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Conversion of an M- group A streptococcus to M+ by transfer of a plasmid containing an M6 gene
Abstract:
An M28-derived group A streptococcal strain deleted for the gene encoding M protein was converted to M+ by introduction of a plasmid carrying emm6, the structural gene for type 6 M protein from strain D471. The reconstituted M+ strain, JRS2, resists phagocytosis in human blood and is opsonized by anti-M6 hyperimmune serum, but not by anti-M28 serum. Immunofluorescent microscopy and ELISA demonstrate the presence of M protein on its surface. In addition, JRS2 removes opsonic antibodies from hyperimmune rabbit sera generated by immunization with purified ColiM6 protein and with a synthetic amino-terminal peptide derived from M6. Immunization of rabbits with JRS2 generates opsonic anti-M6 antibodies. These results indicate that the cloned emm6 gene contains the information necessary to convert a phagocytosis-sensitive streptococcus to phagocytosis resistance. Furthermore, it also contains the determinants for M type specificity and those required to elicit opsonic antibodies. It thus appears to determine all the traits associated with M protein.
Insights
Introducing the emm6 gene into Group A Streptococcus converts the bacteria to a fully functional M+ strain. This engineered strain resists phagocytosis and elicits specific opsonic antibodies, demonstrating the gene
Area of Science:
- Microbiology
- Immunology
- Molecular Biology
Background:
- M protein is a major virulence factor in Group A Streptococcus (GAS), mediating resistance to phagocytosis.
- Understanding the genetic basis of M protein function is crucial for developing effective vaccines and treatments.
- Previous studies have implicated M protein in GAS pathogenesis, but its complete functional determinants were not fully elucidated.
Purpose of the Study:
- To investigate whether the cloned emm6 gene alone is sufficient to confer M protein-associated traits to a non-M-producing GAS strain.
- To determine if the emm6 gene encodes all necessary information for phagocytosis resistance, M type specificity, and antibody elicitation.
Main Methods:
- Genetic manipulation of a Group A Streptococcus strain (M28-derived) to delete the endogenous M protein gene.
- Introduction of a plasmid containing the emm6 gene (encoding M protein type 6) into the modified strain.
- Assessment of phagocytosis resistance in human blood, opsonization with specific antisera, immunofluorescence microscopy, ELISA, and antibody absorption assays.
Main Results:
- The reconstituted M+ strain (JRS2) demonstrated resistance to phagocytosis and was opsonized by anti-M6 serum, confirming M protein surface expression.
- JRS2 effectively removed opsonic antibodies specific for M6 protein and its peptide, indicating functional M6 protein presence.
- Immunization with JRS2 generated opsonic antibodies against M6 protein, further validating the functional expression of M protein.
Conclusions:
- The cloned emm6 gene is sufficient to restore full M+ phenotype, including resistance to phagocytosis, in a non-M-producing GAS strain.
- The emm6 gene encodes all determinants for M type specificity and the ability to elicit opsonic antibodies.
- This study confirms that the emm6 gene alone confers all known functional traits associated with M protein in Group A Streptococcus.