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.

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.

Genomic Analyses Identify Manganese Homeostasis as a Driver of Group B Streptococcal Vaginal Colonization.

Group B Streptococcus (GBS) is associated with severe infections in utero and in newborn populations, including pneumonia, sepsis, and meningitis. GBS vaginal colonization of the pregnant mother is an important prerequisite for transmission to the newborn and the development of neonatal invasive disease; however, our understanding of the factors required for GBS persistence and ascension in the female reproductive tract (FRT) remains limited. Here, we utilized a GBS mariner transposon (Krmit) mutant library previously developed by our group and identified underrepresented mutations in 535 genes that contribute to survival within the vaginal lumen and colonization of vaginal, cervical, and uterine tissues. From these mutants, we identified 47 genes that were underrepresented in all samples collected, including mtsA, a component of the mtsABC locus, encoding a putative manganese (Mn2+)-dependent ATP-binding cassette transporter. RNA sequencing analysis of GBS recovered from the vaginal tract also revealed a robust increase of mtsA expression during vaginal colonization. We engineered an ΔmtsA mutant strain and found by using inductively coupled plasma mass spectrometry that it exhibited decreased concentrations of intracellular Mn2+, confirming its involvement in Mn2+ acquisition. The ΔmtsA mutant was significantly more susceptible to the metal chelator calprotectin and to oxidative stressors, including both H2O2 and paraquat, than wild-type (WT) GBS. We further observed that the ΔmtsA mutant strain exhibited a significant fitness defect in comparison to WT GBS in vivo by using a murine model of vaginal colonization. Taken together, these data suggest that Mn2+ homeostasis is an important process contributing to GBS survival in the FRT. IMPORTANCE 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. We identified several necessary systems for initial colonization of the vaginal lumen and penetration into the reproductive tissues via transposon mutagenesis sequencing. We determined that mutations in mtsA, the gene encoding a protein putatively involved in manganese (Mn2+) transport, were significantly underrepresented in all in vivo samples collected. We also show that mtsA contributes to Mn2+ acquisition and GBS survival during metal limitation by calprotectin, a metal-chelating protein complex. We further demonstrate that a mutant lacking mtsA is hypersusceptible to oxidative stress induced by both H2O2 and paraquat and has a severe fitness defect compared to WT GBS in the murine vaginal tract. This work reveals the importance of Mn2+ homeostasis at the host-pathogen interface in the FRT.

Carbon catabolite repression on the Rgg2/3 quorum sensing system in Streptococcus pyogenes is mediated by PTSMan and Mga.

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 PTSMan , Mga, and QS, and further demonstrate that Mga is a central regulatory nexus for integrating nutritional status 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.

Hemoglobin Induces Early and Robust Biofilm Development in Streptococcus pneumoniae by a Pathway That Involves comC but Not the Cognate comDE Two-Component System.

Streptococcus pneumoniae grows in biofilms during both asymptomatic colonization and infection. Pneumococcal biofilms on abiotic surfaces exhibit delayed growth and lower biomass and lack the structures seen on epithelial cells or during nasopharyngeal carriage. We show here that adding hemoglobin to the medium activated unusually early and vigorous biofilm growth in multiple S. pneumoniae serotypes grown in batch cultures on abiotic surfaces. Human blood (but not serum, heme, or iron) also stimulated biofilms, and the pore-forming pneumolysin, ply, was required for this induction. S. pneumoniae transitioning from planktonic into sessile growth in the presence of hemoglobin displayed an extensive transcriptome remodeling within 1 and 2 h. Differentially expressed genes included those involved in the metabolism of carbohydrates, nucleotides, amino acid, and lipids. The switch into adherent states also influenced the expression of several regulatory systems, including the comCDE genes. Inactivation of comC resulted in 67% reduction in biofilm formation, while the deletion of comD or comE had limited or no effect, respectively. These observations suggest a novel route for CSP-1 signaling independent of the cognate ComDE two-component system. Biofilm induction and the associated transcriptome remodeling suggest hemoglobin serves as a signal for host colonization in pneumococcus.

Identification of Zinc-Dependent Mechanisms Used by Group B Streptococcus To Overcome Calprotectin-Mediated Stress.

