Deletion of gene encoding the nucleoid-associated protein H-NS unmasks hidden regulatory connections in El Tor biotype Vibrio cholerae

Abstract

Hypervirulent atypical El Tor biotype Vibrio cholerae O1 isolates harbour mutations in the DNA-binding domain of the nucleoid-associated protein H-NS and the receiver domain of the response regulator VieA. Here, we provide two examples in which inactivation of H-NS in El Tor biotype vibrios unmasks hidden regulatory connections. First, deletion of the helix-turn-helix domain of VieA in an hns mutant background diminished biofilm formation and exopolysaccharide gene expression, a function that phenotypically opposes its phosphodiesterase activity. Second, deletion of vieA in an hns mutant diminished the expression of σ^E, a virulence determinant that mediates the envelope stress response. hns mutants were highly sensitive to envelope stressors compared to wild-type. However, deletion of vieA in the hns mutant restored or exceeded wild-type resistance. These findings suggest an evolutionary path for the emergence of hypervirulent strains starting from nucleotide sequence diversification affecting the interaction of H-NS with DNA.

Full-Text

The current cholera pandemic is characterized by the predominance of Vibrio cholerae O1 of the El Tor biotype, which exhibits a low case-fatality rate compared to former classical biotype pandemic strains. Cholera continues to be a significant global health threat aggravated by the emergence of wave 3 hypervirulent atypical El Tor biotype vibrios [1, 2]. Genome sequencing has shown that most atypical El Tor environmental and clinical isolates (including 47 isolates from the 2010 Haiti outbreak [3]) harbour single nucleotide polymorphisms (SNPs) in the regulatory genes hns and vieA [1, 2, 4] encoding the histone-like nucleoid structuring protein (H-NS) and the response regulator VieA, respectively. The SNPs identified in hns and vieA resulted in G107S and L79F amino acid substitutions within their DNA binding and receiver domains, respectively [4]. Though the effect of these amino acid substitutions on protein function is not obvious, sequential transfer of these SNPs into the prototype wave 1 V. cholerae strain N16961 increased virulence in an additive manner [5]. Moreover, atypical El Tor strains carrying the hns SNPs exhibited phenotypes typical of hns mutants such as overexpression of cholera toxin and haemolysin [4, 6, 7]. Taken together, the ubiquity of SNPs in hns and vieA in wave 3 strains [3] and their stepwise contribution to virulence [4, 5] show that these mutational changes are significant to the observed evolution of El Tor biotype vibrios. However, the mechanism by which the regulatory function of these genes is interconnected remains unknown.

H-NS compacts and organizes the bacterial nucleoid and functions as a transcriptional repressor with a bias for laterally acquired genes [8, 9]. It also modulates the expression of numerous core genome genes by either binding to AT-rich sequences or affecting DNA shape [10]. RNA-Seq and chromatin immunoprecipitation (ChIP)-Seq in V. cholerae of the El Tor biotype showed that H-NS regulates 18 % of all predicted genes and associates with 6.3 % of the genome [7, 11]. In this biotype, H-NS was shown to repress virulence gene expression, biofilm development and rpoE, this latter gene encoding the extra-cytoplasmic RNA polymerase sigma subunit σ^E [6, 7, 12]. In addition, H-NS repressed vieA itself, which is part of an operon encoding the VieSAB signal transduction system [7]. This system consists of the hybrid sensor kinase VieS, the response regulator VieA and the auxiliary protein VieB. The VieA protein exhibits helix-turn-helix (HTH) and EAL domains, the latter acting as a cyclic diguanylate (c-di-GMP) phosphodiesterase (PDE) [13]. VieS acts through VieA to regulate gene expression by altering the c-di-GMP pool, while VieB associates with VieS to inhibit VieA phosphorylation [14, 15].

The vieSAB operon is expressed in classical biotype V. cholerae to regulate ≈ 10 % of the genome [16]. In this biotype, VieSAB negatively regulates the expression of genes required to make the biofilm exopolysacchide matrix [16–18]. In the El Tor biotype, the vieSAB operon is almost silent [7] due to its combined repression by H-NS and quorum sensing [19]. Consequently, vieA mutants do not exhibit consistent phenotypes in El Tor biotype vibrios. Nevertheless, the additive effect of introducing mutations in hns and vieA on the virulence of a prototype wave 1 El Tor strain [5] suggests that changes in H-NS activity could unmask hidden VieA regulatory connections. Thus, in this study we examine the role of VieA on (i) vibrio exopolysaccharide gene expression and (ii) the envelope stress response in an El Tor biotype hns mutant in which the vieSAB operon is expressed.

