modulation of transcriptional regulators is an important control point for motility and the coordination of multiple phenotypes. Another consideration is that in the presence of bile, dysregulation may occur and the roles of other messenger molecules, such as pppGpp and c-di-GMP, become more important for influencing gene expression [62, 67]. In addition, strain N16961 is a naturally occurring HapR mutant, and probably exhibits phenotypic differences due to altered HapR-related networks.

Importantly, our genetic and phenotypic data provide evidence that although individual regulatory networks governing environmentally important and virulence-associated phenotypes such as motility, protease production and biofilm formation in V. cholerae may overlap among diverse strains, the regulation of virulence gene expression (e.g. T3SS) may not always overlap in using conserved transcriptional regulatory control elements such as DksA. One interpretation is that a wide margin of variability in gene regulation can be tolerated and that depending on the nature of acquired pathogenicity islands, phenotypic outcomes for environmental adaptation may be moderated independently of pathogenic mechanisms. In addition, given that we have uncovered a relationship between DksA activity and bile modulation of gene expression for specific phenotypes, it will be interesting to conduct future studies to determine whether the genetic networks and target genes of the stringent response and bile response of V. cholerae strains intersect, perhaps via c-di-GMP levels and coordinated effects on HapR activities.