Nutritional immunity is an elegant host mechanism used to starve invading pathogens of necessary nutrient metals. Calprotectin, a metal-binding protein, is produced abundantly by neutrophils and is found in high concentrations within inflammatory sites during infection. Group B Streptococcus (GBS) colonizes the gastrointestinal and female reproductive tracts and is commonly associated with severe invasive infections in newborns such as pneumonia, sepsis, and meningitis. Although GBS infections induce robust neutrophil recruitment and inflammation, the dynamics of GBS and calprotectin interactions remain unknown. Here, we demonstrate that disease and colonizing isolate strains exhibit susceptibility to metal starvation by calprotectin. We constructed a mariner transposon (Krmit) mutant library in GBS and identified 258 genes that contribute to surviving calprotectin stress. Nearly 20% of all underrepresented mutants following treatment with calprotectin are predicted metal transporters, including known zinc systems. As calprotectin binds zinc with picomolar affinity, we investigated the contribution of GBS zinc uptake to overcoming calprotectin-imposed starvation. Quantitative reverse transcriptase PCR (qRT-PCR) revealed a significant upregulation of genes encoding zinc-binding proteins, adcA, adcAII, and lmb, following calprotectin exposure, while growth in calprotectin revealed a significant defect for a global zinc acquisition mutant (ΔadcAΔadcAIIΔlmb) compared to growth of the GBS wild-type (WT) strain. Furthermore, mice challenged with the ΔadcAΔadcAIIΔlmb mutant exhibited decreased mortality and significantly reduced bacterial burden in the brain compared to mice infected with WT GBS; this difference was abrogated in calprotectin knockout mice. Collectively, these data suggest that GBS zinc transport machinery is important for combatting zinc chelation by calprotectin and establishing invasive disease.IMPORTANCE Group B Streptococcus (GBS) asymptomatically colonizes the female reproductive tract but is a common causative agent of meningitis. GBS meningitis is characterized by extensive infiltration of neutrophils carrying high concentrations of calprotectin, a metal chelator. To persist within inflammatory sites and cause invasive disease, GBS must circumvent host starvation attempts. Here, we identified global requirements for GBS survival during calprotectin challenge, including known and putative systems involved in metal ion transport. We characterized the role of zinc import in tolerating calprotectin stress in vitro and in a mouse model of infection. We observed that a global zinc uptake mutant was less virulent than the parental GBS strain and found calprotectin knockout mice to be equally susceptible to infection by wild-type (WT) and mutant strains. These findings suggest that calprotectin production at the site of infection results in a zinc-limited environment and reveals the importance of GBS metal homeostasis to invasive disease.

Hemoglobin stimulates vigorous growth of Streptococcus pneumoniae and shapes the pathogen's global transcriptome.

Streptococcus pneumoniae (Spn) must acquire iron from the host to establish infection. We examined the impact of hemoglobin, the largest iron reservoir in the body, on pneumococcal physiology. Supplementation with hemoglobin allowed Spn to resume growth in an iron-deplete medium. Pneumococcal growth with hemoglobin was unusually robust, exhibiting a prolonged logarithmic growth, higher biomass, and extended viability in both iron-deplete and standard medium. We observed the hemoglobin-dependent response in multiple serotypes, but not with other host proteins, free iron, or heme. Remarkably, hemoglobin induced a sizable transcriptome remodeling, effecting virulence and metabolism in particular genes facilitating host glycoconjugates use. Accordingly, Spn was more adapted to grow on the human α - 1 acid glycoprotein as a sugar source with hemoglobin. A mutant in the hemoglobin/heme-binding protein Spbhp-37 was impaired for growth on heme and hemoglobin iron. The mutant exhibited reduced growth and iron content when grown in THYB and hemoglobin. In summary, the data show that hemoglobin is highly beneficial for Spn cultivation in vitro and suggest that hemoglobin might drive the pathogen adaptation in vivo. The hemoglobin receptor, Spbhp-37, plays a role in mediating the positive influence of hemoglobin. These novel findings provide intriguing insights into pneumococcal interactions with its obligate human host.

Phosphotransferase System Uptake and Metabolism of the ß-Glucoside Salicin Impact Group A Streptococcal Bloodstream Survival and Soft Tissue Infection.