The construction of Δhns, ΔvieA, ΔvieSAB, ΔhnsΔvieA and ΔhnsΔvieSAB mutants has been described previously [7]. The above mutants were constructed in the genetic background of the El Tor biotype strain C7258ΔlacZ and the classical biotype strain O395ΔlacZ [7]. Similarly, the construction of vieA-, rpoE-, vpsA,-vpsL, vpsR- and vpsT-lacZ promoter fusions is described in [7, 12]. In addition, we constructed a vieA mutant lacking the VieA C-terminal HTH domain. To this end, the suicide vector pCVDVieA^ΔHTH [19] containing a 3′- truncated vieA allele was transferred by conjugation from Escherichia coli strain SM10λpir [20] to V. cholerae strain AJB80 (Δhns :: km ΔlacZ) [21]. Then, the vieA^ΔHTH mutant was isolated by allelic exchange and sucrose selection as previously described [21].

We first examined the capacity of the above mutants to form biofilms in 96-well polystyrene microtitre plates. Strains were allowed to form biofilms in Luria–Bertani (LB) medium and adherent vibrios were measured using the crystal violet staining method described previously [12]. The amount of crystal violet-stained material (OD₅₇₀) was normalized by bacterial growth (OD₆₀₀). As anticipated from previous studies [12, 18], Fig. 1(a) shows that deletion of vieA or hns enhanced biofilm formation in classical biotype V. cholerae. Deletion of hns and the entire vieSAB operon, which removes the inhibitory protein VieB, had an almost additive effect (Fig. 1a). This result is expected from the lack of VieA elevating the c-di-GMP pool, plus the hns mutation increasing the expression of the c-di-GMP receptor protein and biofilm activator VpsT [11]. Consistent with the silencing of vieA by H-NS and quorum sensing in El Tor biotype vibrios [19], its deletion did not affect biofilm formation (Fig. 1a). Surprisingly, deletion of vieA in the El Tor biotype hns background diminished biofilm formation (Fig. 1a). To further substantiate this result, we tested the expression of vpsA- and vpsL-lacZ promoter fusions in El Tor biotype Δhns, ΔhnsΔvieSAB and ΔhnsΔvieA mutants (Fig. 1b). β-Galactosidase was measured using the chromogenic substrate ortho-nitrophenyl-β-galactoside as an indicator of promoter activity and expressed as Miller units [22]. vpsA and vpsL are the first genes of the two operons (vpsABCDEFGHIJK and vpsLMNOPQ) required for the biosynthesis and export of the V. cholerae biofilm exopolysacchide matrix [23]. As shown in Fig. 1(b), deletion of vieA or vieSAB in the hns mutant significantly diminished the transcription of vpsA and vpsL. Furthermore, deletion of the DNA sequence encoding the VieA C-terminal HTH domain fully recapitulated the effect of deleting the entire vieSAB operon (Fig. 1b). The expression of vps genes is activated by the transcription factors VpsR and VpsT, which act to displace H-NS from vps promoters [11]. In Fig. 1(b) we show that deletion of DNA sequences encoding the VieA HTH domain diminished VpsR and VpsT expression. We note that the exact vieA^ΔHTH allele constructed for this study was found to encode an enzymatically active protein that negatively affected biofilm formation [18]. Thus, the function of VieA unmasked in the El Tor hns mutant appears to require DNA binding. We have not been able to demonstrate binding of VieA to the vpsR and vpsT promoters in vitro. We hypothesize that VieA regulation of VpsR and/or VpsT is either (i) indirect or (ii) VieA requires phosphorylation by VieS to bind DNA. We note that deletion of vieA in the El Tor hns mutant exhibited the opposite effect on biofilm formation compared to its inactivation in classical biotype vibrios. In the classical biotype, the negative effect of VieA on biofilm formation is explained by its PDE activity [14]. We suggest that the positive effect of VieA on biofilm formation in El Tor biotype Δhns vibrios, mediated by its HTH domain, overrides its negative effect on the c-di-GMP pool mediated by its EAL domain. Further studies are required to fully dissect the role of the VieA functional domains in the behaviour of El Tor biotype vibrios.

Fig. 1.

