M-RELATED PROTEIN (MRP) CONTRIBUTES TO GROUP-A STREPTOCOCCAL RESISTANCE TO PHAGOCYTOSIS BY HUMAN GRANULOCYTES

The M protein has been postulated to be a major group A streptococcal (GAS) virulence factor because of its contribution to the bacterial re sistance to opsonophagocytosis. Direct evidence of this was only provi ded for GAS strains which expressed a single M protein, The majority o f GAS express additional, structurally similar M-related proteins, Mrp and Enn, which have been described as IgG- and IgA-binding proteins. To determine the involvement of Mrp and M protein in phagocytosis resi stance, the mrp and emm genes from serotypes M2, M4, and M49 as well a s from M-untypeable strain 64/14 were insertionally inactivated, The m rp and emm mutants were subjected to direct bactericidal assays, As ju dged by numbers of surviving colony-forming units, all mutant strains with the exception of the mrp4 mutant exhibited reduced multiplication factors as compared to the isogenic wild-type strains, Subsequent ana lysis of phagocytosis by flow cytometry, measuring association of BCEC F/AM-labelled bacteria and granulocytes, paralleled the results from d irect bactericidal assays regardless of whether isolated granulocytes or whole blood were utilized. Resistant wild-type GAS strains bound to less than 24% of granulocytes, whereas phagocytosis-sensitive control s attached to more than 90% of the white blood cells. 40 to 60% of the granulocytes associated with the mrp and emm mutants within 1 h of co -incubation. Kinetic data suggested that attachment to granulocytes pr oceeds faster for emm mutants than for corresponding mrp mutants. By a dding a dihydro-rhodamine123 stain and measuring fluorescence induced by oxidative burst, the experimental data suggested that bacteria boun d to granulocytes were also engulfed and integrated into phagolysosome s. Thus, these data indicated that, if present, both mrp and emm gene products contribute to phagocytosis resistance by decreasing bacterial binding to granulocytes.