Streptococcus pyogenes (group A Streptococcus [GAS]), a major human-specific pathogen, relies on efficient nutrient acquisition for successful infection within its host. The phosphotransferase system (PTS) couples the import of carbohydrates with their phosphorylation prior to metabolism and has been linked to GAS pathogenesis. In a screen of an insertional mutant library of all 14 annotated PTS permease (EIIC) genes in MGAS5005, the annotated ß-glucoside PTS transporter (bglP) was found to be crucial for GAS growth and survival in human blood and was validated in another M1T1 GAS strain, 5448. In 5448, bglP was shown to be in an operon with a putative phospho-ß-glucosidase (bglB) downstream and a predicted antiterminator (licT) upstream. Using defined nonpolar mutants of the ß-glucoside permease (bglP) and ß-glucosidase enzyme (bglB) in 5448, we showed that bglB, not bglP, was important for growth in blood. Furthermore, transcription of the licT-blgPB operon was found to be repressed by glucose and induced by the ß-glucoside salicin as the sole carbon source. Investigation of the individual bglP and bglB mutants determined that they influence in vitro growth in the ß-glucoside salicin; however, only bglP was necessary for growth in other non-ß-glucoside PTS sugars, such as fructose and mannose. Additionally, loss of BglP and BglB suggests that they are important for the regulation of virulence-related genes that control biofilm formation, streptolysin S (SLS)-mediated hemolysis, and localized ulcerative lesion progression during subcutaneous infections in mice. Thus, our results indicate that the ß-glucoside PTS transports salicin and its metabolism can differentially influence GAS pathophysiology during soft tissue infection.

The Arginine Deiminase Pathway Impacts Antibiotic Tolerance during Biofilm-Mediated Streptococcus pyogenes Infections.

Bacterial biofilms are responsible for a variety of serious human infections and are notoriously difficult to treat due to their recalcitrance to antibiotics. Further work is necessary to elicit a full understanding of the mechanism of this antibiotic tolerance. The arginine deiminase (ADI) pathway is responsible for bacterial pH maintenance and is highly expressed during biofilm growth in multiple bacterial species. Using the group A Streptococcus (GAS) as a model human pathogen, the ADI pathway was demonstrated to contribute to biofilm growth. The inability of antibiotics to reduce GAS populations when in a biofilm was demonstrated by in vitro studies and a novel animal model of nasopharyngeal infection. However, disruption of the ADI pathway returned GAS biofilms to planktonic levels of antibiotic sensitivity, suggesting the ADI pathway is influential in biofilm-related antibiotic treatment failure and provides a new strategic target for the treatment of biofilm infections in GAS and potentially numerous other bacterial species.IMPORTANCE Biofilm-mediated bacterial infections are a major threat to human health because of their recalcitrance to antibiotic treatment. Through the study of Streptococcus pyogenes, a significant human pathogen that is known to form antibiotic-tolerant biofilms, we demonstrated the role that a bacterial pathway known for responding to acid stress plays in biofilm growth and antibiotic tolerance. This not only provides some insight into antibiotic treatment failure in S. pyogenes infections but also, given the widespread nature of this pathway, provides a potentially broad target for antibiofilm therapies. This discovery has the potential to impact the treatment of many different types of recalcitrant biofilm infections.

Protocols for Tn-seq Analyses in the Group A Streptococcus.

Transposon-sequencing (Tn-seq) has revolutionized forward-genetic analyses to study genotype-phenotype associations and interrogate bacterial cell physiology. The Tn-seq approach allows the en masse monitoring of highly complex mutant libraries, leveraging massive parallel DNA sequencing as a means to characterize the composition of these mutant pools on a genome-scale with unprecedented nucleotide-level high resolution. In this chapter, we present step-by-step protocols for Tn-seq analyses in the human pathogen Streptococcus pyogenes (Group A Streptococcus or GAS) using the mariner-based Krmit transposon. We detail how to generate highly complex Krmit mutant libraries in GAS and the en masse production of Krmit insertion tags for Illumina sequencing of the transposon-genome junctions for Tn-seq analyses. Most of the protocols presented here were developed and implemented using the S. pyogenes M1T1 serotype clinical isolate 5448, but they have been successfully applied to multiple GAS serotypes as well as other pathogenic Streptococci.

The scfCDE Operon Encodes a Predicted ABC Importer Required for Fitness and Virulence during Group A Streptococcus Invasive Infection.

