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.

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.

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.

The Transcriptional Regulator CpsY Is Important for Innate Immune Evasion in Streptococcus pyogenes.

As an exclusively human pathogen, Streptococcus pyogenes (the group A streptococcus [GAS]) has specifically adapted to evade host innate immunity and survive in multiple tissue niches, including blood. GAS can overcome the metabolic constraints of the blood environment and expresses various immunomodulatory factors necessary for survival and immune cell resistance. Here we present our investigation of one such factor, the predicted LysR family transcriptional regulator CpsY. The encoding gene, cpsY, was initially identified as being required for GAS survival in a transposon-site hybridization (TraSH) screen in whole human blood. CpsY is homologous with transcriptional regulators of Streptococcus mutans (MetR), Streptococcus iniae (CpsY), and Streptococcus agalactiae (MtaR) that regulate methionine transport, amino acid metabolism, resistance to neutrophil-mediated killing, and survival in vivo Our investigation indicated that CpsY is involved in GAS resistance to innate immune cells of its human host. However, GAS CpsY does not manifest the in vitro phenotypes of its homologs in other streptococcal species. GAS CpsY appears to regulate a small set of genes that is markedly different from the regulons of its homologs. The differential expression of these genes depends on the growth medium, and CpsY modestly influences their expression. The GAS CpsY regulon includes known virulence factors (mntE, speB, spd, nga [spn], prtS [SpyCEP], and sse) and cell surface-associated factors of GAS (emm1, mur1.2, sibA [cdhA], and M5005_Spy0500). Intriguingly, the loss of CpsY in GAS does not result in virulence defects in murine models of infection, suggesting that CpsY function in immune evasion is specific to the human host.

The fruRBA Operon Is Necessary for Group A Streptococcal Growth in Fructose and for Resistance to Neutrophil Killing during Growth in Whole Human Blood.

Bacterial pathogens rely on the availability of nutrients for survival in the host environment. The phosphoenolpyruvate-phosphotransferase system (PTS) is a global regulatory network connecting sugar uptake with signal transduction. Since the fructose PTS has been shown to impact virulence in several streptococci, including the human pathogen Streptococcus pyogenes(the group A Streptococcus[GAS]), we characterized its role in carbon metabolism and pathogenesis in the M1T1 strain 5448. Growth in fructose as a sole carbon source resulted in 103 genes affected transcriptionally, where the frulocus (fruRBA) was the most induced. Reverse transcriptase PCR showed that fruRBA formed an operon which was repressed by FruR in the absence of fructose, in addition to being under carbon catabolic repression. Growth assays and carbon utilization profiles revealed that although the entire fruoperon was required for growth in fructose, FruA was the main transporter for fructose and also was involved in the utilization of three additional PTS sugars: cellobiose, mannitol, and N-acetyl-D-galactosamine. The inactivation of sloR, a fruA homolog that also was upregulated in the presence of fructose, failed to reveal a role as a secondary fructose transporter. Whereas the ability of both ΔfruR and ΔfruB mutants to survive in the presence of whole human blood or neutrophils was impaired, the phenotype was not reproduced in murine whole blood, and those mutants were not attenuated in a mouse intraperitoneal infection. Since the ΔfruA mutant exhibited no phenotype in the human or mouse assays, we propose that FruR and FruB are important for GAS survival in a human-specific environment.

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.

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.