Protein regions important for plasminogen activation and inactivation of α₂‐antiplasmin in the surface protease Pla of Yersinia pestis

Abstract

SummaryThe plasminogen activator, surface protease Pla, of the plague bacterium Yersinia 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, a 2 -antiplasmin, and the bacteria were found to inactivate a 2 -antiplasmin. In contrast, only poor plasminogen activation and no cleavage of a 2 -antiplasmin was observed with recombinant bacteria expressing the homologous gene ompT from E. coli. A b-barrel topology model for Pla and OmpT predicted 10 transmembrane b-strands and five surface-exposed loops L1±L5. Hybrid Pla± OmpT proteins were created by substituting each of the loops between Pla and OmpT. Analysis of the hybrid molecules suggested a critical role of L3 and L4 in the substrate specificity of Pla towards plasminogen and a 2 -antiplasmin. Substitution analysis at 25 surface-located residues showed the importance of the conserved residues H101, H208, D84, D86, D206 and S99 for the proteolytic activity of Pla-expressing recombinant E. coli. The mature a-Pla of 292 amino acids was processed into b-Pla by an autoprocessing cleavage at residue K262, and residues important for the self-recognition of Pla were identified. Prevention of autoprocessing of Pla, however, had no detectable effect on plasminogen activation or cleavage of a 2 -antiplasmin. Cleavage of a 2 -antiplasmin and plasminogen activation were influenced 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 residue K217 for R217 in L4 of OmpT and also by substituting the entire L3 with that from Pla. This simple modification of the surface loops and the substrate specificity of OmpT exemplifies the evolution of a housekeeping protein into a virulence factor by subtle mutations at critical protein regions. We propose that inactivation of a 2 -antiplasmin by Pla of Y. pestis promotes uncontrolled proteolysis and contributes to the invasive character of plague.

Identification of essential acidic residues of outer membrane protease OmpT supports a novel active site

“…Attempts to crystallize OmpT in complex with a substrate analogue are currently under way. Prior to submission of this paper, we became aware of a very recent publication on omptin Pla of Y. pestis by Kukkonen et al [33] and His 101 were proposed to be involved in substrate interaction [33]. Now that we have solved the crystal structure of OmpT and generated mutagenesis data on all conserved Ser, His, Asp and Glu residues in OmpT, we have a sound basis for our novel active site model, which does not involve a nucleophilic serine.…”

Capsular Antigen Fraction 1 and Pla Modulate the Susceptibility of Yersinia pestis to Pulmonary Antimicrobial Peptides Such as Cathelicidin

“…The Pla protease is an essential virulence factor for the invasion and dissemination of Y. pestis from the periphery. Although Pla was known to have additional substrates, such as the C3 complement factor and ␣ 2 -antiplasmin (33,57), the identification and characterization of its proteolytic inactivation of CAMPs herein are novel. Pla belongs to a family of outer membrane aspartate proteases, designated omptins, in Enterobacteriaceae (32,64).…”

“…After delivery through a fleabite, Y. pestis quickly spreads to the local lymph node, in a process that is facilitated by Psa and the plasminogen activator protein (Pla) (13). Pla is a bacterial outer membrane protein that acts as a protease that cleaves plasminogen to plasmin and degrades ␣ 2 -antiplasmin, a plasmin inhibitor (6,33,57). Pla is also a mediator of bacterial binding to extracellular matrix proteins such as laminin (35).…”

Characterizing the virulence factor YapE in Yersinia pestis.

“…Pla is able to autoprocess itself into three forms, alpha, beta, and gamma. These forms of Pla consist of α-Pla (43%), β-Pla (30%) and γ-Pla (16%) [99]. α-Pla corresponds to the full-size mature Pla where β and γ-Pla are the subsequent processing of α-Pla [100] [97,101].…”

“…Pla, a member of the omptin family of enterobacterial surface protein, is a multifunctional protein that can activate the mammalian plasma proenzyme plasminogen into plasmin [49]. In addition to being able to activate plasminogen into plasmin, Pla also cleaves the complement C3 component [52], modifies bacterium-produced Yops (Yersinia outer proteins) [96,97], has the ability to activate adhesins (YapE) and proteolytically inactivates α2-antiplasmin [98,99].…”