As a strict human pathogen, Streptococcus pyogenes (group A Streptococcus, or GAS) causes a wide range of infections, from superficial to life-threatening diseases, upon dissemination. Thus, it is necessary to gain a better understanding of how GAS successfully overcomes host-mediated challenges and infects various host niches. We previously identified subcutaneous fitness (scf) genes in the clinically relevant wild-type (WT) GAS M1T1 5448 strain that are critical for fitness during murine soft-tissue infection at both 24 h and 48 h postinfection. The uncharacterized locus scfCDE was transcribed as an operon and is predicted to encode an ABC importer for nutrient uptake (e.g., amino acids). Individual scfCDE deletion mutants grew comparably to WT 5448 in rich medium but exhibited reduced fitness during competitive growth in murine soft tissue and in nutrient-limiting chemically defined medium (CDM). A deletion of the permease gene scfD resulted in a monoculture growth defect in CDM that could be rescued by addition of excess peptides, suggesting a role as an amino acid importer. Interestingly, the ΔscfC substrate-binding and ΔscfD permease mutants, but not the ΔscfE ATPase mutant, were highly attenuated in murine soft tissue. Moreover, all three genes were required for GAS survival in human blood, indicating their impact is not limited to superficial infections. As such, scfCDE plays an integral role in enhancing GAS adaptation during localized infection as well as dissemination to deeper host environments. Since scfCDE is conserved throughout Firmicutes, this work may contribute to the development of therapeutic strategies against GAS and other Gram-positive pathogens.

Discovery of glycerol phosphate modification on streptococcal rhamnose polysaccharides.

Cell wall glycopolymers on the surface of Gram-positive bacteria are fundamental to bacterial physiology and infection biology. Here we identify gacH, a gene in the Streptococcus pyogenes group A carbohydrate (GAC) biosynthetic cluster, in two independent transposon library screens for its ability to confer resistance to zinc and susceptibility to the bactericidal enzyme human group IIA-secreted phospholipase A2. Subsequent structural and phylogenetic analysis of the GacH extracellular domain revealed that GacH represents an alternative class of glycerol phosphate transferase. We detected the presence of glycerol phosphate in the GAC, as well as the serotype c carbohydrate from Streptococcus mutans, which depended on the presence of the respective gacH homologs. Finally, nuclear magnetic resonance analysis of GAC confirmed that glycerol phosphate is attached to approximately 25% of the GAC N-acetylglucosamine side-chains at the C6 hydroxyl group. This previously unrecognized structural modification impacts host-pathogen interaction and has implications for vaccine design.

Streptococcal Lancefield polysaccharides are critical cell wall determinants for human Group IIA secreted phospholipase A2 to exert its bactericidal effects.

Human Group IIA secreted phospholipase A2 (hGIIA) is an acute phase protein with bactericidal activity against Gram-positive bacteria. Infection models in hGIIA transgenic mice have suggested the importance of hGIIA as an innate defense mechanism against the human pathogens Group A Streptococcus (GAS) and Group B Streptococcus (GBS). Compared to other Gram-positive bacteria, GAS is remarkably resistant to hGIIA activity. To identify GAS resistance mechanisms, we exposed a highly saturated GAS M1 transposon library to recombinant hGIIA and compared relative mutant abundance with library input through transposon-sequencing (Tn-seq). Based on transposon prevalence in the output library, we identified nine genes, including dltA and lytR, conferring increased hGIIA susceptibility. In addition, seven genes conferred increased hGIIA resistance, which included two genes, gacH and gacI that are located within the Group A Carbohydrate (GAC) gene cluster. Using GAS 5448 wild-type and the isogenic gacI mutant and gacI-complemented strains, we demonstrate that loss of the GAC N-acetylglucosamine (GlcNAc) side chain in the ΔgacI mutant increases hGIIA resistance approximately 10-fold, a phenotype that is conserved across different GAS serotypes. Increased resistance is associated with delayed penetration of hGIIA through the cell wall. Correspondingly, loss of the Lancefield Group B Carbohydrate (GBC) rendered GBS significantly more resistant to hGIIA-mediated killing. This suggests that the streptococcal Lancefield antigens, which are critical determinants for streptococcal physiology and virulence, are required for the bactericidal enzyme hGIIA to exert its bactericidal function.

Glucose Levels Alter the Mga Virulence Regulon in the Group A Streptococcus.

