Role in proinflammatory response of YghJ, a secreted metalloproteasefrom neonatal septicemic Escherichia coli

Abstract

Neonatal sepsis is the invasion of microbial pathogens into blood stream and is associated with a sys-temic inflammatory response with production and release of a wide range of inflammatory mediators.The increased serum levels of cytokines were found to correlate with the severity and mortality in courseof sepsis. There have been no reports on the role of microbial proteases in stimulation of proinflamma-tory response in neonatal sepsis. We have identified YghJ, a secreted metalloprotease from a neonatalsepticemic Escherichia coli (NSEC) isolate. The protease was partially purified from culture supernatant bysuccessive anion and gel filtration chromatography. MS/MS peptide sequencing of the protease showedhomology with YghJ. YghJ was cloned, expressed and purified in pBAD TOPO expression vector. YghJ wasfound to be proteolytically active against Methoxysuccinyl Ala-Ala-Pro-Met-p-nitroanilide oligopeptidesubstrate, but not against casein and gelatin. YghJ showed optimal activity at pH 7–8 and at temperatures37–40◦C. YghJ showed clear changes in cellular morphologies of Int407, HT-29 and HEK293 cells. YghJstimulated the secretion of cytokines IL-1�, IL-1� and TNF-� in murine macrophages (RAW 264.7) andIL-8 from human intestinal epithelial cells (HT-29). YghJ also down-regulated the production of anti-inflammatory cytokines such as IL-10. YghJ is present in both septicemic (78%) and fecal E. coli isolates(54%). However, expression and secretion of YghJ is significantly higher among the septicemic (89%) thanthe fecal isolates (33%). This is the first study to show the role of a microbial protease, YghJ in triggeringproinflammatory response in NSEC