SITE-DIRECTED MUTAGENESIS OF STREPTOCOCCAL PLASMIN RECEPTOR PROTEIN (PLR) IDENTIFIES THE C-TERMINAL LYS(334) AS ESSENTIAL FOR PLASMIN BINDING, BUT MUTATION OF THE PLR GENE DOES NOT REDUCE PLASMIN BINDING TO GROUP-A STREPTOCOCCI

Plasmin(ogen) binding is a common property of many pathogenic bacteria including group A streptococci. Previous analysis of a putative plasm in receptor protein. Plr, from the group A streptococcal strain 64/14 revealed that it is a glyceraldehyde-3-phosphate dehydrogenase and tha t the plr gene is present on the chromosome as a single copy. This stu dy continues the functional characterization of Plr as a plasmin recep tor. Attempts at insertional inactivation of the plr gene suggested th at this single-copy gene may be essential for cell viability. Therefor e, an alternative strategy was applied to manipulate this gene in vivo . Site-directed mutagenesis of Plr revealed that a C-terminal lysyl re sidue is required for wild-type levels of plasmin binding. Mutated Plr proteins expressed in Escherichia coil demonstrated reduced plasmin-b inding activity yet retained glyceraldehyde-3-phosphate dehydrogenase activity, A novel integration vector was constructed to precisely repl ace the wild-type copy of the plr gene with these mutations. Isogenic streptococcal strains expressing altered Plr bound equivalent amounts of plasmin as wild-type streptococci. These data suggest that Plr does not function as a unique plasmin receptor, and underscore the need to identify other plasmin-binding structures on group A streptococci and to assess the importance of the plasminogen system in pathogenesis by inactivation of plasminogen activators and the use of appropriate ani mal models.