Kevin S. McIver

Opportunistic pathogens must coordinate virulence-factor production with changing conditions encountered during colonization and infection. Group B Streptococcus (GBS), a common human opportunistic pathogen, uses the CovRS two-component system to regulate virulence, but how the CovS sensor kinase is tuned to environmental conditions remains unclear. Here, we identify CflP, a noncatalytic membrane protein of the CPBP family, as an accessory regulator of CovS. CflP associates with CovS and promotes CovS-dependent phosphorylation of the response regulator CovR, thereby repressing virulence-associated genes, including the β-hemolysin/cytolysin operon. Loss of CflP also enhances cytotoxicity toward human immune cells. CflP acts independently of Abx1, a previously identified homologous CPBP protein that promotes CovS phosphatase activity. CflP abundance is regulated post-transcriptionally by Mg²⁺ availability, linking an environmental cue to CovRS signaling. Comparative genomic analysis revealed distinct patterns of natural variation in these CovS regulators: cflP is predominantly maintained as an intact gene, although specific amino acid substitutions can compromise CflP abundance, whereas abx1 is frequently disrupted by recurrent frameshifts. Together, these findings reveal an accessory regulatory layer that tunes CovS activity in response to environmental conditions and whose natural variation may contribute to strain-dependent differences in CovRS regulation. More broadly, our findings illustrate how noncatalytic membrane proteins can modulate bacterial signaling to balance virulence-factor production with adaptation to changing host environments. IMPORTANCE: Group B Streptococcus (GBS) commonly colonizes the human gastrointestinal and vaginal tracts without causing disease but can cause life-threatening infections in newborns and adults. How GBS senses environmental conditions to regulate the expression of host-damaging factors remains poorly understood. We identify CflP, a membrane protein that regulates the CovRS signaling system, a central regulator of GBS virulence. CflP activates the CovS sensor kinase, promoting CovR phosphorylation and repression of genes encoding host-damaging factors. We further show that Mg²⁺ availability regulates CflP protein abundance, linking an environmental signal to CovRS activity. These findings suggest a mechanism by which GBS tunes its virulence potential in response to environmental conditions, restraining expression of host-damaging factors when nutrients are abundant while potentially permitting their induction when Mg²⁺ becomes limiting.

Group A Streptococcus (GAS) causes various human diseases linked to virulome expression predominantly regulated by the two-component system (TCS), CovR/S. Here, we demonstrate that asparagine (Asn) presence in a minimal chemically defined medium increases virulence gene expression in a CovR-dependent fashion. It also decreases the transcription of asparagine synthetase (AsnA), the ABC transporter responsible for Asn uptake (GlnPQ), and that of the hemolysin toxins responsible for scavenging Asn from the host. Metabolomics data show that Asn availability increases intracellular ADP/ATP ratio, which enhances phosphatase activity in structurally related CovS sensors and is probably responsible for the Asn-mediated decrease in CovR phosphorylation. Mutants deficient in AsnA, GlnPQ, asparaginase, (AsnB) activities are attenuated in a mouse model of human GAS invasive soft tissue infection. The similarity between the mechanisms of Asn-mediated regulation of GAS virulence and tumor growth suggests that, as in cancer, components maintaining Asn homeostasis could be targeted for anti-GAS treatments.

Group B Streptococcus (GBS) is a Gram-positive pathobiont that commonly colonizes the gastrointestinal and lower female genital tracts but can cause sepsis and pneumonia in newborns and is a leading cause of neonatal meningitis. Despite the resulting disease severity, the pathogenesis of GBS is not completely understood, especially during the early phases of infection. To investigate GBS factors necessary for bloodstream survival, we performed a transposon (Tn) mutant screen in our bacteremia infection model using a GBS mariner transposon mutant library previously developed by our group. We identified significantly underrepresented mutations in 623 genes that contribute to survival in the blood, including those encoding known virulence factors such as capsule, the β-hemolysin, and inorganic metal ion transport systems. Most of the underrepresented genes have not been previously characterized or studied in GBS, including gloA and gloB, which are homologs for genes involved in methylglyoxal (MG) detoxification. MG is a byproduct of glycolysis and a highly reactive toxic aldehyde that is elevated in immune cells during infection. Here, we observed MG sensitivity across multiple GBS isolates and confirmed that gloA contributes to MG tolerance and invasive GBS infection. We show specifically that gloA contributes to GBS survival in the presence of neutrophils and depleting neutrophils in mice abrogates the decreased survival and infection of the gloA mutant. The requirement of the glyoxalase pathway during GBS infection suggests that MG detoxification is important for bacterial survival during host-pathogen interactions. IMPORTANCE: A transposon-mutant screen of group B Streptococcus (GBS) in a bacteremia mouse model of infection revealed virulence factors known to be important for GBS survival such as the capsule, β-hemolysin/cytolysin, and genes involved in metal homeostasis. Many uncharacterized factors were also identified including genes that are part of the metabolic pathway that breaks down methylglyoxal (MG). The glyoxalase pathway is the most ubiquitous metabolic pathway for MG breakdown and is only a two-step process using glyoxalase A ( gloA ) and B ( gloB ) enzymes. MG is a highly reactive byproduct of glycolysis and is made by most cells. Here, we show that in GBS, the first enzyme in the glyoxalase pathway, encoded by gloA , contributes to MG resistance and blood survival. We further demonstrate that GloA contributes to GBS survival against neutrophils in vitro and in vivo and, therefore, is an important virulence factor required for invasive infection.