The VieSAB system positively affects biofilm formation and vibrio exopolysaccharide (vps) gene expression in the El Tor biotype hns genetic background. (a). Biofilm formation. Strains O395ΔlacZ (classical, Wt) and C7258ΔlacZ (El Tor, Wt) and their isogenic mutants were allowed to form biofilms as described in the text. Each bar represents the average of six independent cultures. Wt versus mutants (open bars) and Δhns versus double mutants (filled bars) were compared using an unpaired T-test (*P<0.05, **P<0.01). Error bars indicate the standard deviation (STDEV). (b). Expression of vps genes in El Tor biotype hns and vieA mutants. Strains C7258ΔlacZ (Wt), AJB80 (C7258ΔlacZΔhns) and isogenic ΔhnsΔvieA,ΔhnsΔvieSAB and Δhns vieA^ΔHTH mutants containing vpsA, vpsL-, vpsR- or vpsT-lacZ promoter fusions were grown to stationary phase in LB medium at 37 °C. β-Galactosidase activity (Miller units) was measured as an indicator of promoter function. Each bar represents the average of six independent cultures. Δhns versus double mutants was compared using an unpaired T-test (*P<0.01). Error bars indicate the STDEV.

A second example in which deletion of hns unmasks a silent regulatory connection relates to the envelope stress response mediated by the extra-cytoplasmic RNA polymerase alternative sigma factor σ^E [7] encoded by rpoE. In V. cholerae, σ^E is required for virulence in the suckling mouse cholera model [24]. Transcription of rpoE is initiated at two promoters: an upstream σ⁷⁰-dependent promoter (P₁) and a downstream σ^E-dependent promoter (P₂) [24]. Upon the occurrence of an envelope stress, σ^E is released from the cell membrane and activates its own P₂ promoter to make more σ^E [25]. A previous micro-array study showed that a classical biotype vieA mutant expressed diminished rpoE [16]. During infection, V. cholerae is subject to envelope stresses by bile and antimicrobial peptides, which compromise outer membrane integrity and induce the expression of rpoE [26]. Here we found that the activity of a vieA-lacZ promoter fusion was increased from 724±56 to 1306±85 Miller units (n=6) in classical biotype strain O395ΔlacZ grown to stationary phase in LB medium containing 0.05 % (w/v) of the bile salt sodium deoxycholate (Sigma Chemical Co.). Taken together, these findings suggest a connection between VieA and the envelope stress response in classical biotype vibrios. As noted above, deletion of vieA does not produce discernible phenotypes in the El Tor biotype. Thus, we tested the effect of deleting vieA on rpoE expression in the El Tor biotype hns mutant. To this end, we introduced promoter fusions encompassing both rpoEσ⁷⁰- and σ^E-dependent promoters (rpoE^P1P2-lacZ) and the rpoE-dependent promoter alone (rpoE^P2-lacZ) in El Tor biotype Δhns and ΔhnsΔvieA mutants. As previously reported, deletion of hns significantly enhanced both promoter activities compared to the wild-type strain (Fig. 2a). As also shown in Fig. 2(a), deletion of vieA from the hns mutant resulted in diminished activity of the rpoEσ^E-dependent promoter. These results identify VieA as a key participant of the envelope stress response in the El Tor biotype. We previously showed that the lack of H-NS in this biotype induces an endogenous envelope stress due to the overexpression of multiple outer membrane proteins [7], a condition known to induce the expression of rpoE [27]. Thus, we investigated the response of hns and vieA mutants to sodium deoxycholate and polymyxin B, two agents that interact with the outer membrane to induce envelope stress. To this end, we tested the growth of wild-type, Δhns, ΔvieA and ΔhnsΔvieA isogenic mutants at different concentrations of the above agents. Overnight cultures of each mutant were diluted 1 : 1000 in fresh LB medium and 0.1 ml of each dilution were added to microtitre plates containing 0.1 ml of twofold dilutions series of sodium deoxycholate (2.5 %) and polymyxin B (200 µg ml⁻¹) in LB medium. The plates were incubated at 37 °C for 16 h and growth was determined by reading the absorbance at 600 nm. The endpoint absorbance readings were normalized by the value obtained in the absence of the corresponding agents to cancel out intrinsic growth differences between the strains tested (relative growth). As shown in Fig. 2(b), the wild-type strain C7258ΔlacZ and its isogenic vieA mutant did not significantly differ in their sensitivity to sodium deoxycholate. This result is consistent with vieA being almost silent in the El Tor biotype [7, 16, 19]. In contrast, the hns mutant was significantly more sensitive to sodium deoxycholate compared to the wild-type and vieA strains (Fig. 2b). This result suggests that overexpression of rpoE in the hns mutant (partly due to derepression of vieA) is deleterious, rendering vibrios poorly responsive and more sensitive to subsequent membrane damage caused by sodium deoxycholate. Interestingly, the ΔhnsΔvieA double mutant exhibited similar or enhanced resistance to sodium deoxycholate compared to wild-type. This observation suggests that lowering rpoE expression by elimination of vieA in the hns genetic background has a compensatory effect that restores responsiveness and resistance to sodium deoxycholate. To further examine this behaviour, we tested the sensitivity of wild-type and mutant strains to a second envelope stressor, polymyxin B. Consistent with the minimal inhibitory concentration of polymyxin B for El Tor biotype V. cholerae reported in [28], all strains tested were resistant to this antibiotic at concentrations lower than 50 µg ml⁻¹ (Fig. 2c). No significant differences could be demonstrated between the wild-type and vieA mutant across the entire concentration range tested (Fig. 2c). Similarly to that observed for sodium deoxycholate, the hns mutant was more sensitive to polymyxin B at high concentrations while subsequent deletion of vieA restored or exceeded the wild-type resistance of V. cholerae to this antibiotic. Taken together, these results suggest a possible evolutionary path for the emergence of wave 3 hypervirulent atypical El Tor biotype strains harbouring mutations in hns and vieA. It is well established that deletion of hns results in elevated expression of major virulence factors such as cholera toxin and the toxin co-regulated pilus, as well as the pore-forming haemolysin/cytolysin (HlyA) and the multifunctional autoprocessing repeats-in toxin (MARTX) [6, 7]. However, a disadvantage of hns mutants in the human gut is their elevated susceptibility to envelope stressors such as bile and antimicrobial peptides (Fig. 2b, c). Thus, our results suggest that secondary mutations blocking VieA signalling could restore wild-type resistance to the above envelope stressors, resulting in an overall hypervirulent phenotype.

