Surfactant dysfunction and lung injury due to the E. coli virulence factor hemolysin in a rat pneumonia model

Departments of 1 Medicine and 2 Microbiology, 3 The Witebsky Center for Microbial Pathogenesis; 4 Veterans Administration Western New York Healthcare System; Departments of 5 Anesthesiology, 6 Pathology, and 7 Pediatrics, 8 Center of Excellence in Bioinformatics and Life Sciences, State University of New York at Buffalo, Buffalo; and Departments of 9 Pediatrics, 10 Environmental Medicine, and 11 Biomedical Engineering, University of Rochester, Rochester, New York Submitted 24 August 2006 ; accepted in final form 27 October 2006 This study tests the hypothesis that the virulence factor hemolysin (Hly) expressed by extraintestinal pathogenic Escherichia coli contributes to surfactant dysfunction and lung injury in a rat model of gram-negative pneumonia. Rats were instilled intratracheally with CP9 (wild type, Hly-positive), CP9 hlyA (Hly-minus), CP9 /pEK50 (supraphysiological Hly), or purified LPS. At 6 h postinfection, rats given CP9 had a decreased percentage content of large surfactant aggregates in cell-free bronchoalveolar lavage (BAL), decreased large aggregate surface activity, decreased Pa O 2 /F I O 2 ratio, increased BAL albumin/protein levels, and increased histological evidence of lung injury compared with rats given CP9 hlyA or LPS. In addition, rats given CP9/ pEK50 or CP9 had decreased large aggregate surface activity, decreased Pa O 2 /F I O 2 ratios, and increased BAL albumin/protein levels at 2 h postinfection compared with rats given CP9 hlyA . The severity of permeability lung injury based on albumin/protein levels in BAL at 2 h was ordered as CP9 /pEK50 > CP9 > CP9 hlyA > normal saline controls. Total lung titers of bacteria were increased at 6 h in rats given CP9 vs. CP9 hlyA , but bacterial titers were not significantly different at 2 h, indicating that increased surfactant dysfunction and lung injury were associated with Hly as opposed to bacterial numbers per se. Further studies in vitro showed that CP9 could directly lyse transformed pulmonary epithelial cells (H441 cells) but that indirect lysis of H441 cells secondary to Hly-induced neutrophil lysis did not occur. Together, these data demonstrate that Hly is an important direct mediator of surfactant dysfunction and lung injury in gram-negative pneumonia. lung surfactant dysfunction; Escherichia coli ; bacterial virulence Address for reprint requests and other correspondence: T. A. Russo, Dept. of Medicine, Division of Infectious Diseases, SUNY at Buffalo, 3435 Main St., Biomedica