Streptococcus pyogenes (Group A Streptococcus , GAS) is a Gram-positive bacterium that inflicts both superficial and life-threatening diseases on its human host. Analysis of fitness using a transposon mutant library revealed that genes predicted to be involved in vitamin B ₆ acquisition are associated with fitness in whole human blood. Vitamin B ₆ is essential for all life and is important for many cellular functions. In several streptococcal species, it has been shown that mutants in B ₆ acquisition exhibited reduced virulence phenotypes and were attenuated during infection. In GAS, B ₆ acquisition is believed to be controlled by the pdxR-pdxKU locus, where PdxR is a positive regulator of pdxKU , which encodes for a B ₆ -substrate kinase and permease, respectively. Mutants in the regulator (Δ pdxR ) and salvage machinery (Δ pdxKU ) both exhibited modest growth defects when grown in oxygenated conditions with limited vitamin B ₆ precursors. ∆ pdxR and ∆ pdxKU mutants also exhibited an impaired ability to survive when challenged with whole human or mouse blood. This defect was characterized by reduced survival in the presence of human neutrophil-like HL60s, primary polymorphonuclear leukocytes, and antimicrobial peptide LL-37. Promoter analysis showed that PdxR is an autoregulator and activated pdxKU in the absence of B ₆ . Interestingly, ∆ pdxR and ∆ pdxKU mutants were not attenuated in mouse models of infection, suggesting a species-specific impact on virulence. Overall, it appears that pdxR-pdxKU is associated with GAS vitamin B ₆ metabolism as well as pathogen survival during encounters with the human innate immune system. IMPORTANCE: Bacterial pathogens such as Streptococcus pyogenes (Group A Streptococcus , GAS) must be able to obtain needed nutrients in their human host. Vitamin B ₆ or pyridoxal 5’ phosphate is essential for all life and is important for many cellular functions. In other streptococcal pathogens, B ₆ acquisition has been shown to be important for their ability to cause disease. Here, we show that loss of the putative vitamin B ₆ salvage pathway locus pdxR-pdxKU affects GAS pathogenesis when encountering innate immune responses from phagocytic neutrophils and antimicrobial peptides within the host. pdxR- pdxKU may contribute to oxygen tolerance through B ₆ ; however, there appear to be other mechanisms for salvaging vitamin B ₆ . Overall, pdxR-pdxKU is associated with GAS resistance to the human innate immune response and oxygen tolerance and contributes modestly to B ₆ metabolism.

Group B Streptococcus (GBS) is a Gram-positive pathobiont that commonly colonizes the gastrointestinal and lower female genital tracts but can cause sepsis and pneumonia in newborns and is a leading cause of neonatal meningitis. Despite the resulting disease severity, the pathogenesis of GBS is not completely understood, especially during the early phases of infection. To investigate GBS factors necessary for blood stream survival, we performed a transposon (Tn) mutant screen in our bacteremia infection model using a GBS mariner transposon mutant library previously developed by our group. We identified significantly underrepresented mutations in 623 genes that contribute to survival in the blood, including those encoding known virulence factors such as capsule, the β-hemolysin, and inorganic metal ion transport systems. Most of the underrepresented genes have not been previously characterized or studied in GBS, including gloA and gloB , which are homologs for genes involved in methylglyoxal (MG) detoxification. MG is a byproduct of glycolysis and a highly reactive toxic aldehyde that is elevated in immune cells during infection. Here, we observed MG sensitivity across multiple GBS isolates and confirm that gloA contributes to MG tolerance and invasive GBS infection. We show specifically that gloA contributes to GBS survival in the presence of neutrophils and depleting neutrophils in mice abrogates the decreased survival and infection of the gloA mutant. The requirement of the glyoxalase pathway during GBS infection suggests that MG detoxification is important for bacterial survival during host-pathogen interactions.

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.

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.

Morbidity and mortality associated with GBS begin with colonization of the female reproductive tract (FRT). To date, our understanding of the factors required for GBS persistence in this environment remain limited.

Streptococcus pyogenes, also known as group A Streptococcus or GAS, is a human‐restricted pathogen causing a diverse array of infections. The ability to adapt to different niches requires GAS to adjust gene expression in response to environmental cues. We previously identified the abundance of biometals and carbohydrates led to natural induction of the Rgg2/3 cell–cell communication system (quorum sensing, QS). Here we determined the mechanism by which the Rgg2/3 QS system is stimulated exclusively by mannose and repressed by glucose, a phenomenon known as carbon catabolite repression (CCR). Instead of carbon catabolite protein A, the primary mediator of CCR in Gram‐positive bacteria; CCR of Rgg2/3 requires the PTS regulatory domain (PRD)‐containing transcriptional regulator Mga. Deletion of Mga led to carbohydrate‐independent activation of Rgg2/3 by down‐regulating rgg3, the QS repressor. Through phosphoablative and phosphomimetic substitutions within Mga PRDs, we demonstrated that selective phosphorylation of PRD1 conferred repression of the Rgg2/3 system. Moreover, given the carbohydrate specificity mediating Mga‐dependent governance over Rgg2/3, we tested mannose‐specific PTS components and found the EIIA/B subunit ManL was required for Mga‐dependent repression. These findings provide newfound connections between PTS^Man, Mga, and QS, and further demonstrate that Mga is a central regulatory nexus for integrating nutritional status and virulence.

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.