Many bacterial pathogens coordinately regulate genes encoding important metabolic pathways during disease progression, including the phosphoenolpyruvate (PEP)-phosphotransferase system (PTS) for uptake of carbohydrates. The Gram-positive Group A Streptococcus (GAS) is a pathogen that infects multiple tissues in the human host. The virulence regulator Mga in GAS can be phosphorylated by the PTS, affecting Mga activity based on carbohydrate availability. Here, we explored the effects of glucose availability on the Mga regulon. RNA-seq was used to identify transcriptomic differences between the Mga regulon grown to late log phase in the presence of glucose (THY) or after glucose has been expended (C media). Our results revealed a correlation between the genes activated in C media with those known to be repressed by CcpA, indicating that C media mimics a non-preferred sugar environment. Interestingly, we found very little overlap in the Mga regulon from GAS grown in THY versus C media beyond the core virulence genes. We also observed an alteration in the phosphorylation status of Mga, indicating that the observed media differences in the Mga regulon may be directly attributed to glucose levels. Thus, these results support an in vivo link between glucose availability and virulence regulation in GAS.

Route of Glucose Uptake in the Group a Streptococcus Impacts SLS-Mediated Hemolysis and Survival in Human Blood.

The transport and metabolism of glucose has been shown to have far reaching consequences in the transcriptional profile of many bacteria. As glucose is most often the preferred carbon source for bacteria, its presence in the environment leads to the repression of many alternate carbohydrate pathways, a condition known as carbon catabolite repression (CCR). Additionally, the expression of many virulence factors is also dependent on the presence of glucose. Despite its importance, little is known about the transport routes of glucose in the human pathogen Streptococcus pyogenes. Considering that Streptococcus pyogenes is an important human pathogen responsible for over 500,000 deaths every year, we characterized the routes of glucose transport in an effort to understand its importance in GAS pathogenesis. Using a deletion of glucokinase (ΔnagC) to block utilization of glucose imported by non-PTS pathways, we determined that of the two glucose transport pathways in GAS (PTS and non-PTS), the non-PTS pathway played a more significant role in glucose transport. However, the expression of both pathways is linked by a currently unknown mechanism, as blocking the non-PTS uptake of glucose reduces ptsI (EI) expression. Similar to the effects of the deletion of the PTS pathway, lack of the non-PTS pathway also leads to the early activity of Streptolysin S. However, this early activity did not adversely or favorably affect survival of ΔnagC in whole human blood. In a subcutaneous murine infection model, ΔnagC-infected mice showed increased lesion severity at the local site of infection; although, lesion size and dissemination from the site of infection was similar to wild type. Here, we show that glucose transport in GAS is primarily via a non-PTS pathway. The route of glucose transport differentially affects the survival of GAS in whole human blood, as well as the lesion size at the local site of infection in a murine skin infection model.

Endopeptidase PepO Regulates the SpeB Cysteine Protease and Is Essential for the Virulence of Invasive M1T1 Streptococcus pyogenes.

Streptococcus pyogenes (group A Streptococcus [GAS]) causes a wide range of human infections. The pathogenesis of GAS infections is dependent on the temporal expression of numerous secreted and surface-associated virulence factors that interact with host proteins. Streptococcal pyrogenic exotoxin B (SpeB) is one of the most extensively studied toxins produced by GAS, and the coordinate growth phase-dependent regulation of speB expression is linked to disease severity phenotypes. Here, we identified the endopeptidase PepO as a novel growth phase-dependent regulator of SpeB in the invasive GAS M1 serotype strain 5448. By using transcriptomics followed by quantitative reverse transcriptase PCR and Western blot analyses, we demonstrate through targeted mutagenesis that PepO influences growth phase-dependent induction of speB gene expression. Compared to wild-type and complemented mutant strains, we demonstrate that the 5448ΔpepO mutant strain is more susceptible to killing by human neutrophils and is attenuated in virulence in a murine model of invasive GAS infection. Our results expand the complex regulatory network that is operating in GAS to control SpeB production and suggest that PepO is a virulence requirement during GAS M1T1 strain 5448 infections.IMPORTANCE Despite the continuing susceptibility of S. pyogenes to penicillin, this bacterial pathogen remains a leading infectious cause of global morbidity and mortality. A particular subclone of the M1 serotype (M1T1) has persisted globally for decades as the most frequently isolated serotype from patients with invasive and noninvasive diseases in Western countries. One of the key GAS pathogenicity factors is the potent broad-spectrum cysteine protease SpeB. Although there has been extensive research interest on the regulatory mechanisms that control speB gene expression, its genetic regulation is not fully understood. Here, we identify the endopeptidase PepO as a new regulator of speB gene expression in the globally disseminated M1T1 clone and as being essential for virulence.

