AN M-PROTEIN WITH A SINGLE C-REPEAT PREVENTS PHAGOCYTOSIS OF STREPTOCOCCUS-PYOGENES - USE OF A TEMPERATURE-SENSITIVE SHUTTLE VECTOR TO DELIVER HOMOLOGOUS SEQUENCES TO THE CHROMOSOME OF S-PYOGENES

The major virulence factor of the important human pathogen Streptococc us pyogenes is the M protein, which prevents phagocytosis of the bacte rium. In different strains of streptococci, there are over 80 serologi cally different M proteins and there are additional M-like proteins, s ome of which bind immunoglobulins. Although the sequence of the M mole cules differs among different S. pyogenes strains, all M proteins, and some of the immunoglobulin-binding molecules, have at least two copie s of the C repeat region. We describe construction of a deletion mutat ion in S. pyogenes, which has only one C repeat copy, and show that th e mutant strain is still resistant to phagocytosis. The mutation was c onstructed in vitro and used to replace the resident emm allele in an S. pyogenes strain. To facilitate homologous recombination into the st reptococcal chromosome, we adapted a shuttle vector which is temperatu re sensitive for replication in Gram-positive bacteria but not in Gram -negative hosts. This new method for delivery of a homologous DNA frag ment to the S. pyogenes chromosome is efficient and reproducible and s hould be of general use.