Abstract
Inactivating mutations in the control of virulence two-component regulatory system (covRS) often account for the hypervirulent phenotype in severe, invasive group A streptococcal (GAS) infections. As CovR represses production of the anti-phagocytic hyaluronic acid capsule, high level capsule production is generally considered critical to the hypervirulent phenotype induced by CovRS inactivation. There have recently been large outbreaks of GAS strains lacking capsule, but there are currently no data on the virulence of covRS-mutated, acapsular strains in vivo. We investigated the impact of CovRS inactivation in acapsular serotype M4 strains using a wild-type (M4-SC-1) and a naturally-occurring CovS-inactivated strain (M4-LC-1) that contains an 11bp covS insertion. M4-LC-1 was significantly more virulent in a mouse bacteremia model but caused smaller lesions in a subcutaneous mouse model. Over 10% of the genome showed significantly different transcript levels in M4-LC-1 vs. M4-SC-1 strain. Notably, the Mga regulon and multiple cell surface protein-encoding genes were strongly upregulated–a finding not observed for CovS-inactivated, encapsulated M1 or M3 GAS strains. Consistent with the transcriptomic data, transmission electron microscopy revealed markedly altered cell surface morphology of M4-LC-1 compared to M4-SC-1. Insertional inactivation of covS in M4-SC-1 recapitulated the transcriptome and cell surface morphology. Analysis of the cell surface following CovS-inactivation revealed that the upregulated proteins were part of the Mga regulon. Inactivation of mga in M4-LC-1 reduced transcript levels of multiple cell surface proteins and reversed the cell surface alterations consistent with the effect of CovS inactivation on cell surface composition being mediated by Mga. CovRS-inactivating mutations were detected in 20% of current invasive serotype M4 strains in the United States. Thus, we discovered that hypervirulent M4 GAS strains with covRS mutations can arise in an acapsular background and that such hypervirulence is associated with profound alteration of the cell surface.
Highlights
Invasive bacterial infections afflict >100,000 persons per year in the United States alone
We further study these strains to discover that CovS inactivation leading to a hypervirulent phenotype can occur in an acapsular background during human infection and results in vast transcriptional changes, including genes encoding key cell surface proteins whose transcript levels are not altered by CovS inactivation in serotype M1T1 group A streptococcal (GAS)
Dr Lancefield described that passage of GAS in mice resulted in hypervirulence some 100 years ago [7], it was not until the early 2000s that inactivating mutations of the CovRS system were identified as the major cause driving the emergence of the GAS hypervirulent phenotype [22]
Summary
Invasive bacterial infections afflict >100,000 persons per year in the United States alone. 100 years ago, Rebecca Lancefield made the key observation that some, but not all, GAS strains increase in virulence after passage in mice and, once achieved, the hypervirulent phenotype persists [7]. The epidemic serotype M1 clone, called M1T1, which is often identified as the most common cause of GAS infection worldwide, readily converts to a hypervirulent phenotype in mice whereas the serotype M1 clone that M1T1 GAS replaced, does not [16, 17]. Studies over the past 15 years have established that GAS hypervirulence is mainly due to mutations in the control of virulence (covRS) two-component gene regulatory system [18,19,20,21,22]. Inasmuch as phosphorylated CovR represses a number of GAS virulence factors, such CovS mutations result in decreased CovR phosphorylation and increased GAS virulence factor production [23,24,25,26,27]
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