Characterization of the StcE protease activity of Escherichia coli O157:H7.
The StcE zinc metalloprotease is secreted by enterohemorrhagic Escherichia coli (EHEC) O157:H7 and contributes to intimate adherence of this bacterium to host cells, a process essential for mammalian colonization. StcE has also been shown to localize the inflammatory regulator C1 esterase inhibitor (C1-INH) to cell membranes. We tried to more fully characterize StcE activity to better understand its role in EHEC pathogenesis. StcE was active at pH 6.1 to 9.0, in the presence of NaCl concentrations ranging from 0 to 600 mM, and at 4 degrees C to 55 degrees C. Interestingly, antisera against StcE or C1-INH did not eliminate StcE cleavage of C1-INH. Treatment of StcE with the proteases trypsin, chymotrypsin, human neutrophil elastase, and Pseudomonas aeruginosa elastase did not eliminate StcE activity against C1-INH. After StcE was kept at 23 degrees C for 65 days, it exhibited full proteolytic activity, and it retained 30% of its original activity after incubation for 8 days at 37 degrees C. Together, these results show the StcE protease is a stable enzyme that is probably active in the environment of the colon. Additionally, k(cat)/K(m) data showed that StcE proteolytic activity was 2.5-fold more efficient with the secreted mucin MUC7 than with the complement regulator C1-INH. This evidence supports a model which includes two roles for StcE during infection, in which StcE acts first as a mucinase and then as an anti-inflammatory agent by localizing C1-INH to cell membranes.
Grys
Thomas E
TE
Department of Medical Microbiology & Immunology, University of Wisconsin-Madison, Room 481 MSC, 1300 University Ave., Madison, WI 53706, USA.
Walters
Laura L
LL
Welch
Rodney A
RA
D000907:Antibodies, Bacterial / Q000276:immunology; D002918:Chymotrypsin / Q000494:pharmacology; D050718:Complement C1 Inhibitor Protein / Q000378:metabolism; D004927:Escherichia coli Infections / Q000382:microbiology; D019453:Escherichia coli O157 / Q000201:enzymology* / Q000276:immunology; D029968:Escherichia coli Proteins / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D019272:Leukocyte Elastase / Q000494:pharmacology; D008666:Metalloendopeptidases / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D009077:Mucins; D010196:Pancreatic Elastase / Q000494:pharmacology; D011550:Pseudomonas aeruginosa / Q000201:enzymology; D012471:Salivary Proteins and Peptides; D013696:Temperature; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology
D000907:Antibodies, Bacterial; D050718:Complement C1 Inhibitor Protein; D029968:Escherichia coli Proteins; C083238:MUC7 protein, human; D009077:Mucins; D012471:Salivary Proteins and Peptides; D002918:Chymotrypsin; D010196:Pancreatic Elastase; D019272:Leukocyte Elastase; D014357:Trypsin; D008666:Metalloendopeptidases; C465555:StcE protein, E coli