Site-directed mutagenesis of streptococcal plasmin receptor protein (Plr) identifies the C-terminal Lys334 as essential for plasmin binding, but mutation of the plr gene does not reduce plasmin binding to group A streptococci

Abstract

Plasmin(ogen) binding is a common property of many pathogenic bacteria including group A streptococci. Previous analysis of a putative plasmin receptor protein, Plr, from the group A streptococcal strain 64/14 revealed that it is a glyceraldehyde-3-phosphate dehydrogenase and that the plr gene is present on the chromosome as a single copy. This study continues the functional characterization of Plr as a plasmin receptor. Attempts at insertional inactivation of the plr gene suggested that this single-copy gene may be essential for cell viability. Therefore, an alternative strategy was applied to manipulate this gene in who. Site-directed mutagenesis of Plr revealed that a C-terminal lysyl residue is required for wild-type levels of plasmin binding. Mutated Plr proteins expressed in Escherichia coli demonstrated reduced plasmin-binding activity yet retained glyceraldehyde-3-phosphate dehydrogenase activity. A novel integration vector was constructed to precisely replace the wild-type copy of the plr gene with these mutations. lsogenic 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 animal models.

Plasminogen binding by oral streptococci from dental plaque and inflammatory lesions

“…Previous studies have shown that group A streptococci bind plasminogen by four surface receptors: group A streptococcal M-like protein, enolase, GAPDH and most recently PAM-related protein Prp (Pancholi & Fischetti, 1998;Winram & Lottenberg, 1998;Walker et al, 2005;Sanderson-Smith et al, 2007). Enolase and GAPDH are known to contain lysine residues (Derbise et al, 2004) and are, therefore, efficient binders of plasminogen.…”

Binding and Activation of Human Plasminogen by Mycobacterium tuberculosis

“…Examples are the glyceraldehyde-3-phosphate dehydrogenase of group A streptococci (25) and the cell surface lipoprotein of B. burgdorferi (3). Moreover, site-directed mutagenesis of the streptococus plasminogen receptor confirmed that C-terminal lysyl residues are required for binding (26). In this regard it is worth pointing out that the M. tuberculosis genome database (2) documents the existence of an ␣-enolase (Rv1023) and a glyceraldehyde-3-phosphate dehydrogenase (Rv2982c) with Cterminal lysyl residues.…”

Inhibition of Cell Surface Export of Group A Streptococcal Anchorless Surface Dehydrogenase Affects Bacterial Adherence and Antiphagocytic Properties

“…In GAS, glucose is metabolized solely by the glycolytic pathway, as GAS lacks the tricarbolicacetic acid (TCA)-metabolic cycle and the Entner-Doudoroff alternative metabolic pathway (1,2,13,29,43). The multifunctional SDH is encoded by a single essential gene (33,45). The gene knockout approach to prevent SDH expression in GAS, therefore, is not feasible.…”

“…The gene knockout approach to prevent SDH expression in GAS, therefore, is not feasible. The site-directed mutagenesis strategies that allow alteration in the specific functional domains without disturbing the conformational structure responsible for the catalytic function have been successfully employed to create Plg-binding enolase-and SDH/Plr-specific deletion mutants (3,12,45). Since the putative transport system responsible for exporting the anchorless metabolic surface enzymes is not known, it has not been possible to create a GAS mutant in which the anchorless enzyme is selectively retained in the cytoplasm and not expressed on the surface.…”