Fig. 2.

VieA enhances the expression of the alternative RNA polymerase sigma subunit σ^E. (a). Strains C7258ΔlacZ (Wt), AJB80 (C7258ΔlacZΔhns) and isogenic ΔhnsΔvieA mutants containing rpoE^P1P2- and rpoE^P2-lacZ promoter fusions were grown to stationary phase in LB medium at 37 °C, and β-galactosidase activity (Miller units) was measured as an indicator of promoter function. Each bar represents the average of six independent cultures. Δhns versus double mutants was compared using an unpaired T-test (*P<0.01). Error bars indicate the STDEV. (b). Strains C7258ΔlacZ (Wt) and isogenic ΔvieA, Δhns, ΔhnsΔvieA mutants were grown in the presence of different concentrations of sodium deoxycholate. (c). Strains C7258ΔlacZ (Wt) and isogenic ΔvieA, Δhns, ΔhnsΔvieA mutants were grown in the presence of different concentrations of polymyxin B. The data shown in panels (b and c) are the average of three independent cultures tested in triplicate. Absorbance readings at increasing concentrations of each envelope stressor were normalized by the reading in the absence of sodium deoxycholate or polymyxin B (relative growth). Significance: *ΔhnsΔvieA>Δhns, P<0,01; ** Δhns<Wt, P<0.01.

This report also suggests that H-NS coverage of the core genome could result in the silencing of multiple regulatory connections. In Fig. 3 we illustrate this function as it applies to the H-NS – VieSAB regulatory axis. The simplest mechanism by which H-NS could exert this function is by binding to promoter elements of genes for which an anti-repression mechanism has not evolved (or has not been discovered). A similar mechanism has been used to explain H-NS protection of DNA sequences from mariner transposon insertion [29]. Based on the ubiquity of H-NS and its paralogues, we suggest that the masking of regulatory connections by nucleoid-associated proteins is a widespread phenomenon with evolutionary implications. Hidden regulatory pathways could become operative through natural selection of mutations tempering the DNA binding activity of nucleoid-associated proteins.

Fig. 3.

Silencing of regulatory pathways in El Tor biotype V. cholerae by the nucleoid associated protein H-NS. H-NS represses vieA, vps genes and σ^E expression. Repression of vps genes by H-NS is antagonized when the c-di-GMP pool is elevated [11]. In the absence of H-NS, vieA is expressed to diminish the c-di-GMP pool through its EAL domain; enhance VpsT and VpsR expression through its HTH domain, and augment σ^E transcription. We suggest that in El Tor biotype vibrios, the positive effect of VieA on vps expression mediated by its HTH-domain is larger than its negative effect on the c-di-GMP pool mediated by its EAL domain. Novel regulatory connections that are silent in the presence of H-NS are shadowed. These connections could be enabled through the natural selection of mutations tempering H-NS DNA binding activity. Symbols: →, positive regulation; ⊥, negative regulation.