title: Role in proinflammatory response of YghJ, a secreted metalloprotease from neonatal septicemic Escherichia coli.
abstract: Neonatal sepsis is the invasion of microbial pathogens into blood stream and is associated with a systemic 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 course of sepsis. There have been no reports on the role of microbial proteases in stimulation of proinflammatory response in neonatal sepsis. We have identified YghJ, a secreted metalloprotease from a neonatal septicemic Escherichia coli (NSEC) isolate. The protease was partially purified from culture supernatant by successive anion and gel filtration chromatography. MS/MS peptide sequencing of the protease showed homology with YghJ. YghJ was cloned, expressed and purified in pBAD TOPO expression vector. YghJ was found to be proteolytically active against Methoxysuccinyl Ala-Ala-Pro-Met-p-nitroanilide oligopeptide substrate, but not against casein and gelatin. YghJ showed optimal activity at pH 7-8 and at temperatures 37-40°C. YghJ showed clear changes in cellular morphologies of Int407, HT-29 and HEK293 cells. YghJ stimulated the secretion of cytokines IL-1α, IL-1β and TNF-α in murine macrophages (RAW 264.7) and IL-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%) than the fecal isolates (33%). This is the first study to show the role of a microbial protease, YghJ in triggering proinflammatory response in NSEC.
authors.affiliation: Division of Pathophysiology, National Institute of Cholera and Enteric Diseases (NICED), Kolkata-10, India.
authors.forename: Rima
authors.initials: R
authors.lastname: Tapader
authors.affiliation: Centre for Infectious Diseases & Control, School of Biosciences and Technology, VIT University, Vellore 632014, India.
authors.forename: Dipro
authors.initials: D
authors.lastname: Bose
authors.affiliation: Infectious Diseases and Immunology Division, CSIR-Indian Institute of Chemical Biology, Kolkata-32, India.
authors.forename: Pallabi
authors.initials: P
authors.lastname: Basu
authors.affiliation: Division of Biochemistry, NICED, Kolkata-10, India.
authors.forename: Moumita
authors.initials: M
authors.lastname: Mondal
authors.affiliation: Division of Pathophysiology, National Institute of Cholera and Enteric Diseases (NICED), Kolkata-10, India.
authors.forename: Ayan
authors.initials: A
authors.lastname: Mondal
authors.affiliation: Division of Biochemistry, NICED, Kolkata-10, India.
authors.forename: Nabendu Sekhar
authors.initials: NS
authors.lastname: Chatterjee
authors.affiliation: Division of Clinical Medicine, NICED, Kolkata-10, India.
authors.forename: Pujarini
authors.initials: P
authors.lastname: Dutta
authors.affiliation: Division of Bacteriology, NICED, Kolkata-10, India.
authors.forename: Sulagna
authors.initials: S
authors.lastname: Basu
authors.affiliation: Infectious Diseases and Immunology Division, CSIR-Indian Institute of Chemical Biology, Kolkata-32, India.
authors.forename: Rupak K
authors.initials: RK
authors.lastname: Bhadra
authors.affiliation: Division of Pathophysiology, National Institute of Cholera and Enteric Diseases (NICED), Kolkata-10, India. Electronic address: apal7364@rediffmail.com.
authors.forename: Amit
authors.initials: A
authors.lastname: Pal
mesh_terms: D000818:Animals; D002460:Cell Line; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D016207:Cytokines / Q000378:metabolism; D004795:Enzyme Stability; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology* / Q000302:isolation & purification / Q000472:pathogenicity*; D004927:Escherichia coli Infections / Q000382:microbiology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D045726:Metalloproteases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D000071074:Neonatal Sepsis / Q000382:microbiology*; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry; D013696:Temperature; D037521:Virulence Factors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D016207:Cytokines; D029968:Escherichia coli Proteins; D037521:Virulence Factors; D045726:Metalloproteases; C587633:YghJ protein, E coli
keywords: Cytotoxicity; Metalloprotease YghJ; Neonatal sepsis; Neonatal septicemic Escherichia coli (NSEC); Proinflammatory response