CovS inactivation reduces CovR promoter binding at diverse virulence factor encoding genes in group A Streptococcus.

Nicola Horstmann,Kevin S Myers,Anthony R Flores,Samuel A Shelburne Iii,Chau Nguyen Tran,Carlos Javier Orihuela

doi:10.1371/journal.ppat.1010341

Abstract

The control of virulence gene regulator (CovR), also called caspsule synthesis regulator (CsrR), is critical to how the major human pathogen group A Streptococcus fine-tunes virulence factor production. CovR phosphorylation (CovR~P) levels are determined by its cognate sensor kinase CovS, and functional abrogating mutations in CovS can occur in invasive GAS isolates leading to hypervirulence. Presently, the mechanism of CovR-DNA binding specificity is unclear, and the impact of CovS inactivation on global CovR binding has not been assessed. Thus, we performed CovR chromatin immunoprecipitation sequencing (ChIP-seq) analysis in the emm1 strain MGAS2221 and its CovS kinase deficient derivative strain 2221-CovS-E281A. We identified that CovR bound in the promoter regions of nearly all virulence factor encoding genes in the CovR regulon. Additionally, direct CovR binding was observed for numerous genes encoding proteins involved in amino acid metabolism, but we found limited direct CovR binding to genes encoding other transcriptional regulators. The consensus sequence AATRANAAAARVABTAAA was present in the promoters of genes directly regulated by CovR, and mutations of highly conserved positions within this motif relieved CovR repression of the hasA and MGAS2221_0187 promoters. Analysis of strain 2221-CovS-E281A revealed that binding of CovR at repressed, but not activated, promoters is highly dependent on CovR~P state. CovR repressed virulence factor encoding genes could be grouped dependent on how CovR~P dependent variation in DNA binding correlated with gene transcript levels. Taken together, the data show that CovR repression of virulence factor encoding genes is primarily direct in nature, involves binding to a newly-identified DNA binding motif, and is relieved by CovS inactivation. These data provide new mechanistic insights into one of the most important bacterial virulence regulators and allow for subsequent focused investigations into how CovR-DNA interaction at directly controlled promoters impacts GAS pathogenesis.

Highlights

The capacity of bacteria to cause infection is closely linked to their ability to modulate gene expression in response to environmental stimuli
The OmpR/PhoB family member control of virulence regulator (CovR) is the master virulence factor controller in group A Streptococcus (GAS), a bacterium which commonly causes a diverse array of human infections
Our findings show that control of virulence gene regulator (CovR)-repressed virulence factor encoding genes are directly regulated by CovR and that CovS inactivation markedly reduces CovR binding at CovR-repressed promoters

Summary

Introduction

The capacity of bacteria to cause infection is closely linked to their ability to modulate gene expression in response to environmental stimuli. The ability of GAS to adapt to changing environmental conditions is fundamental to its pathogenicity and is facilitated by the coordinated expression of a large array of virulence factors. Model organisms such as Escherichia coli and Bacillus subtilis use alternative sigma factors as a major mechanism of controlling gene expression [2]. There are thirteen conserved TCS in GAS, the control of virulence (CovRS) system, known as capsule synthesis regulator (CsrRS), has long been recognized as the most important to GAS pathogenesis given its impact on a diverse array of critical virulence factor encoding genes [5,6]. In addition to its role in GAS pathogenesis, CovR is highly conserved amongst β-hemolytic streptococci, and CovR homologues are present in a wide range of important human pathogens, such as Staphylococcus aureus [11,12,13]

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