title: E. coli virulence factor hemolysin induces neutrophil apoptosis and necrosis/lysis in vitro and necrosis/lysis and lung injury in a rat pneumonia model.
abstract: Enteric gram-negative bacilli, such as Escherichia coli are the most common cause of nosocomial pneumonia. In this study a wild-type extraintestinal pathogenic strain of E. coli (ExPEC)(CP9) and isogenic derivatives deficient in hemolysin (Hly) and cytotoxic necrotizing factor (CNF) were assessed in vitro and in a rat model of gram-negative pneumonia to test the hypothesis that these virulence factors induce neutrophil apoptosis and/or necrosis/lysis. As ascertained by in vitro caspase-3/7 and LDH activities and neutrophil morphology, Hly mediated neutrophil apoptosis at lower E. coli titers (1 x 10(5-6) cfu) and necrosis/lysis at higher titers (> or =1 x 10(7) cfu). Data suggest that CNF promotes apoptosis but not necrosis or lysis. We also demonstrate that annexin V/7-amino-actinomycin D staining was an unreliable assessment of apoptosis using live E. coli. The use of caspase-3/7 and LDH activities and neutrophil morphology supported the notion that necrosis, not apoptosis, was the primary mechanism by which neutrophils were affected in our in vivo gram-negative pneumonia model using live E. coli. In addition, in vivo studies demonstrated that Hly mediates lung injury. Neutrophil necrosis was not observed when animals were challenged with purified lipopolysaccharide, demonstrating the importance of using live bacteria. These findings establish that Hly contributes to ExPEC virulence by mediating neutrophil toxicity, with necrosis/lysis being the dominant effect of Hly on neutrophils in vivo and by lung injury. Whether Hly-mediated lung injury is due to neutrophil necrosis, a direct effect of Hly, or both is unclear.
authors.affiliation: Dept. of Medicine, Div. of Infectious Diseases, 3435 Main St., Biomedical Research Bldg., Rm. 141, University at Buffalo, Buffalo, New York 14214, USA. trusso@acsu.buffalo.edu
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Russo
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Davidson
authors.forename: Stacy A
authors.initials: SA
authors.lastname: Genagon
authors.forename: Natalie M
authors.initials: NM
authors.lastname: Warholic
authors.forename: Ulrike
authors.initials: U
authors.lastname: Macdonald
authors.forename: Patrick D
authors.initials: PD
authors.lastname: Pawlicki
authors.forename: Janet M
authors.initials: JM
authors.lastname: Beanan
authors.forename: Ruth
authors.initials: R
authors.lastname: Olson
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Holm
authors.forename: Paul R
authors.initials: PR
authors.lastname: Knight
mesh_terms: D000818:Animals; D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects*; D001427:Bacterial Toxins / Q000494:pharmacology*; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases / Q000378:metabolism; D004195:Disease Models, Animal; D029968:Escherichia coli Proteins / Q000494:pharmacology*; D006460:Hemolysin Proteins / Q000494:pharmacology*; D066298:In Vitro Techniques; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008168:Lung / Q000187:drug effects*; D009336:Necrosis; D009504:Neutrophils / Q000201:enzymology / Q000473:pathology*; D018410:Pneumonia, Bacterial / Q000382:microbiology*; D051381:Rats; D020318:Rats, Long-Evans
chemical_list: D017304:Annexin A5; D001427:Bacterial Toxins; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins; C036507:Hlya protein, E coli; C060296:cytotoxic necrotizing factor type 1; D007770:L-Lactate Dehydrogenase; C505410:Casp3 protein, rat; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases