Mutational analysis of the group A streptococcal operon encoding streptolysin S and its virulence role in invasive infection

The pathogen group A Streptococcus (GAS) produces a wide spectrum of infections including necrotizing fasciitis (NF). Streptolysin S (SLS) produces the hallmark beta-haemolytic phenotype produced by GAS. The nine-gene GAS locus (sagA-sagI) resembling a bacteriocin biosynthetic operon is necessary and sufficient for SLS production. Using precise, in-frame allelic exchange mutagenesis and single-gene complementation, we show sagA, sagB, sagC, sagD, sagE, sagF and sagG are each individually required for SLS production, and that sagE may further serve an immunity function. Limited site-directed mutagenesis of specific amino acids in the SagA prepropeptide supports the designation of SLS as a bacteriocin-like toxin. No significant pleotrophic effects of sagA deletion were observed on M protein, capsule or cysteine protease production. In a murine model of NF, the SLS-negative M1T1 GAS mutant was markedly diminished in its ability to produce necrotic skin ulcers and spread to the systemic circulation. The SLS toxin impaired phagocytic clearance and promoted epithelial cell cytotoxicity, the latter phenotype being enhanced by the effects of M protein and streptolysin O. We conclude that all genetic components of the sag operon are required for expression of functional SLS, an important virulence factor in the pathogenesis of invasive M1T1 GAS infection.     

MicroRegulators come of age in senescence

Cellular senescence was first reported five decades ago as a state of long-term growth inhibition in viable, metabolically active cells cultured in vitro. However, evidence that senescence occurs in vivo and underlies pathophysiologic processes has only emerged over the past few years. Coincident with this increased knowledge, understanding of the mechanisms that control senescent-cell gene expression programs has also recently escalated. Such mechanisms include a prominent group of regulatory factors (miRNA), a family of small, noncoding RNAs that interact with select target mRNAs and typically repress their expression. Here, we review recent reports that miRNAs are key modulators of cellular senescence, and we examine their influence upon specific senescence-regulatory proteins. We discuss evidence that dysregulation of miRNA-governed senescence programs underlies age-associated diseases, including cancer.     

Individual genetic variations directly effect polarization of cytokine responses to superantigens associated with streptococcal sepsis: implications for customized patient care

Host immunogenetic variations strongly influence the severity of group A streptococcus sepsis by modulating responses to streptococcal superantigens (Strep-SAgs). Although HLA-II-DR15/DQ6 alleles strongly protect against severe sepsis, HLA-II-DR14/DR7/DQ5 alleles significantly increase the risk for toxic shock syndrome. We found that, regardless of individual variations in TCR-Vβ repertoires, the presentation of Strep-SAgs by the protective HLA-II-DR15/DQ6 alleles significantly attenuated proliferative responses to Strep-SAgs, whereas their presentation by the high-risk alleles augmented it. Importantly, HLA-II variations differentially polarized cytokine responses to Strep-SAgs: the presentation of Strep-SAgs by HLA-II-DR15/DQ6 alleles elicited significantly higher ratios of anti-inflammatory cytokines (e.g., IL-10) to proinflammatory cytokines (e.g., IFN-γ) than did their presentation by the high-risk HLA-II alleles. Adding exogenous rIL-10 significantly attenuated responses to Strep-SAgs presented by the high-risk HLA-II alleles but did not completely block the response; instead, it reduced it to a level comparable to that seen when these superantigens were presented by the protective HLA-II alleles. Furthermore, adding neutralizing anti-IL-10 Abs augmented Strep-SAg responses in the presence of protective HLA-II alleles to the same level as (but no higher than) that seen when the superantigens were presented by the high-risk alleles. Our findings provide a molecular basis for the role of HLA-II allelic variations in modulating streptococcal sepsis outcomes and suggest the presence of an internal control mechanism that maintains superantigen responses within a defined range, which helps to eradicate the infection while attenuating pathological inflammatory responses that can inflict more harm than the infection itself.     

DNase Sda1 provides selection pressure for a switch to invasive group A streptococcal infection

Most invasive bacterial infections are caused by species that more commonly colonize the human host with minimal symptoms. Although phenotypic or genetic correlates underlying a bacterium's shift to enhanced virulence have been studied, the in vivo selection pressures governing such shifts are poorly understood. The globally disseminated M1T1 clone of group A Streptococcus (GAS) is linked with the rare but life-threatening syndromes of necrotizing fasciitis and toxic shock syndrome. Mutations in the GAS control of virulence regulatory sensor kinase (covRS) operon are associated with severe invasive disease, abolishing expression of a broad-spectrum cysteine protease (SpeB) and allowing the recruitment and activation of host plasminogen on the bacterial surface. Here we describe how bacteriophage-encoded GAS DNase (Sda1), which facilitates the pathogen's escape from neutrophil extracellular traps, serves as a selective force for covRS mutation. The results provide a paradigm whereby natural selection exerted by the innate immune system generates hypervirulent bacterial variants with increased risk of systemic dissemination.