Characterization of the StcE Protease Activity of Escherichia coli O157:H7

Abstract

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°C to 55°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°C for 65 days, it exhibited full proteolytic activity, and it retained 30% of its original activity after incubation for 8 days at 37°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 antiinflammatory agent by localizing C1-INH to cell membranes.Enterohemorrhagic E. coli (EHEC) O157:H7 strains cause infections that result in diarrhea and hemorrhagic colitis, which can progress to hemolytic-uremic syndrome (17). These bacteria belong to a group of pathogens that form attaching and effacing (A/E) lesions, which are characterized by intimate adherence to the host cells and effacement of the microvilli (9, 17). The StcE protease produced by EHEC is secreted by the type II secretion system (15) that is encoded 3Ј of stcE on the pO157 virulence plasmid (2). Our laboratory has recently demonstrated that StcE contributes to intimate adherence of EHEC to host cells (12), a process essential for colonization of the terminal rectum of the bovine host (18). We have also shown that StcE cleaves C1 esterase inhibitor (C1-INH), a regulator of multiple inflammatory pathways (15), and localizes it to host cell surfaces (14).The current model for StcE activity (12) proposes that initially, StcE allows passage of EHEC through the oral cavity by cleaving the salivary glycoproteins that are responsible for bacterial aggregation. Similarly, in the colon, StcE cleaves the glycoproteins that protect the intestinal epithelial surface, allowing EHEC to come into close contact with host cell membranes, where components of the locus of enterocyte effacement mediate formation of the characteristic A/E lesions. Later during infection, Shiga toxins and other virulence factors compromise the intestinal epithelial and endothelial barriers, allowing blood with its complement effectors into the intestinal lumen (9, 17). At this point, StcE localizes C1-...

Norwegian Sheep Are an Important Reservoir for Human-Pathogenic Escherichia coli O26:H11

“…This is in agreement with our findings, because isolates from all four HUS patients included in the present study carried stx and were assigned to group A. Another characteristic seen in approximately half of the human and ovine group A isolates was the presence of stcE O103 , a homologue of stcE (39) that promotes the formation of A/E lesions and inhibits the inflammatory system (31,51). This result therefore further supports the pathogenic potential of the group A isolates.…”

EspP, a Serine Protease of EnterohemorrhagicEscherichia coli, Impairs Complement Activation by Cleaving Complement Factors C3/C3b and C5

“…A zinc metalloprotease, StcE of E. coli O157:H7, was reported to cleave C1-esterase inhibitor and mucus-related glycoproteins (11,16). Later, it was proposed that StcE mediates binding of C1-esterase inhibitor to bacterial and host cell surfaces, protecting them from complement-mediated lysis (11,17). However, the interference of these and other virulence factors with the complement cascade is not completely understood.…”

“…For example, a 56-kDa protease of Serratia marcescens was reported to inactivate the chemotactic activity of C5a (19). A zinc metalloprotease, StcE of E. coli O157:H7, was reported to cleave C1-esterase inhibitor and mucus-related glycoproteins (11,16). Later, it was proposed that StcE mediates binding of C1-esterase inhibitor to bacterial and host cell surfaces, protecting them from complement-mediated lysis (11,17).…”

Mecanismos de virulencia de Escherichia coli enteropatógena

“…• Otros factores de virulencia de ECST: Adicionalmente, el plásmido pO157 de ECST codifica genes para catalasa-peroxidasa (katP) 56 , adhesina ToxB 57 , metaloproteasa dependiente de zinc (StcE) 58-61 y para la enterohemolisina (Ehx) 59 , cuyas funciones están principalmente enfocadas a mediar la unión a la célula hospedera, degradación de mucinas y glicoproteínas, regular mecanismos de inflamación y citotoxicidad, entre otras [56][57][58][59][60][61][62][63] .…”