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 S. 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 A 2 . Subsequent structural and phylogenetic analysis of the GacH extracellular domain revealed that GacH represents a new class of glycerol phosphate (GroP) transferase. We detected the presence of GroP in the GAC as well as the Serotype c Carbohydrate (SCC) from S. mutans, which depended on the presence of the respective gacH homologs. Finally, NMR analysis of GAC confirmed that GroP is attached to approximately 30% 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.

Streptococcus pyogenes (group A Streptococcus [GAS]) is an obligate human pathogen responsible for a spectrum of human disease states. Metallobiology of human pathogens is revealing the fundamental role of metals in both nutritional immunity leading to pathogen starvation and metal poisoning of pathogens by innate immune cells. Spy0980 (MntE) is a paralog of the GAS zinc efflux pump CzcD. Through use of an isogenic mntE deletion mutant in the GAS serotype M1T1 strain 5448, we have elucidated that MntE is a manganese-specific efflux pump required for GAS virulence. The 5448ΔmntE mutant had significantly lower survival following infection of human neutrophils than did the 5448 wild type and the complemented mutant (5448ΔmntE::mntE). Manganese homeostasis may provide protection against oxidative stress, explaining the observed ex vivo reduction in virulence. In the presence of manganese and hydrogen peroxide, 5448ΔmntE mutant exhibits significantly lower survival than wild-type 5448 and the complemented mutant. We hypothesize that MntE, by maintaining homeostatic control of cytoplasmic manganese, ensures that the peroxide response repressor PerR is optimally poised to respond to hydrogen peroxide stress. Creation of a 5448ΔmntE-ΔperR double mutant rescued the oxidative stress resistance of the double mutant to wild-type levels in the presence of manganese and hydrogen peroxide. This work elucidates the mechanism for manganese toxicity within GAS and the crucial role of manganese homeostasis in maintaining GAS virulence.

Cell wall glycopolymers on the surface of Gram-positive bacteria are fundamental to bacterial physiology and infection biology. These structures have also gained interest as vaccine antigens, in particular for the human pathogens Group A Streptococcus (GAS) and Streptococcus mutans. Streptococcal cell wall glycopolymers are considered to be functional homologues of wall teichoic acids but surprisingly lack the biologically-relevant and characteristic anionic charge. Here we identify gacH, a gene of unknown function in the GAS 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. To understand the underlying mechanism of these phenotypes, we determined the structure of the extracellular domain of GacH and discover that it represents a new family of glycerol phosphate (GroP) transferases. Importantly, we demonstrate the presence of GroP in both the GAC and the homologous Serotype c Carbohydrate (SCC) from S. mutans, which is conferred by gacH and sccH products, respectively. NMR analysis of GAC released from cell wall by non-destructive methods reveals that approximately 30% of the GAC GlcNAc side-chains are modified by GroP at the C6 hydroxyl group. This previously unrecognized structural modification impacts host-pathogen interaction and has implications for vaccine design.Graphical abstract

Streptococcus pyogenes (group A Streptococcus [GAS]) is an obligate human pathogen responsible for a broad spectrum of human disease. GAS has a requirement for metal homeostasis within the human host and, as such, tightly modulates metal uptake and efflux during infection. Metal acquisition systems are required to combat metal sequestration by the host, while metal efflux systems are essential to protect against metal overload poisoning. Here, we investigated the function of PmtA (PerRregulated metal transporter A), a P 1B-4 -type ATPase efflux pump, in invasive GAS M1T1 strain 5448. We reveal that PmtA functions as a ferrous iron [Fe(II)] efflux system. In the presence of high Fe(II) concentrations, the 5448ΔpmtA deletion mutant exhibited diminished growth and accumulated 5-fold-higher levels of intracellular Fe(II) than did the wild type and the complemented mutant. The 5448ΔpmtA deletion mutant also showed enhanced susceptibility to killing by the Fe-dependent antibiotic streptonigrin as well as increased sensitivity to hydrogen peroxide and superoxide. We suggest that the PerRmediated control of Fe(II) efflux by PmtA is important for bacterial defense against oxidative stress. PmtA represents an exemplar for an Fe(II) efflux system in a host-adapted Gram-positive bacterial pathogen.KEYWORDS iron efflux, PmtA, oxidative stress response, PerR, group A Streptococcus, Streptococcus pyogenes D efense against peroxide is recognized as one of the most widespread stress responses in prokaryotes. A characteristic of the peroxide stress response is the increased expression of peroxidases that can directly remove hydrogen peroxide as well as the induction of systems that can repair damaged proteins and nucleic acids (1, 2). Streptococcus pyogenes (group A Streptococcus [GAS]) coordinates oxidative stress responses through the action of the regulator PerR, which functions as a repressor of oxidative stress defense genes under normal conditions through binding to DNA at per boxes upstream of these genes (3, 4). This stable complex typically exists with Fe as a prosthetic group; in the presence of hydrogen peroxide, Fe causes metal-catalyzed oxidation and damage of the complex, leading to its dissociation and the subsequent transcription of peroxide response genes (3-6). In GAS, the PerR-regulated oxidative stress response encompasses both direct mechanisms, involving the detoxification of reactive oxygen species (ROS), and indirect mechanisms, which involve the repair of biomolecules damaged by oxidative stress. Direct mechanisms involving the enzymatic detoxification of reactive oxygen species are achieved through alkyl hydroperoxidases and glutathione peroxidases (1, 2, 5) as well as a single superoxide dismutase, SodA, which is Mn dependent (7). An example of an indirect response mechanism is PolAI, a DNA polymerase that repairs oxidatively damaged DNA (8).