The Streptococcus pyogenes stand-alone regulator RofA exhibits characteristics of a PRD-containing virulence regulator.

Streptococcus pyogenes [group A streptococcus (GAS)] is a human pathogen capable of infecting diverse tissues. To successfully infect these sites, GAS must detect available nutrients and adapt accordingly. The phosphoenolpyruvate transferase system (PTS) mediates carbohydrate uptake and metabolic gene regulation to adapt to the nutritional environment. Regulation by the PTS can occur through phosphorylation of transcriptional regulators at conserved PTS-regulatory domains (PRDs). GAS has several PRD-containing stand-alone regulators with regulons encoding both metabolic genes and virulence factors [PRD-containing virulence regulators (PCVRs)]. One is RofA, which regulates the expression of virulence genes in multiple GAS serotypes. It was hypothesized that RofA is phosphorylated by the PTS in response to carbohydrate levels to coordinate virulence gene expression. In this study, the RofA regulon of M1T1 strain 5448 was determined using RNA sequencing. Two operons were consistently differentially expressed across growth in the absence of RofA; the pilus operon was downregulated, and the capsule operon was upregulated. This correlated with increased capsule production and decreased adherence to keratinocytes. Purified RofA-His was phosphorylated in vitro by PTS proteins EI and HPr, and phosphorylated RofA-FLAG was detected in vivo when GAS was grown in low-glucose C medium. Phosphorylated RofA was not observed when C medium was supplemented 10-fold with glucose. Mutations of select histidine residues within the putative PRDs contributed to the in vivo phosphorylation of RofA, although phosphorylation of RofA was still observed, suggesting other phosphorylation sites exist in the protein. Together, these findings support the hypothesis that RofA is a PCVR that may couple sugar metabolism with virulence regulation.

The Impacts of msaABCR on sarA-Associated Phenotypes Are Different in Divergent Clinical Isolates of Staphylococcus aureus.

The staphylococcal accessory regulator (sarA) plays an important role in Staphylococcus aureus infections, including osteomyelitis, and the msaABCR operon has been implicated as an important factor in modulating expression of sarA Thus, we investigated the contribution of msaABCR to sarA-associated phenotypes in the S. aureus clinical isolates LAC and UAMS-1. Mutation of msaABCR resulted in reduced production of SarA and a reduced capacity to form a biofilm in both strains. Biofilm formation was enhanced in a LAC msa mutant by restoring the production of SarA, but this was not true in a UAMS-1 msa mutant. Similarly, extracellular protease production was increased in a LAC msa mutant but not a UAMS-1 msa mutant. This difference was reflected in the accumulation and distribution of secreted virulence factors and in the impact of extracellular proteases on biofilm formation in a LAC msa mutant. Most importantly, it was reflected in the relative impact of mutating msa as assessed in a murine osteomyelitis model, which had a significant impact in LAC but not in UAMS-1. In contrast, mutation of sarA had a greater impact on all of these in vitro and in vivo phenotypes than mutation of msaABCR, and it did so in both LAC and UAMS-1. These results suggest that, at least in osteomyelitis, it would be therapeutically preferable to target sarA rather than msaABCR to achieve the desired clinical result, particularly in the context of divergent clinical isolates of S. aureus.

Infection of Primary Human Alveolar Macrophages Alters Staphylococcus aureus Toxin Production and Activity.

Pulmonary pathogens encounter numerous insults, including phagocytic cells designed to degrade bacteria, while establishing infection in the human lung. Staphylococcus aureus is a versatile, opportunistic pathogen that can cause severe pneumonia, and methicillin-resistant isolates are of particular concern. Recent reports present conflicting data regarding the ability of S. aureus to survive and replicate within macrophages. However, due to use of multiple strains and macrophage sources, making comparisons between reports remains difficult. Here, we established a disease-relevant platform to study innate interactions between S. aureus and human lungs. Human precision-cut lung slices (hPCLS) were subjected to infection by S. aureus LAC (methicillin-resistant) or UAMS-1 (methicillin-sensitive) isolates. Additionally, primary human alveolar macrophages (hAMs) were infected with S. aureus, and antibacterial activity was assessed. Although both S. aureus isolates survived within hAM phagosomes, neither strain replicated efficiently in these cells. S. aureus was prevalent within the epithelial and interstitial regions of hPCLS, with limited numbers present in a subset of hAMs, suggesting that the pathogen may not target phagocytic cells for intracellular growth during natural pulmonary infection. S. aureus-infected hAMs mounted a robust inflammatory response that reflected natural human disease. S. aureus LAC was significantly more cytotoxic to hAMs than UAMS-1, potentially due to isolate-specific virulence factors. The bicomponent toxin Panton-Valentine leukocidin was not produced during intracellular infection, while alpha-hemolysin was produced but was not hemolytic, suggesting that hAMs alter toxin activity. Overall, this study defined a new disease-relevant infection platform to study S. aureus interaction with human lungs and to define virulence factors that incapacitate pulmonary cells.

Loss of rpoE Encoding the δ-Factor of RNA Polymerase Impacts Pathophysiology of the Streptococcus pyogenes M1T1 Strain 5448.

