Null Mutations of Group A Streptococcus Orphan Kinase RocA: Selection in Mouse Infection and Comparison with CovS Mutations in Alteration of In Vitro and In Vivo Protease SpeB Expression and Virulence

Abstract

Group A Streptococcus (GAS) acquires mutations of the virulence regulator CovRS in human and mouse infections, and these mutations result in the upregulation of virulence genes and the downregulation of the protease SpeB. To identify in vivo mutants with novel phenotypes, GAS isolates from infected mice were screened by enzymatic assays for SpeB and the platelet-activating factor acetylhydrolase Sse, and a new type of variant that had enhanced Sse expression and normal levels of SpeB production was identified (the variants had a phenotype referred to as enhanced Sse activity [Sse Aϩ ] and normal SpeB activity [SpeB Aϩ ]). Sse Aϩ SpeB Aϩ variants had transcript levels of CovRS-controlled virulence genes comparable to those of a covS mutant but had no covRS mutations. Genome resequencing of an Sse Aϩ SpeB Aϩ isolate identified a C605A nonsense mutation in orphan kinase gene rocA, and 6 other Sse Aϩ SpeB Aϩ isolates also had nonsense mutations or small indels in rocA. RocA and CovS mutants had similar levels of enhancement of the expression of CovRS-controlled virulence genes at the exponential growth phase; however, mutations of RocA but not mutations of CovS did not result in the downregulation of speB transcription at stationary growth phase or in subcutaneous infection of mice. GAS with RocA and CovS mutations caused greater enhancement of the expression of hasA than spyCEP in mouse skin infection than wild-type GAS did. RocA mutants ranked between wild-type GAS and CovS mutants in skin invasion, inhibition of neutrophil recruitment, and virulence in subcutaneous infection of mice. Thus, GAS RocA mutants can be selected in subcutaneous infections in mice and exhibit gene expression patterns and virulences distinct from those of CovS mutants. The findings provide novel information for understanding GAS fitness mutations in vivo, virulence gene regulation, in vivo gene expression, and virulence.KEYWORDS CovRS, PAF acetylhydrolase, RocA, SpeB, Streptococcus pyogenes, group A Streptococcus, in vivo expression, mutation, virulence, virulence regulation T he human pathogen group A Streptococcus (GAS) commonly causes pharyngitis and superficial skin infections. GAS also causes severe invasive infections, such as necrotizing fasciitis and sepsis. The most recently available data indicate that from 2005 to 2012 severe invasive infections in the United States were most frequently associated with GAS of the M protein serotypes M1, M12, M28, M89, and M3 (1), and the currently circulating M1 GAS belongs to the pandemic M1T1 clone (2). GAS produces an

RocA Is an Accessory Protein to the Virulence-Regulating CovRS Two-Component System in Group A Streptococcus

“…2). The altered virulence factor expression profile due to rocA mutation results in an enhanced ability to cause invasive infections, as is evident from previous animal infection data (22,24,44) and also our blood bactericidal assays (Fig. 1B).…”

“…While serotype M3 and M18 GAS are the only known serotypes that are exclusively rocA mutant, individual isolates of other serotypes have also been identified as rocA mutant (22-25, 44, 47, 48). Data from several studies are consistent with rocA mutant strains being selected for from rocA ϩ parental isolates, and that selection primarily occurs during invasive GAS infection (44,(47)(48)(49). This mirrors the more than 15 years worth of data concerning the selection of covR or covS mutant strains during invasive GAS infections (27)(28)(29)(30)(31)(32)(33)(34)50).…”

“…The mutation of any one of the rocA, covR, and covS genes results in a virulence factor expression profile that promotes invasive disease, but undoubtedly there are important regulatory and virulence differences. The abundance of CovRϳP appears to be a key correlate of invasive infection virulence, as there is an inverse correlation between levels of CovRϳP (parental [wild type {WT}] Ͼ rocA mutant Ͼ covS mutant Ͼ covR mutant) and virulence (parental [WT] Ͻ rocA mutant Ͻ covS mutant Ͻ covR mutant) (30,44). While not as extensively studied (32, 51, 52), we hypothesize that there is a positive correlation between the abundance of CovRϳP and GAS virulence during noninvasive infections (parental [WT] Ͼ rocA mutant Ͼ covS mutant Ͼ covR mutant).…”

RocA Has Serotype-Specific Gene Regulatory and Pathogenesis Activities in Serotype M28 Group A Streptococcus

“…Streptococcal secreted esterase (SSE) is a known secreted GAS virulence factor that hydrolyzes platelet-activating factor (PAF) (80). Thus, SSE activity in culture supernatants collected at mid-exponential growth phase was assayed with the PAF acetylhydrolase assay kit (Cayman Chemical, Ann Arbor, MI), according to the manufacturer's instructions with minor modifications (31). GAS strains were grown in triplicate in THY to mid-exponential growth phase.…”

“…Similarly, RocA is inactivated in all serotype M3 strains by a single nucleotide deletion that introduces a frameshift mutation, and restoration of RocA by introduction of the serotype M1 wild-type rocA allele decreases M3 GAS virulence in a mouse model of bacteremia (26)(27)(28). Deletion of rocA in serotype M1, M3, M6, M14, M18, and M89 GAS results in increased expression of virulence factors known to be regulated by the CovRS system (26)(27)(28)(29)(30)(31)(32). Although the molecular mechanism for RocA function has not been determined, RocA increases phosphorylation of the DNA binding response regulator CovR in the presence of its cognate sensor histidine kinase CovS, resulting in CovR activation (28,30).…”