Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Fri, 14 Dec 2018 17:35:41 DksA-HapR-RpoS axis regulates haemagglutinin protease production in Vibrio cholerae Pallabi Basu, Ritesh Ranjan Pal,† Shreya Dasgupta‡ and Rupak K. Bhadra* 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 DdksA mutant (DdksA Vc ) 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 (DksA Vc ) positively regulates HapR at both the transcriptional and post-transcriptional levels. We show that in DdksA Vc 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 DdksA Vc cells compared to that of wild-type strain, suggesting another layer of complex regulation of HapR by DksA Vc . 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 DdksA Vc cells. Since HAP expression through HapR and RpoS is stationary phase-specific in V. cholerae, it appears that DksA Vc 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 DksA Vc is sitting at the top of the hierarchy of regulation of expression of HAP in V. cholerae. INTRODUCTION In order to survive and grow in any environment, micro- organisms must regulate the expression of a wide variety of genes involving multiple genetic circuits. For pathogens, the situation is further complicated due to the presence of multi- ple virulence genes and their coordinated expression in a host environment. Since the evolution of bacterial pathogens is believed to be due to the acquisition of virulence genes/cas- settes by innocuous progenitor cells, it is, therefore, highly expected that survival, growth and virulence should also be under critical regulations interlinking many ancestral gene regulatory networks. Inside the host, pathogens face a plethora of physicochemical challenges, including nutritional scarcity. Nutritional insufficiency is an important signal in any living organism and about which our knowledge is still limited. Nutritional deficiency in prokaryotes evokes the stringent response, a well-studied regulatory network involving several genes, and the situation is efficiently managed through the generation of two intracellular small molecules, guanosine 3¢-diphosphate 5¢-triphosphate (pppGpp) and guanosine 3¢,5¢-bis(diphosphate) (ppGpp), together called (p)ppGpp [1– 4]. However, several lines of evidence suggest that (p)ppGpp alone is unable to control gene expression; instead, a 17.5 kDa small protein molecule, called DksA, is needed as a co-factor [5, 6]. Interestingly, like (p)ppGpp, DksA also binds with the secondary channel of RNA polymerase and thus it is an unusual transcription factor [5–8]. Apart from regulation of the stringent response, DksA has been shown to be needed for expression of several virulence genes in different bacterial pathogens [9–15]. DksA has been shown to be involved in the regulation of quorum sensing (QS) in the opportunistic human pathogen Pseudo- monas aeruginosa [11, 16]. In Vibrio cholerae, QS is a highly regulated process [17]. At low cell density, the intracellular concentration of QS master regulator HapR is low, which allows cells to express major virulence determinants like Received 28 December 2016; Accepted 30 March 2017 Author affiliation: Infectious Diseases and Immunology Division, CSIR-Indian Institute of Chemical Biology, Kolkata-700 032, India. *Correspondence: Rupak K. Bhadra, rupakbhadra@iicb.res.in Keywords: DksA; HapR; RpoS; HAP; CsrBCD; Fis; stationary phase. Abbreviations: CT, cholera toxin; HAP, haemagglutinin protease; HCD, high cell density; LA, Luria agar; LB, Luria broth; ONPG, O-nitrophenyl-b-D-gal- actopyranoside; ppGpp, guanosine 3¢,5¢-bis(diphosphate); pppGpp, guanosine 3¢-diphosphate 5¢-triphosphate; Qrr, quorum regulatory RNA; qRT-PCR, quantitative reverse transcriptase-PCR; QS, quorum sensing; TCP, toxin co-regulated pilus. †Present address: Department of Microbiology and Molecular Genetics, Hebrew University of Jerusalem, Israel. ‡Present address: Department of Clinical Medicine, National Institute of Cholera and Enteric Diseases, India. One supplementary table is available with the online Supplementary Material. RESEARCH ARTICLE Basu et al., Microbiology 2017;163:900–910 DOI 10.1099/mic.0.000469 000469 ã 2017 The Authors 900