DksA-HapR-RpoS axis regulates haemagglutinin protease production in Vibrio cholerae

Abstract

DksA acts as a co-factor for the intracellular small signalling molecule ppGpp during the stringent response. We recently reported that the expression of the haemagglutinin protease (HAP), which is needed for shedding of the cholera pathogen Vibrio cholerae during the late phase of infection, is significantly downregulated in V. cholerae ∆dksA mutant (∆dksAVc) cells. So far, it has been shown that HAP production by V. cholerae cells is critically regulated by HapR and also by RpoS. Here, we provide evidence that V. cholerae DksA (DksAVc) positively regulates HapR at both the transcriptional and post-transcriptional levels. We show that in ∆dksAVc cells the CsrB/C/D sRNAs, required for the maintenance of intracellular levels of hapR transcripts during the stationary growth, are distinctly downregulated. Moreover, the expression of exponential phase regulatory protein Fis, a known negative regulator of HapR, was found to continue even during the stationary phase in ∆dksAVc cells compared to that of wild-type strain, suggesting another layer of complex regulation of HapR by DksAVc. Extensive reporter construct-based and quantitative reverse-transcriptase PCR (qRT-PCR) analyses supported that RpoS is distinctly downregulated at the post-transcriptional/translational levels in stationary phase-grown ∆dksAVc cells. Since HAP expression through HapR and RpoS is stationary phase-specific in V. cholerae, it appears that DksAVc is also a critical stationary phase regulator for fine tuning of the expression of HAP. Moreover, experimental evidence provided in this study clearly supports that DksAVc is sitting at the top of the hierarchy of regulation of expression of HAP in V. cholerae.

Functional Insights Into the Role of gppA in (p)ppGpp Metabolism of Vibrio cholerae

“…For reverse transcriptase PCR (RT-PCR) assay, V. cholerae cells were grown in LB at an OD 600 value of ∼1.0 followed by extraction of total cellular RNA using TRI Reagent (Sigma-Aldrich, United States) essentially as described previously (Pal et al, 2012;Basu et al, 2017;Basu and Bhadra, 2019). Purity and quantitation of prepared RNA were done spectrophotometrically.…”

Regulatory Hierarchies Controlling Virulence Gene Expression in Shigella flexneri and Vibrio cholerae

“…The HapR and AphA proteins play reciprocal roles in the response of V. cholerae to cell density: AphA operates at low cell density and HapR takes over the role of master regulator at high cell density. At low cell density, AphA production is upregulated by sRNAs and then AphA and Hfq-dependent sRNAs repress production of HapR ( Lenz et al, 2005 ; Lenz and Bassler, 2007 ; Rutherford et al, 2011 ; Basu et al, 2017 ) (Figure 4 ). These (redundant) sRNA molecules are four Quorum Regulatory RNAs (Qrr) and they act with Hfq to repress production of HapR.…”

“…V. cholerae also has a GTP-binding protein called CgtA that modulates the function of SpoT, repressing the stringent response by keeping ppGpp levels low ( Raskin et al, 2007 ). DksA regulates the production of the hemagglutinin/protease (HAP) that is involved in the shedding stage of the V. cholerae infection ( Basu et al, 2017 ). HAP production is controlled by HapR and RpoS with DksA controlling HapR production positively at the transcriptional and post-transcriptional levels.…”

“…The post-transcriptional regulation involves Fis-dependent Quorum Regulatory RNAs (Qrr) sRNAs and the levels of these regulatory RNA molecules are strongly reduced in the absence of DksA ( Lenz et al, 2005 ; Lenz and Bassler, 2007 ). Furthermore, DksA is required for normal levels of RpoS production in stationary phase V. cholerae cells ( Basu et al, 2017 ). The counterpart to CgtA in E. coli (and Shigella ) is the evolutionarily highly conserved Obg GTPase ( Dewachter et al, 2016 ).…”

“…HapR and quorum sensing have been reported to enhance the production of RpoS ( Joelsson et al, 2007 ) and RpoS plays a positive role in the expression of the HapR-dependent V. cholerae hapA gene, encoding a hemagglutinin/protease ( Silva and Benitez, 2004 ) (Figure 4 ). This enzyme plays an important part in the bacterial shedding phase that occurs toward the end of the infection ( Basu et al, 2017 ). Transcription of the rpoS gene is repressed by the VpsT transcription factor, in association with the second messenger cyclic-di-GMP, when V. cholerae is within biofilm ( Wang et al, 2014 ) (Figure 5 ).…”