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 Streptococcus pyogenes is the M protein, which prevents phagocytosis of the bacterium. In different strains of streptococci, there are over 80 serologically different M proteins and there are additional M-like proteins, some of which bind immunoglobulins. Although the sequence of the M molecules differs among different S. pyogenes strains, all M proteins, and some of the immunoglobulin-binding molecules, have at least two copies of the C repeat region. We describe construction of a deletion mutation in S. pyogenes, which has only one C repeat copy, and show that the mutant strain is still resistant to phagocytosis. The mutation was constructed in vitro and used to replace the resident emm allele in an S. pyogenes strain. To facilitate homologous recombination into the streptococcal chromosome, we adapted a shuttle vector which is temperature sensitive for replication in Gram-positive bacteria but not in Gram-negative hosts. This new method for delivery of a homologous DNA fragment to the S. pyogenes chromosome is efficient and reproducible and should be of general use.
Perez-Casal
J
J
Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia 30322.
Price
J A
JA
Maguin
E
E
Scott
J R
JR
D000483:Alleles; D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000235:genetics*; D002352:Carrier Proteins; D002876:Chromosomes, Bacterial; D005798:Genes, Bacterial; D005813:Genes, Synthetic; D005822:Genetic Vectors; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010587:Phagocytosis; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid; D017384:Sequence Deletion; D012689:Sequence Homology, Nucleic Acid; D013297:Streptococcus pyogenes / Q000235:genetics* / Q000472:pathogenicity; D013696:Temperature; D014169:Transformation, Bacterial; D014774:Virulence / Q000235:genetics
D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; D011993:Recombinant Fusion Proteins; C014689:streptococcal M protein