| The purpose of this project was to survey over one thousand clinical isolates of gram negative bacteria for plasminogen activation-like (Pla-like) activity. The isolates were cultured, and whole cell lysates were screened using a two-stage activity assay. Their activities were compared to medical records of patients from whom each isolate was obtained to determine whether high Pla-like activity alone might confer high virulence. With the number of medical records analyzed and the diversity of bacteria sampled, no direct correlation could be established between the level of Pla-like activity and specific patient symptoms. However a strong correlation exists between percent of sequence homology to the Pla protease of Yersinia pestis (the agent of plague) and the potential of the organism to cause disseminated infections. In a plot of log10 cell-specific Pla-like activity versus the percent sequence homology to Y. pestis Pla, a linear relationship (R2 = 0.9599) was observed indicating that Pla-like properties are important for plasminogen activity per se . The high-activity, high-dissemination potential cause and effect relationship was supported by Klebsiella pneumoniae, the third most frequent cause of hospital-acquired bacteremia. One member of a subset of high activity K. pneumoniae strains had the highest activity of all bacteria tested from infectious sources, other than Y. pestis. The relationship was also supported by Enterobacter cloacae, a highly clinically significant bacterium, currently one of the leading causes of hospital-acquired sepsis, that also had very high Pla-like activity. Cloning of a Pla-like Enterobacter protease revealed the highest homology to Y. pestis Pla yet known. Also cloned was a highly active Pla-like protease (12 times higher activity than the mean infective E. coli) from Enterohemorrhagic E. coli (EHEC), a gastrointestinal agent also known to cause multi-organ failure. The EHEC Pla-like protease was 99.9% similar to the subsequently published Protease VII gene of the complete sequencing of the EHEC genome. Another gastrointestinal agent Salmonella, also a cause of bacteremia, has high Pla-like activity. The localized (non disseminated) dysentery agent Shigella had low Pla-like activity. Because Shigella has a Pla-like homolog, yet has low activity, this suggests that Pla homologs appear to have multifunctional roles in virulence.; Pseudomonas aeruginosa is currently the fourth leading cause of hospital acquired sepsis. In the survey for Pla-like activities, we also found proteases with plasmin-like activities from P. aeruginosa . Seventy-three percent of a random collection of these isolates had low plasmin-like activity, while only 22% of corneal infective P. aeruginosa had equally low activities. This highly significant difference (p = 0.000013) indicates that strains with very low plasmin-like activity are rare among the corneal infection-causing isolates. Biochemical isolation and characterization of this protease indicated it is a serine protease, with an amino terminal sequence similar to a previously characterized Protease IV, a lysyl endopeptidase (lep). However our protease had a lower Km and higher Kcat, and over 1000-fold greater activity than the previously characterized protease. These kinetic data suggest that the increased activity difference from published reports is due more to kinetics than to protease production. Overall, the data extend our knowledge of Pla-like and plasmin-like proteases in gram negative bacteria, and strongly support a role for bacterial proteases in virulence. |