Streptococcus pyogenes, also known as the Group A Streptococcus (GAS), is a Gram-positive bacterial pathogen of major clinical significance. Despite remaining relatively susceptible to conventional antimicrobial therapeutics, GAS still causes millions of infections and hundreds of thousands of deaths each year worldwide. Thus, a need for prophylactic and therapeutic interventions for GAS is in great demand. In this study, we investigated the importance of the gene encoding the delta (δ) subunit of the GAS RNA polymerase, rpoE, for its impact on virulence during skin and soft-tissue infection. A defined 5448 mutant with an insertionally-inactivated rpoE gene was defective for survival in whole human blood and was attenuated for both disseminated lethality and lesion size upon mono-culture infection in mouse soft tissue. Furthermore, the mutant had reduced competitive fitness when co-infected with wild type (WT) 5448 in the mouse model. We were unable to attribute this attenuation to any observable growth defect, although colony size and the ability to grow at higher temperatures were both affected when grown with nutrient-rich THY media. RNA-seq of GAS grown in THY to late log phase found that mutation of rpoE significantly impacted (>2-fold) the expression of 429 total genes (205 upregulated, 224 downregulated), including multiple virulence and "housekeeping" genes. The arc operon encoding the arginine deiminase (ADI) pathway was the most upregulated in the rpoE mutant and this could be confirmed phenotypically. Taken together, these findings demonstrate that the delta (δ) subunit of RNA polymerase is vital in GAS gene expression and virulence.

PRD-Containing Virulence Regulators (PCVRs) in Pathogenic Bacteria.

Bacterial pathogens rely on a complex network of regulatory proteins to adapt to hostile and nutrient-limiting host environments. The phosphoenolpyruvate phosphotransferase system (PTS) is a conserved pathway in bacteria that couples transport of sugars with phosphorylation to monitor host carbohydrate availability. A family of structurally homologous PTS-regulatory-domain-containing virulence regulators (PCVRs) has been recognized in divergent bacterial pathogens, including Streptococcus pyogenes Mga and Bacillus anthracis AtxA. These paradigm PCVRs undergo phosphorylation, potentially via the PTS, which impacts their dimerization and their activity. Recent work with predicted PCVRs from Streptococcus pneumoniae (MgaSpn) and Enterococcus faecalis (MafR) suggest they interact with DNA like nucleoid-associating proteins. Yet, Mga binds to promoter sequences as a homo-dimeric transcription factor, suggesting a bi-modal interaction with DNA. High-resolution crystal structures of 3 PCVRs have validated the domain structure, but also raised additional questions such as how ubiquitous are PCVRs, is PTS-mediated histidine phosphorylation via potential PCVRs widespread, do specific sugars signal through PCVRs, and do PCVRs interact with DNA both as transcription factors and nucleoid-associating proteins? Here, we will review known and putative PCVRs based on key domain and functional characteristics and consider their roles as both transcription factors and possibly chromatin-structuring proteins.

Limiting protease production plays a key role in the pathogenesis of the divergent clinical isolates of Staphylococcus aureus LAC and UAMS-1.

Using the USA300, methicillin-resistant Staphylococcus aureus strain LAC, we previously examined the impact of regulatory mutations implicated in biofilm formation on protease production and virulence in a murine sepsis model. Here we examined the impact of these mutations in the USA200, methicillin-sensitive strain UAMS-1. Mutation of agr, mgrA, rot, sarA and sigB attenuated the virulence of UAMS-1. A common characteristic of codY, rot, sigB, and sarA mutants was increased protease production, with mutation of rot having the least impact followed by mutation of codY, sigB and sarA, respectively. Protein A was undetectable in conditioned medium from all four mutants, while extracellular nuclease was only present in the proteolytically cleaved NucA form. The abundance of high molecular weight proteins was reduced in all four mutants. Biofilm formation was reduced in codY, sarA and sigB mutants, but not in the rot mutant. Eliminating protease production partially reversed these phenotypes and enhanced biofilm formation. This was also true in LAC codY, rot, sarA and sigB mutants. Eliminating protease production enhanced the virulence of LAC and UAMS-1 sarA, sigB and rot mutants in a murine sepsis model but did not significantly impact the virulence of the codY mutant in either strain. Nevertheless, these results demonstrate that repressing protease production plays an important role in defining critical phenotypes in diverse clinical isolates of S. aureus and that Rot, SigB and SarA play critical roles in this regard.

Impact of Staphylococcus aureus regulatory mutations that modulate biofilm formation in the USA300 strain LAC on virulence in a murine bacteremia model.

Staphylococcus aureus causes acute and chronic forms of infection, the latter often associated with formation of a biofilm. It has previously been demonstrated that mutation of atl, codY, rot, sarA, and sigB limits biofilm formation in the USA300 strain LAC while mutation of agr, fur, and mgrA has the opposite effect. Here we used a murine sepsis model to assess the impact of these same loci in acute infection. Mutation of agr, atl, and fur had no impact on virulence, while mutation of mgrA and rot increased virulence. In contrast, mutation of codY, sarA, and sigB significantly attenuated virulence. Mutation of sigB resulted in reduced accumulation of AgrA and SarA, while mutation of sarA resulted in reduced accumulation of AgrA, but this cannot account for the reduced virulence of sarA or sigB mutants because the isogenic agr mutant was not attenuated. Indeed, as assessed by accumulation of alpha toxin and protein A, all of the mutants we examined exhibited unique phenotypes by comparison to an agr mutant and to each other. Attenuation of the sarA, sigB and codY mutants was correlated with increased production of extracellular proteases and global changes in extracellular protein profiles. These results suggest that the inability to repress the production of extracellular proteases plays a key role in attenuating the virulence of S. aureus in acute as well as chronic, biofilm-associated infections, thus opening up the possibility that strategies aimed at the de-repression of protease production could be used to broad therapeutic advantage. They also suggest that the impact of codY, sarA, and sigB on protease production occurs via an agr-independent mechanism.