Distinct residues of plasminogen activator affect plasminogen cleavage and Yop delivery in Yersinia pestis

Jones, Christina, Suleyman Felek, Joshua Thomson, and Eric Krukonis

Yersinia pestis, the causative agent of plague, utilizes plasminogen activator (Pla) to activate the host’s circulating plasminogen into plasmin via proteolysis as well as adhere to extracellular matrix (ECM) proteins and host cells. Once generated, plasmin degrades fibrin clots at the point of infection and allows the bacteria to disseminate to regional lymph nodes and eventually the bloodstream. Pla-mediated cell adhesion on the other hand can facilitate delivery of cytotoxic Yop proteins to host cells via a type 3 secretion system (T3SS). Pla has a β-barrel structure containing five short extracellular loops. We altered various amino acid residues in two of these loops to determine their roles in proteolysis, ECM binding and Yop delivery. While some Pla residues previously defined as critical for cleavage of plasminogen were also important for plasminogen binding (D86, S99, H101, D206), other residues maintained relatively strong plasminogen binding despite being largely or completely unable to cleave the substrate (T96, D97, H208, R211), suggesting those residues are especially critical for proteolysis. Binding of Pla mutants to the ECM protein laminin indicated some distinct residues important for binding. Finally, some residues required for the proteolytic activity of Pla hinder Yop delivery (D86, R211) suggesting these residues, while locking proteolytic substrates in place for cleavage, may impede binding to other substrates.