The Integrative Conjugative Element ICESpyM92 Contributes to Pathogenicity of Emergent Antimicrobial-Resistant emm92 Group A Streptococcus.

Antimicrobial resistance-encoding mobile genetic elements (MGEs) may contribute to the disease potential of bacterial pathogens. We previously described the association of Group A Streptococcus (GAS) derived from invasive disease with increasingly frequent antimicrobial resistance (AMR). We hypothesized that a 65-kb AMR-encoding MGE (ICESpyM92), highly conserved among closely related emergent invasive emm92 GAS, contributes to GAS disease potential. Here, we provide evidence that a combination of ICESpyM92- and core genome-dependent differential gene expression (DGE) contributes to invasive disease phenotypes of emergent emm92 GAS. Using isogenic ICESpyM92 mutants generated in distinct emm92 genomic backgrounds, we determined the presence of ICESpyM92 enhances GAS virulence in a mouse subcutaneous infection model. Measurement of in vitro and ex vivo DGE indicates ICESpyM92 influences GAS global gene expression in a background-dependent manner. Our study links virulence and AMR on a unique MGE via MGE-related DGE and highlights the importance of investigating associations between AMR-encoding MGEs and pathogenicity.

Population Genomics Reveals Distinct Temporal Association with the Emergence of ST1 Serotype V Group B Streptococcus and Macrolide Resistance in North America.

Identified in the 1970s as the leading cause of invasive bacterial disease in neonates and young infants, group B Streptococcus (GBS) is now also recognized as a significant cause of morbidity and mortality among adults with underlying medical conditions and the elderly. Concomitant with the increasing incidence of GBS invasive disease in adults is the rise of resistance among GBS isolates to second line antibiotics. Previous research shows that among serotype V GBS, one of the most common capsular types causing adult invasive disease, sequence type 1 (ST1), accounts for an overwhelming majority of adult invasive disease isolates and frequently harbors macrolide resistance. In this study, using whole-genome sequencing data from strains isolated in the United States and Canada over a 45-year period, we examined the association of antimicrobial resistance with the emergence of invasive serotype V ST1 GBS. Our findings show a strong temporal association between increased macrolide resistance and the emergence of serotype V ST1 GBS subpopulations that currently co-circulate to cause invasive disease in adults and young infants. ST1 GBS subpopulations are defined, in part, by the presence of macrolide resistance genes in mobile genetic elements. Increased frequency of macrolide resistance-encoding mobile genetic elements among invasive GBS ST1 strains suggests the presence of such elements contributes to GBS virulence. Our work provides a foundation for the investigation of genetic features contributing to the increasing prevalence and pathogenesis of serotype V GBS in adult invasive disease.

The LiaFSR Transcriptome Reveals an Interconnected Regulatory Network in Group A Streptococcus.

The mechanisms by which bacteria sense the host environment and alter gene expression are poorly understood. LiaFSR is a gene regulatory system unique to Gram-positive bacteria, including group A Streptococcus (GAS), and responds to cell envelope stress. We previously showed that LiaF acts as an inhibitor to LiaFSR activation in GAS. To better understand gene regulation associated with LiaFSR activation, we performed RNA sequencing on isogenic deletion mutants fixed in a LiaFSR "always on" (ΔliaF) or "always off" (ΔliaR) state. Transcriptome analyses of ΔliaF and ΔliaR in GAS showed near perfect inverse correlation, including the gene encoding the global transcriptional regulator SpxA2. In addition, mutant transcriptomes included genes encoding multiple virulence factors and showed substantial overlap with the CovRS regulon. Chromatin immunoprecipitation quantitative PCR demonstrated direct spxA2 gene regulation following activation of the response regulator, LiaR. High SpxA2 levels as a result of LiaFSR activation were directly correlated with increased CovR-regulated virulence gene transcription. Furthermore, consistent with known virulence gene repression by phosphorylated CovR, elevated SpxA2 levels were inversely correlated with CovR phosphorylation. Despite increased transcription of several virulence factors, ΔliaF (high SpxA2) exhibited a paradoxical virulence phenotype in both in vivo mouse and ex vivo human blood models of disease. Likewise, despite decreased virulence factor transcription with ΔliaR (low SpxA2), increased virulence was observed in an in vivo mouse model of disease-a phenotype attributable, in part, to known SpxA2-associated speB transcription. Our findings provide evidence of a critical role of LiaFSR in sensing the host environment and suggest a potential mechanism for gene regulatory system cross talk shared by many Gram-positive pathogens.

ExPortal and the LiaFSR Regulatory System Coordinate the Response to Cell Membrane Stress in Streptococcus pyogenes.

LiaFSR is a gene regulatory system important for response to cell membrane stress in Gram-positive bacteria but is minimally studied in the important human pathogen group A Streptococcus (GAS). Using immunofluorescence and immunogold electron microscopy, we discovered that LiaF (a membrane-bound repressor protein) and LiaS (a sensor kinase) reside within the GAS membrane microdomain (ExPortal). Cell envelope stress induced by antimicrobials resulted in ExPortal disruption and activation of the LiaFSR system. The only human antimicrobial peptide whose presence resulted in ExPortal disruption and LiaFSR activation was the alpha-defensin human neutrophil peptide 1 (hNP-1). Elimination of membrane cardiolipin through targeted gene deletion resulted in loss of LiaS colocalization with the GAS ExPortal and activation of LiaFSR, whereas LiaF membrane localization was unaffected. Isogenic mutants lacking either LiaF or LiaS revealed a critical role of LiaF in ExPortal integrity. Thus, LiaF and LiaS colocalize with the GAS ExPortal by distinct mechanisms, further supporting codependence. These are the first data identifying a multicomponent signal system within the ExPortal, thereby providing new insight into bacterial intramembrane signaling in GAS that may serve as a paradigm for Gram-positive bacteria.IMPORTANCE Bacterial two-component systems sense and induce transcriptional changes in response to environmental stressors, including antimicrobials and human antimicrobial peptides. Since the stresses imposed by the host's defensive responses may act as markers of specific temporal stages of disease progression or host compartments, pathogens often coordinately regulate stress response programs with virulence factor expression. The mechanism by which bacteria recognize these stresses and subsequently induce transcriptional responses remains not well understood. In this study, we showed that LiaFSR senses cell envelope stress through colocalization of LiaF and LiaS with the group A Streptococcus (GAS) ExPortal and is activated in direct response to ExPortal disruption by antimicrobials or human antimicrobial peptides. Our studies shed new light on the sensing of cell envelope stress in Gram-positive bacteria and may contribute to the development of therapies targeting these processes.