Regulating gene expression during infection is critical to the ability of pathogens to circumvent the immune response and cause disease. This is true for the group A Streptococcus (GAS), a pathogen that causes both invasive (e.g., necrotizing fasciitis) and noninvasive (e.g., pharyngitis) diseases. The control of virulence (CovRS) two-component system has a major role in regulating GAS virulence factor expression. The regulator of cov (RocA) protein, which is a predicted kinase, functions in an undetermined manner through CovRS to alter gene expression and reduce invasive disease virulence. Here, we show that the ectopic expression of a truncated RocA derivative, harboring the membrane-spanning domains but not the dimerization or HATPase domain, is sufficient to complement a rocA mutant strain. Coupled with a previous bioinformatic study, the data are consistent with RocA being a pseudokinase. RocA reduces the ability of serotype M1 GAS isolates to express capsule and to evade killing in human blood, phenotypes that are not observed for M3 or M18 GAS due to isolates of these serotypes naturally harboring mutant rocA alleles. In addition, we found that varying the RocA concentration attenuates the regulatory activity of Mg 2ϩ and the antimicrobial peptide LL-37, which positively and negatively regulate CovS function, respectively. Thus, we propose that RocA is an accessory protein to the CovRS system that influences the ability of GAS to modulate gene expression in response to host factors. A model of how RocA interacts with CovRS, and of the regulatory consequences of such activity, is presented.

The group A Streptococcus (GAS) causes diseases that range from mild (e.g. pharyngitis) to severely invasive (e.g. necrotizing fasciitis). Strain-and serotypespecific differences influence the ability of isolates to cause individual diseases. At the center of this variability is the CovR/S two-component system and the accessory protein RocA. Through incompletely defined mechanisms, CovR/S and RocA repress the expression of more than a dozen immunomodulatory virulence factors. Alleviation of this repression is selected for during invasive infections, leading to the recovery of covR, covS or rocA mutant strains. Here, we investigated how RocA promotes CovR/S activity, identifying that RocA is a pseudokinase that interacts with CovS. Disruption of CovS kinase or phosphatase activities abolishes RocA function, consistent with RocA acting through the modulation of CovS activity. We also identified, in conflict with a previous study, that the RocA regulon includes the secreted protease-encoding gene speB. Finally, we discovered an inverse correlation between the virulence of wild-type, rocA mutant, covS mutant and covR mutant strains during invasive infection and their fitness in an ex vivo upper respiratory tract model. Our data inform on mechanisms that control GAS disease potential and provide an explanation for observed strain-and serotype-specific variability in RocA function.

Isolates of a given bacterial pathogen often display phenotypic variation, and this can negatively impact public health, for example, by reducing the efficacy of preventative measures. Here, we identify that the human pathogen group A (GAS;) expresses pili on its cell surface in a serotype-specific manner. Specifically, we show that serotype M3 GAS isolates, which are nonrandomly associated with causing particularly severe and lethal invasive infections, produce negligible amounts of pili relative to serotype M1 and M49 isolates. Performance of an interserotype transcriptome comparison (serotype M1 versus serotype M3) was instrumental in this discovery. We also identified that the transcriptional regulator Nra positively regulates pilus expression in M3 GAS isolates and that the low level of pilus expression of these isolates correlates with a low level of transcription. Finally, we discovered that the phenotypic consequences of low levels of pilus expression by M3 GAS isolates are a reduced ability to adhere to host cells and an increased ability to survive and proliferate in human blood. We propose that an enhanced ability to survive in human blood, in part due to reduced pilus expression, is a contributing factor in the association of serotype M3 isolates with highly invasive infections. In conclusion, our data show that GAS isolates express pili in a serotype-dependent manner and may inform vaccine development, given that pilus proteins are being discussed as possible GAS vaccine antigens.

Background Bacterial infections following childbirth—so-called puerperal infections—cause morbidity in 5%–10% of all new mothers. At low frequency, the infection can spread to the blood, resulting in life-threatening sepsis known as puerperal sepsis. Pathogens causing puerperal sepsis include group A Streptococcus (GAS), and epidemiological analyses have identified isolates of a single serotype, M28, as being nonrandomly associated with cases of puerperal sepsis. The genomes of serotype M28 GAS isolates harbor a 36.3-kb mobile genetic element of apparent group B Streptococcus origin, termed region of difference 2 (RD2). Methods The phenotypic (determined via tissue culture and a vaginal colonization model) and regulatory (determined via RNA sequencing analysis) contributions of RD2 were assessed by comparing parental, RD2 deletion mutant, and complemented mutant serotype M28 GAS strains. Results RD2 affords serotype M28 isolates an enhanced ability to adhere to human vaginal epithelial cells and to colonize the female reproductive tract in a mouse model of infection. In addition, RD2 influences the abundance of messenger RNAs from >100 core chromosomal GAS genes. Conclusions The data are consistent with RD2 directly, via encoded virulence factors, and indirectly, via encoded regulatory proteins, modifying the virulence potential of GAS and contributing to the decades-old association of serotype M28 isolates with cases of puerperal sepsis.

This study investigates the regulatory and phenotypic consequences of a naturally occurring mutation in a strain of the bacterial pathogen the group A Streptococcus (Streptococcus pyogenes). We show that this mutation, which occurs in a regulator-encoding gene, rocA, leads to altered virulence factor expression and reduces the ability of this isolate to survive in human blood. Critically, the blood survival phenotype and the assortment of genes regulated by RocA differ compared to previous studies into RocA activity. The data are consistent with there being strain- or serotype-specific variability in RocA function. Given that phenotypic variants can lead to treatment failures and escape from preventative regimes, our data provide information with regard to a mechanism of phenotypic variation in a prevalent Gram-positive pathogen.