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ABSTRACTIn Gram-positive bacteria, and particularly the Firmicutes, the DNA-dependent RNA polymerase (RNAP) complex contains an additional subunit, termed the d factor, or RpoE. This enigmatic protein has been studied for more than 30 years for various organisms, but its function is still not well understood. In this study, we investigated its role in the major human pathogen Staphylococcus aureus. We showed conservation of important structural regions of RpoE in S. aureusand other species and demonstrated binding to core RNAP that is mediated by the ß and/or ß' subunits. To identify the impact of the d subunit on transcription, we performed transcriptome sequencing (RNA-seq) analysis and observed 191 differentially expressed genes in the rpoEmutant. Ontological analysis revealed, quite strikingly, that many of the downregulated genes were known virulence factors, while several mobile genetic elements (SaPI5 and prophage ?SA3usa) were strongly upregulated. Phenotypically, the rpoEmutant had decreased accumulation and/or activity of a number of key virulence factors, including alpha toxin, secreted proteases, and Panton-Valentine leukocidin (PVL). We further observed significantly decreased survival of the mutant in whole human blood, increased phagocytosis by human leukocytes, and impaired virulence in a murine model of infection. Collectively, our results demonstrate that the d subunit of RNAP is a critical component of the S. aureustranscription machinery and plays an important role during infection.

ABSTRACTHumans commonly carry pathogenic bacteria asymptomatically, but the molecular factors underlying microbial asymptomatic carriage are poorly understood. We previously reported that two epidemiologically unassociated serotype M3 group A Streptococcus(GAS) carrier strains had an identical 12-bp deletion in the promoter of the gene encoding Mga, a global positive gene regulator. Herein, we report on studies designed to test the hypothesis that the identified 12-bp deletion in the mgapromoter alters GAS virulence, thereby potentially contributing to the asymptomatic carrier phenotype. Using allelic exchange, we introduced the variant promoter into a serotype M3 invasive strain and the wild-type promoter into an asymptomatic carrier strain. Compared to strains with the wild-type mgapromoter, we discovered that strains containing the promoter with the 12-bp deletion produced significantly fewer mgaand Mga-regulated gene transcripts. Consistent with decreased mgatranscripts, strains containing the variant mgapromoter were also significantly less virulent in in vivoand ex vivomodels of GAS disease. Further, we provide evidence that the pleiotropic regulator protein CodY binds to the mgapromoter and that the 12-bp deletion in the mgapromoter reduces CodY-mediated mgatranscription. We conclude that the naturally occurring 12-bp deletion in the mgapromoter significantly alters the pathogen-host interaction of these asymptomatic carrier strains. Our findings provide new insight into the molecular basis of the carrier state of an important human pathogen.

The Staphylococcus aureuscyclophilin PpiB is an intracellular peptidyl prolyl cis/transisomerase (PPIase) that has previously been shown to contribute to secreted nuclease and hemolytic activity. In this study, we investigated the contribution of PpiB to S. aureusvirulence.