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

Abstract

Serotype M28 group A streptococcus (GAS) is a common cause of infections such as pharyngitis ("strep throat") and necrotizing fasciitis ("flesh-eating" disease). Relatively little is known about the molecular mechanisms underpinning M28 GAS pathogenesis. Whole-genome sequencing studies of M28 GAS strains recovered from patients with invasive infections found an unexpectedly high number of missense (amino acid-changing) and nonsense (protein-truncating) polymorphisms in (egulator fov), leading us to hypothesize that altered RocA activity contributes to M28 GAS molecular pathogenesis. To test this hypothesis, an isogenic deletion mutant strain was created. Transcriptome sequencing (RNA-seq) analysis revealed that RocA inactivation significantly alters the level of transcripts for 427 and 323 genes at mid-exponential and early stationary growth phases, respectively, including genes for 41 transcription regulators and 21 virulence factors. In contrast, RocA transcriptomes from other GAS M protein serotypes are much smaller and include fewer transcription regulators. The mutant strain had significantly increased secreted activity of multiple virulence factors and grew to significantly higher colony counts under acid stress RocA inactivation also significantly increased GAS virulence in a mouse model of necrotizing myositis. Our results demonstrate that RocA is an important regulator of transcription regulators and virulence factors in M28 GAS and raise the possibility that naturally occurring polymorphisms in in some fashion contribute to human invasive infections caused by M28 GAS strains.

Polymorphisms in Regulator of Cov Contribute to the Molecular Pathogenesis of Serotype M28 Group A Streptococcus

“…45 Our interest in RocA is based on serotype M28 GAS whole-genome sequencing studies of strains collected from human invasive infections. 43,46 Although previous molecular pathogenesis studies bearing on RocA have investigated strains with laboratory-generated gene deletions and protein truncations, 39e44, 47 we discovered that the number of missense (amino acidealtering) and nonsense (protein-truncating) polymorphisms in rocA within the M28 population studied was higher than expected ( Figure 1A). 43,46 We hypothesized that naturally occurring polymorphisms in rocA result in altered RocA function and contribute to the molecular pathogenesis of serotype M28 GAS invasive infections.…”

“…43,46 Although previous molecular pathogenesis studies bearing on RocA have investigated strains with laboratory-generated gene deletions and protein truncations, 39e44, 47 we discovered that the number of missense (amino acidealtering) and nonsense (protein-truncating) polymorphisms in rocA within the M28 population studied was higher than expected ( Figure 1A). 43,46 We hypothesized that naturally occurring polymorphisms in rocA result in altered RocA function and contribute to the molecular pathogenesis of serotype M28 GAS invasive infections. To test this hypothesis, clinical isolates and isogenic mutant strains were used to perform genome-wide transcript analysis [RNA sequencing (RNA-seq)], in vitro virulence factor assays, and mouse and nonhuman primate (NHP) pathogenesis studies.…”

“…Clinical Isolates Grown in Duplicate GAS strains were grown in duplicate at 37 C with 5% CO 2 in Todd-Hewitt broth with 0.2% yeast extract (THY) to midexponential (ME; OD 600 Z 0.5) and early-stationary (ES; OD 600 Z 1.65) growth phases, as previously described. 43,48,49 RNAprotect Bacteria Reagent (Qiagen Inc., Germantown, MD) was added at a 2:1 ratio, and then cells were lysed by ballistic disintegration (FastPrep-96 instrument and lysing matrix B; MP Biomedicals, Santa Ana, CA). RNA was extracted using standard methods (RNeasy kit; Qiagen Inc.), and then RNA quality and quantity were assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) and Qubit (Invitrogen, Carlsbad, CA), respectively.…”

“…Daggers indicate one strain has two polymorphisms in rocA. Adapted from Bernard et al 43 Copyright ª American Society for Microbiology. B and C: Three-dimensional principal component (PC) analysis of the RNA-sequencing data generated with the clinical isolates with naturally occurring rocA polymorphisms, an isogenic rocA deletion mutant (DrocA) strain, and four phylogenetically matched wild-type (WT) strains at midexponential (B) and early-stationary (C) growth phases.…”

Gene fitness landscape of group A streptococcus during necrotizing myositis

“…Of note, rocA and ppiB (peptidyl-prolyl cis-trans isomerase) were identified in both the serotype M1 and M28 strains (Figure 3B, Table S3, Table S5). Inactivation of the rocA gene in an M28 GAS strain was recently shown to significantly increase virulence in a mouse model of necrotizing myositis (66).…”