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YvqE and CovRS of Group A Streptococcus Play a Pivotal Role in Viability and Phenotypic Adaptations to Multiple Environmental Stresses

  • Amonrattana Roobthaisong,

    Affiliations Section of Molecular Craniofacial Embryology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan, Department of Microbiology, Graduate School of Medicine, Kyoto University, Kyoto, Japan

  • Chihiro Aikawa,

    Affiliation Department of Microbiology, Graduate School of Medicine, Kyoto University, Kyoto, Japan

  • Takashi Nozawa,

    Affiliation Department of Microbiology, Graduate School of Medicine, Kyoto University, Kyoto, Japan

  • Fumito Maruyama,

    Affiliation Department of Microbiology, Graduate School of Medicine, Kyoto University, Kyoto, Japan

  • Ichiro Nakagawa

    nakagawa.ichiro.7w@kyoto-u.ac.jp

    Affiliation Department of Microbiology, Graduate School of Medicine, Kyoto University, Kyoto, Japan

YvqE and CovRS of Group A Streptococcus Play a Pivotal Role in Viability and Phenotypic Adaptations to Multiple Environmental Stresses

  • Amonrattana Roobthaisong, 
  • Chihiro Aikawa, 
  • Takashi Nozawa, 
  • Fumito Maruyama, 
  • Ichiro Nakagawa
PLOS
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Abstract

Streptococcus pyogenes (group A Streptococcus, or GAS) is a human pathogen that causes a wide range of diseases. For successful colonization within a variety of host niches, GAS utilizes TCSs to sense and respond to environmental changes and adapts its pathogenic traits accordingly; however, many GAS TCSs and their interactions remain uncharacterized. Here, we elucidated the roles of a poorly characterized TCS, YvqEC, and a well-studied TCS, CovRS, in 2 different GAS strain SSI-1 and JRS4, respectively. Deletion of yvqE and yvqC in JRS4 resulted in lower cell viability and abnormality of cell division when compared to the wild-type strain under standard culture conditions, demonstrating an important role for YvqEC. Furthermore, a double-deletion of yvqEC and covRS in SSI-1 and JRS4 resulted in a significantly impaired ability to survive under various stress conditions, as well as an increased sensitivity to cell wall-targeting antibiotics compared to that observed in either single mutant or wild-type strains suggesting synergistic interactions. Our findings provide new insights into the impact of poorly characterized TCS (YvqEC) and potential synergistic interactions between YvqEC and CovRS and reveal their potential role as novel therapeutic targets against GAS infection.

Introduction

Streptococcus pyogenes (group A Streptococcus; GAS) is an important human pathogen that causes a wide spectrum of diseases, ranging from mild infections of the upper respiratory tract (pharyngitis) and skin (impetigo) to more serious invasive infections such as necrotizing fasciitis and streptococcal toxic shock syndrome (STSS) [1]. To successfully colonize and persist in a number of physiologically distinct host sites, GAS has developed complex mechanisms to cope with various environmental stresses. In many other gram-positive bacteria, sigma factors have been shown to play an essential role in the regulation of virulence genes in response to stress or growth [2]. However, GAS does not encode alternative sigma factors for regulating virulence gene expression [3]. Two-component systems (TCSs) are typically composed of two proteins, a sensor histidine kinase (HK) and its cognate response regulator (RR). TCSs are typically involved in various physiological functions in bacteria, such as survival, motility, metabolism, antibiotic resistance, stress response, and virulence, by sensing changes in the external or internal environment, modulating gene expression in response to a variety of stimuli inside the host cells, and avoiding host immune systems [4].

The available genome sequences indicate the presence of 12 cognate HK and RR pairs that are well conserved in all the strains of GAS [5]. Of these, the CovRS TCS is one of the most thoroughly studied regulatory systems due to its central role in the pathogenesis of GAS. In contrast to most TCSs, the results of several studies have indicated that the CovR/S TCS shows contradictory functions, such a Janus-like behaviour in the regulation of GAS virulence gene expression [6]. Under normal conditions, CovS acts in as a kinase to activate CovR and repress the expression of virulence factors but simultaneously acts as a phosphatase to permit gene expression in response to environmental changes during human infection. Moreover, CovR functions independently, and can still exert some of its regulatory function even in the absence of CovS [7]. CovR/S regulates the expression of about 15% of genes in the serotype M1 GAS genome [8] and mediates a general stress response to changing temperature, pH, and osmolarity [7]. In contrast to CovRS, most of TCSs in GAS, including YvqEC, remain functionally poorly characterized. To our knowledge, only a recent study has primarily focused on YvqE (HK) and reported that yvqE knockout (spy1622) in the GAS M1 clinical strain 1529, isolated from an STSS patient, resulted in growth reduction under acid conditions (pH 6.0) and reduced virulence in a mouse infection model [9]. However, relatively few have studied the YvqEC system in GAS, its physiological functions, underlying mechanisms, and cellular responses.

The YvqEC of GAS shows high sequence homology with LiaSR in Bacillus subtilis [10], Streptococcus pneumoniae [11], Streptococcus mutans [12, 13], Streptococcus agalactiae [14], and Listeria monocytogenes [15], with VraSR in Staphylococcus aureus [16, 17], and with CesSR in Lactococcus lactis [18]. Several studies have reported that the function of these systems seems to be a part of a complex regulatory network that counteracts cell envelope stress by maintaining its integrity under different stress conditions [10, 19], and that all of them are involved in species-specific responses to a variety of cell envelope-damaging agents, including cell wall-targeting antibiotics and antimicrobial peptides [20, 21]. Another recent study reported that deletion of either yvqE or yvqEC in Bacillus thuringiensis increases its susceptibility to vancomycin and suggested that YvqEC system plays a role in vancomycin resistance [22].

Strains SSI-1 and JRS4 are representative of GAS invasive and non-invasive strains, respectively. The invasive STSS strain SSI-1 (serotype M3) has been shown to contain a large-scale genomic rearrangement and differ from other GAS strains [23], suggesting that SSI-1 acquired strain-specific virulence factors. Moreover, the non-invasive isolate JRS4 (serotype M6) [24], is most widely used in studies of bacteria-host cell interactions [25, 26]. In the present study, we sought to characterize the phenotypes associated with a series of yvqE/C and covR/S TCSs mutants in the GAS strains SSI-1 and JRS4