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Characterization of an isogenic mutant of Streptococcus pyogenes Manfredo lacking the ability to make streptococcal
B A Degnan1, M C Fontaine, A H Doebereiner
1School of Clinical Medical Sciences (Rheumatology), Medical School, University of Newcastle, Newcastle upon Tyne NE2 4HH, United Kingdom. pm274@cam.ac.uk
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. A. Degnan, J. M. Palmer, T. Robson, C. E. D. Jones, M. Fischer, M. Glanville, G. D. Mellor, A. G. Diamond, M. A. Kehoe, and J. A. Goodacre, Infect. Immun. 66:3050-3058, 1998). 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.
Insights
Streptococcus pyogenes lacking streptococcal acid glycoprotein (SAGP) showed reduced invasion and impaired survival under acidic conditions. This suggests SAGP, and its arginine deiminase (AD) activity, are crucial for bacterial virulence and acid resistance.
Area of Science:
- Microbiology
- Immunology
- Molecular Biology
Background:
- Streptococcus pyogenes is an important human pathogen.
- Streptococcal acid glycoprotein (SAGP) is a surface protein of S. pyogenes.
- The function of SAGP and its associated enzyme, arginine deiminase (AD), in S. pyogenes pathogenesis is not fully understood.
Purpose of the Study:
- To investigate the role of SAGP and AD activity in S. pyogenes invasion, immune cell modulation, and acid survival.
- To confirm that SAGP possesses AD activity.
Main Methods:
- Construction of an isogenic mutant of S. pyogenes Manfredo lacking SAGP using allelic exchange.
- Enzyme assays of cell extracts for arginine deiminase (AD) activity.
- Inhibition assays of human peripheral blood mononuclear cell proliferation.
- Epithelial cell invasion assays.
- Assessment of bacterial viability under acidic conditions with and without L-arginine.
Main Results:
- The sagp mutant lacked detectable AD activity, confirming SAGP possesses AD activity.
- Wild-type cell extracts inhibited mitogen-stimulated peripheral blood mononuclear cell proliferation, an effect reversed by L-arginine.
- The sagp mutant exhibited a 3-5 fold reduction in epithelial cell invasion compared to wild-type.
- L-arginine significantly increased wild-type S. pyogenes viability at low pH, but not the mutant's.
Conclusions:
- SAGP, possessing arginine deiminase (AD) activity, is essential for S. pyogenes invasion of epithelial cells.
- AD activity contributes to S. pyogenes's ability to survive acidic environments, potentially through L-arginine metabolism.
- These findings highlight SAGP and AD as potential targets for therapeutic intervention against S. pyogenes infections.