Protein regions important for plasminogen activation and inactivation of alpha(2)-antiplasmin in the surface protease Pla of Yersinia pestis

The plasminogen activator, surface protease Pla, of the plague bacterium Ye rsinia pestis is an important virulence factor that enables the spread of Y . pestis from subcutaneous sites into circulation. Pla-expressing Y. pestis and recombinant Escherichia coli formed active plasmin in the presence of the major human plasmin inhibitor, alpha (2)-antiplasmin, and the bacteria were found to inactivate alpha (2)-antiplasmin. In contrast, only poor plas minogen activation and no cleavage of alpha (2)-antiplasmin was observed wi th recombinant bacteria expressing the homologous gene ompT from E. coli. A beta -barrel topology model for Pla and OmpT predicted 10 transmembrane be ta -strands and five surface-exposed loops L1-L5. Hybrid Pla-OmpT proteins were created by substituting each of the loops between Pla and OmpT. Analys is of the hybrid molecules suggested a critical role of L3 and L4 in the su bstrate specificity of Pla towards plasminogen and alpha (2)-antiplasmin. S ubstitution analysis at 25 surface-located residues showed the importance o f the conserved residues H101, H208, D84, D86, D206 and S99 for the proteol ytic activity of Pla-expressing recombinant E. coli. The mature alpha -Pla of 292 amino acids was processed into beta -Pla by an autoprocessing cleava ge at residue K262, and residues important for the self-recognition of Pla were identified. Prevention of autoprocessing of Pla, however, had no detec table effect on plasminogen activation or cleavage of alpha (2)-antiplasmin . Cleavage of alpha (2)-antiplasmin and plasminogen activation were influen ced by residue R211 in L4 as well as by unidentified residues in L3. OmpT, which is not associated with invasive bacterial disease, was converted into a Pla-like protease by deleting residues D214 and P215, by substituting re sidue K217 for R217 in L4 of OmpT and also by substituting the entire L3 wi th that from Pla. This simple modification of the surface loops and the sub strate specificity of OmpT exemplifies the evolution of a housekeeping prot ein into a virulence factor by subtle mutations at critical protein regions . We propose that inactivation of alpha (2)-antiplasmin by Pla of Y. pestis promotes uncontrolled proteolysis and contributes to the invasive characte r of plague.