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M-related protein (Mrp) contributes to group A streptococcal resistance to phagocytosis by human granulocytes
A Podbielski1, N Schnitzler, P Beyhs
1Institute of Medical Microbiology, Hospital of the Technical University (Klinikum RWTH), Aachen, Germany.
Abstract:
The M protein has been postulated to be a major group A streptococcal (GAS) virulence factor because of its contribution to the bacterial resistance to opsonophagocytosis. Direct evidence of this was only provided for GAS strains which expressed a single M protein. The majority of 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 resistance, the mrp and emm genes from serotypes M2, M4, and M49 as well as from M-untypeable strain 64/14 were insertionally inactivated. The mrp and emm mutants were subjected to direct bactericidal assays. As judged 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 analysis of phagocytosis by flow cytometry, measuring association of BCECF/AM-labelled bacteria and granulocytes, paralleled the results from direct 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 controls 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 proceeds faster for emm mutants than for corresponding mrp mutants. By adding a dihydro-rhodamine123 stain and measuring fluorescence induced by oxidative burst, the experimental data suggested that bacteria bound to granulocytes were also engulfed and integrated into phagolysosomes. Thus, these data indicated that, if present, both mrp and emm gene products contribute to phagocytosis resistance by decreasing bacterial binding to granulocytes.
Insights
Group A Streptococcus (GAS) M protein and M-related proteins (Mrp) are key virulence factors. Inactivating their genes reduced bacterial resistance to phagocytosis, indicating their role in immune evasion.
Area of Science:
- Microbiology
- Immunology
- Molecular Biology
Background:
- Group A Streptococcus (GAS) M protein is a major virulence factor, enhancing resistance to phagocytosis.
- Most GAS strains express M-related proteins (Mrp, Enn) alongside M protein, which bind IgG and IgA.
- Previous evidence for M protein's role in phagocytosis resistance was limited to strains with a single M protein.
Purpose of the Study:
- To investigate the role of M protein and Mrp in GAS resistance to phagocytosis.
- To determine if Mrp and M proteins contribute to immune evasion by preventing phagocyte interaction.
Main Methods:
- Insertionally inactivated mrp and emm genes in GAS serotypes M2, M4, M49, and strain 64/14.
- Conducted direct bactericidal assays to assess bacterial survival.
- Utilized flow cytometry to quantify granulocyte-bacterial association and measured oxidative burst via fluorescence.
Main Results:
- Mutant strains lacking mrp or emm genes showed reduced multiplication factors compared to wild-type strains.
- Phagocytosis assays confirmed reduced bacterial binding to granulocytes for mutants (40-60% association) versus wild-type ( <24% association).
- Kinetic analysis indicated faster granulocyte attachment for emm mutants than mrp mutants, and evidence of bacterial engulfment via oxidative burst.
Conclusions:
- Both M protein (emm gene product) and Mrp (mrp gene product) contribute to GAS resistance against phagocytosis.
- These proteins reduce bacterial binding to granulocytes, facilitating immune evasion.
- The findings highlight the importance of both M and Mrp proteins in GAS pathogenesis.