Genome-wide discovery of novel M1T1 group A streptococcal determinants important for fitness and virulence during soft-tissue infection.

The Group A Streptococcus remains a significant human pathogen causing a wide array of disease ranging from self-limiting to life-threatening invasive infections. Epithelium (skin or throat) colonization with progression to the subepithelial tissues is the common step in all GAS infections. Here, we used transposon-sequencing (Tn-seq) to define the GAS 5448 genetic requirements for in vivo fitness in subepithelial tissue. A near-saturation transposon library of the M1T1 GAS 5448 strain was injected subcutaneously into mice, producing suppurative inflammation at 24 h that progressed to prominent abscesses with tissue necrosis at 48 h. The library composition was monitored en masse by Tn-seq and ratios of mutant abundance comparing the output (12, 24 and 48 h) versus input (T0) mutant pools were calculated for each gene. We identified a total of 273 subcutaneous fitness (scf) genes with 147 genes (55 of unknown function) critical for the M1T1 GAS 5448 fitness in vivo; and 126 genes (53 of unknown function) potentially linked to in vivo fitness advantage. Selected scf genes were validated in competitive subcutaneous infection with parental 5448. Two uncharacterized genes, scfA and scfB, encoding putative membrane-associated proteins and conserved among Gram-positive pathogens, were further characterized. Defined scfAB mutants in GAS were outcompeted by wild type 5448 in vivo, attenuated for lesion formation in the soft tissue infection model and dissemination to the bloodstream. We hypothesize that scfAB play an integral role in enhancing adaptation and fitness of GAS during localized skin infection, and potentially in propagation to other deeper host environments.

Using a Concept Inventory to Reveal Student Thinking Associated with Common Misconceptions about Antibiotic Resistance.

Misconceptions, also known as alternate conceptions, about key concepts often hinder the ability of students to learn new knowledge. Concept inventories (CIs) are designed to assess students' understanding of key concepts, especially those prone to misconceptions. Two-tiered CIs include prompts that ask students to explain the logic behind their answer choice. Such two-tiered CIs afford an opportunity for faculty to explore the student thinking behind the common misconceptions represented by their choice of a distractor. In this study, we specifically sought to probe the misconceptions that students hold prior to beginning an introductory microbiology course (i.e., preconceptions). Faculty-learning communities at two research-intensive universities used the validated Host-Pathogen Interaction Concept Inventory (HPI-CI) to reveal student preconceptions. Our method of deep analysis involved communal review and discussion of students' explanations for their CI answer choice. This approach provided insight valuable for curriculum development. Here the process is illustrated using one question from the HPI-CI related to the important topic of antibiotic resistance. The frequencies with which students chose particular multiple-choice responses for this question were highly correlated between institutions, implying common underlying misconceptions. Examination of student explanations using our analysis approach, coupled with group discussions within and between institutions, revealed patterns in student thinking to the participating faculty. Similar application of a two-tiered concept inventory by general microbiology instructors, either individually or in groups, at other institutions will allow them to better understand student thinking related to key concepts in their curriculum.

A PTS EII mutant library in Group A Streptococcus identifies a promiscuous man-family PTS transporter influencing SLS-mediated hemolysis.

The Group A Streptococcus (GAS, Streptococcus pyogenes) is a Gram-positive human pathogen that must adapt to unique host environments in order to survive. Links between sugar metabolism and virulence have been demonstrated in GAS, where mutants in the phosphoenolpyruvate-dependent phosphotransferase system (PTS) exhibited Streptolysin S (SLS)-mediated hemolysis during exponential growth. This early onset hemolysis correlated with an increased lesion size and severity in a murine soft tissue infection model when compared with parental M1T1 MGAS5005. To identify the PTS components responsible for this phenotype, we insertionally inactivated the 14 annotated PTS EIIC-encoding genes in the GAS MGAS5005 genome and subjected this library to metabolic and hemolysis assays to functionally characterize each EIIC. It was found that a few EIIs had a very limited influence on PTS sugar metabolism, whereas others were fairly promiscuous. The mannose-specific EII locus, encoded by manLMN, was expressed as a mannose-inducible operon that exhibited the most influence on PTS sugar metabolism, including mannose. Importantly, components of the mannose-specific EII also acted to prevent the early onset of SLS-mediated hemolysis. Interestingly, these roles were not identical in two different M1T1 GAS strains, highlighting the possible versatility of the PTS to adapt to strain-specific needs.