SUMMARY The ability of a bacterial pathogen to monitor available carbon sources in host tissues provides a clear fitness advantage. In the group A streptococcus (GAS), the virulence regulator Mga contains homology to phosphotransferase system (PTS) regulatory domains (PRDs) found in sugar operon regulators. Here we show that Mga was phosphorylated in vitro by the PTS components EI/HPr at conserved PRD histidines. A ∆ptsI (EI-deficient) GAS mutant exhibited decreased Mga activity. However, PTS-mediated phosphorylation inhibited Mga-dependent transcription of emm in vitro. Using alanine (unphosphorylated) and aspartate (phosphomimetic) mutations of PRD histidines, we establish that a doubly phosphorylated PRD1 phosphomimetic (D/DMga4) is completely inactive in vivo, shutting down expression of the Mga regulon. Although D/DMga4 is still able to bind DNA in vitro, homo-multimerization of Mga is disrupted and the protein is unable to activate trancription. PTS- mediated regulation of Mga activity appears to be important for pathogenesis, as bacteria expressing either nonphosphorylated (A/A) or phosphomimetic (D/D) PRD1 Mga mutants were attenuated in a model of GAS invasive skin disease. Thus, PTS-mediated phosphorylation of Mga may allow the bacteria to modulate virulence gene expression in response to carbohydrate status. Furthermore, PRD-containing virulence regulators (PCVRs) appear to be widespread in Gram-positive pathogens.

Summary 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 to 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. We 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.

bObtaining essential nutrients, such as carbohydrates, is an important process for bacterial pathogens to successfully colonize host tissues. The phosphoenolpyruvate phosphotransferase system (PTS) is the primary mechanism by which bacteria transport sugars and sense the carbon state of the cell. The group A streptococcus (GAS) is a fastidious microorganism that has adapted to a variety of niches in the human body to elicit a wide array of diseases. A ⌬ptsI mutant (enzyme I [EI] deficient) generated in three different strains of M1T1 GAS was unable to grow on multiple carbon sources (PTS and non-PTS). Complementation with ptsI expressed under its native promoter in single copy was able to rescue the growth defect of the mutant. In a mouse model of GAS soft tissue infection, all ⌬ptsI mutants exhibited a significantly larger and more severe ulcerative lesion than mice infected with the wild type. Increased transcript levels of sagA and streptolysin S (SLS) activity during exponential-phase growth was observed. We hypothesized that early onset of SLS activity would correlate with the severity of the lesions induced by the ⌬ptsI mutant. In fact, infection of mice with a ⌬ptsI sagB double mutant resulted in a lesion comparable to that of either the wild type or a sagB mutant alone. Therefore, a functional PTS is not required for subcutaneous skin infection in mice; however, it does play a role in coordinating virulence factor expression and disease progression.T he ability to obtain essential nutrients during an infection is critical for bacterial pathogens to successfully colonize and proliferate within host tissues. One such key process involves the ability to import and catabolize optimal carbon sources such as carbohydrates. Bacteria have evolved elegant regulatory pathways that detect the presence of preferred carbohydrates, repress the utilization of nonpreferred sugars, and regulate their metabolism based on this information flow (1, 2). In fact, many pathogens tightly control the genes involved in carbohydrate utilization and regulation in response to in vivo growth, and these same genes have been shown to be important to the disease process (3-8). Therefore, it is apparent that bacterial pathogens have closely linked their sugar metabolic sensing networks to virulence gene expression during infection.The primary bacterial system coupling the transport of carbohydrates across the cytoplasmic membrane with their phosphorylation is a multiprotein phosphorelay called the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) (9). The PTS is composed of at least three distinct proteins: the cytosolic proteins enzyme I (EI) and Hpr, encoded by ptsI and ptsH, respectively, and the membrane-bound sugar-specific enzyme II (EII) proteins. Each EII consists of one or two integral membranebound domains (EIIC/EIID) that are necessary for sugar translocation, and these domains form complexes with two hydrophilic components (EIIA and EIIB) that are required for substrate phosphorylation (1, 10). EI initiates ...

Streptococcus pyogenes is a Gram‐positive bacterium that strictly infects humans. It is the causative agent of a broad spectrum of diseases accounting for millions of infections and at least 517,000 deaths each year worldwide. It is a nutritionally fastidious organism that ferments sugars to produce lactic acid and has strict requirements for growth. To aid in the study of this organism, this unit describes the growth and maintenance of S. pyogenes. Curr. Protoc. Microbiol. 30:9D.2.1‐9D.2.13. © 2013 by John Wiley & Sons, Inc.