Characterization of an Isogenic Mutant of Streptococcus pyogenes Manfredo Lacking the Ability To Make Streptococcal Acid Glycoprotein

Abstract

An isogenic mutant of Streptococcus pyogenes Manfredo that lacks the ability to make streptococcal acid glycoprotein (SAGP) has been constructed by inserting a deletion in the sagp gene using the method of allelic exchange. An assay of cell extracts (CE) prepared from the wild-type and mutant Manfredo strains for the enzyme arginine deiminase (AD) showed that significant activity was present in wild-type CE but none could be detected in mutant CE. These findings confirm our earlier conclusion that SAGP has AD activity (B. Wild-type CE but not mutant CE potently inhibited human peripheral blood mononuclear cell proliferation in response to phytohemagglutinin, and this inhibition was overcome by the addition of L-arginine to proliferation assay mixtures. Invasion assays showed that the isogenic mutant organisms lacking SAGP, and thus AD activity, were between three and five times less able to enter epithelial cells (Hep-2C and A549) than were the wild-type streptococci. Both wild-type and mutant S. pyogenes bacteria were extremely sensitive to low pH. However, L-arginine (1 mM or above) significantly increased the viability of the wild type but not the isogenic mutant organisms under acidic conditions. The difference in acid susceptibility between wild-type and mutant bacteria may explain the reduced capacity of the isogenic mutant bacteria to invade and survive intracellularly.AStreptococcus pyogenes (group A Streptococcus) is an important human pathogen, infections with which can lead to acute disorders such as pharyngitis, erysipelas, otitis media, and impetigo or to pathogenic sequelae including glomerulonephritis, acute rheumatic fever and reactive arthritis (6,7,21,29,42,53). In the last decade there has been a dramatic resurgence in the incidence of serious streptococcal infections; in particular, the number of cases of streptococcal toxic shock syndrome, bacteremia, and necrotising fasciitis has increased (11). A greater understanding of the ways in which this pathogen interacts with the host, the virulence factors that it produces, and the identification of possible targets for vaccine design are required.During a previous study, it was observed that cell extracts (CE) prepared from a wide range of S. pyogenes strains potently inhibited antigen-, superantigen-, or phytohemagglutinin (PHA)-stimulated human peripheral blood mononuclear cell (PBMC) proliferation in vitro (16,17). Purification of the inhibitory component present in CE of S. pyogenes Manfredo by anion-exchange chromatography followed by gel filtration chromatography yielded a single protein. When sequenced, this protein was found to have an NH 2 -terminal sequence identical to streptococcal acid glycoprotein (SAGP), which had been isolated from S. pyogenes Su (23,24,56,57). SAGP has 31 to 39% amino acid identity to arginine deiminase (AD) from Mycoplasma hominis, Mycoplasma arginini, Pseudomonas putida, and Pseudomonas aeruginosa (3). It was subsequently shown that CE obtained from several group A streptococcal strains all contained AD activit...

Staphylococcus aureus Biofilm Metabolism and the Influence of Arginine on Polysaccharide Intercellular Adhesin Synthesis, Biofilm Formation, and Pathogenesis

“…First, the deamination of arginine by the arginine deiminase pathway results in the extracellular accumulation of ammonia, facilitating bacterial pH homeostasis (2,44,45). Second, the staphylococcal arginine deiminase may function in a capacity similar to that in Streptococcus pyogenes (15); specifically, arginine deiminase may aid in evasion of the host immune response by inhibiting peripheral blood mononuclear cell proliferation (13,14). Hence, one function of arginine may be to induce transcription of the arginine deiminase (arc) operon.…”

Surviving the Acid Test: Responses of Gram-Positive Bacteria to Low pH

“…A protein originally designated streptococcal acid glycoprotein and originally characterized as being an inhibitor of stimulated human peripheral blood mononuclear cell proliferation (73,74) was ultimately identified as an ADI. Its role in acid resistance is demonstrated by a massive 9-log-unit difference in survival between a wild-type strain and an isogenic streptococcal acid glycoprotein-negative mutant after 6 h at pH 4 in the presence of 10 mM arginine (72). This difference in acid sensitivity is likely to be responsible for the observation that this mutant demonstrates a reduced ability to enter and initially survive in epithelial cells.…”

Analysis of the Transcriptome of Group A Streptococcus in Mouse Soft Tissue Infection

“…In turn, fermentation of amino acids produces ammonia, which could beneficially neutralize surrounding pH thereby protecting GAS from acid-induced damage. 96 GAS arginine deaminase activity has been linked to inhibition of human peripheral blood mononuclear cell proliferation. 97 The arginine deiminase system of Streptococcus suis is induced by arginine, elevated temperature, and reduced oxygen tension, and is subject to carbon catabolite repression.…”