Direct Binding and Regulation by Fur and HapR of the Intermediate Regulator and Virulence Factor Genes Within the ToxR Virulence Regulon in Vibrio cholerae.

He Gao,Yiquan Zhang,Jie Li,Jingyun Zhang,Jing Lou,Biao Kan,Qin Qin,Qiannan Shi

doi:10.3389/fmicb.2020.00709

Abstract

Cholera toxin (CT) and toxin coregulated pilus (TCP, TcpA is the major subunit) are two major virulence factors of Vibrio cholerae, both of which play critical roles in developing severe diarrhea in human. Expression of CT and TCP is under the tight control of the regulatory cascade known as the ToxR virulence regulon, which is composed of three regulators ToxR, TcpP, and ToxT. Besides, their expression is also regulated by the quorum sensing (QS) master regulator HapR and the regulatory protein Fur. Though transcription of tcpP, toxT, and/or tcpA are reported to be regulated by HapR and Fur, to date there are no studies to verify their direct regulations. In the present study, we showed that HapR directly repress the transcription of tcpP and tcpA by binding to their promoter regions, and possibly repress toxT transcription in an indirect manner. Fur directly activated the transcription of tcpP, toxT, and tcpA by binding to their promoters. Taking account of the sequential expression of hapR, fur, tcpP, toxT, and tcpA in the different growth phases of V. cholerae, we deduce that at the early mid-logarithmic growth phase, Fur binds to the promoters of tcpP, toxT, and tcpA to activate their transcription; while at the later mid-logarithmic growth phase, HapR can bind to the promoters of tcpP and tcpA to repress their transcription. Our study reveals the new recognition in the virulence regulatory pathways in V. cholerae and suggests the complicated and subtle regulation network with the growth density dependence.

Highlights

Vibrio cholerae is a Gram-negative bacterium that naturally inhabits salty coastal waters and estuaries (Clemens et al, 2017), and some are the causative agent of cholera
Expression of Fur itself was directly and negatively regulated by HapR in V. cholerae (Gao et al, 2018), we speculated that the transcription of tcpP, toxT, and tcpA might be regulated under the collective effects of HapR and Fur in a subtle manner in V. cholerae
The Quantitative PCR (qPCR) assay was employed to investigate the regulatory effects of Fur on the transcription of toxR, tcpP, toxT, and tcpA, and the results showed that the mRNA levels of tcpP, toxT, and tcpA were obviously decreased in fur relative to WT (Figure 4A), while that of toxR manifested no obvious difference between fur and WT (Supplementary Figure S2A)

Summary

Introduction

Vibrio cholerae is a Gram-negative bacterium that naturally inhabits salty coastal waters and estuaries (Clemens et al, 2017), and some are the causative agent of cholera. Fur/HapR Regulate V. cholerae Virulence (Merritt and Hol, 1995) It can enhance the concentration of intracellular cyclic AMP, which causes an imbalance in electrolyte transport across the intestinal epithelial cell membrane, resulting in the secretion of water and electrolytes into the bowel accompanied by severe watery diarrhea, which may lead to death without timely treatment (Clemens et al, 2017). TCP, the subunit of which is encoded by tcpA (in the tcpABQCRDSTEF operon), is essential for colonization of V. cholerae in the small intestine at the early stage of infection (Kirn et al, 2000) It functions as the receptor for the CTX (Waldor and Mekalanos, 1996)

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Results

Conclusion