RESUMEN
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), CP9hlyA (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(O2)/FiO2) ratio, increased BAL albumin/protein levels, and increased histological evidence of lung injury compared with rats given CP9hlyA or LPS. In addition, rats given CP9/pEK50 or CP9 had decreased large aggregate surface activity, decreased Pa(O2)/FiO2) ratios, and increased BAL albumin/protein levels at 2 h postinfection compared with rats given CP9hlyA. The severity of permeability lung injury based on albumin/protein levels in BAL at 2 h was ordered as CP9/pEK50 > CP9 > CP9hlyA > normal saline controls. Total lung titers of bacteria were increased at 6 h in rats given CP9 vs. CP9hlyA, 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.
Asunto(s)
Toxinas Bacterianas/farmacología , Modelos Animales de Enfermedad , Proteínas de Escherichia coli/farmacología , Proteínas Hemolisinas/farmacología , Pulmón/metabolismo , Neumonía Bacteriana/metabolismo , Surfactantes Pulmonares/metabolismo , Animales , Lavado Broncoalveolar , Pulmón/efectos de los fármacos , Lesión Pulmonar , Neutrófilos/patología , Neumonía Bacteriana/patología , Ratas , Ratas Long-EvansRESUMEN
Infections due to extraintestinal pathogenic E. coli (ExPEC) result in significant morbidity, mortality and increased healthcare costs. An efficacious vaccine against ExPEC would be desirable. In this report, we explore the use of killed-whole E. coli as a vaccine immunogen. Given the diversity of capsule and O-antigens in ExPEC, we have hypothesized that alternative targets are viable vaccine candidates. We have also hypothesized that immunization with a genetically engineered strain that is deficient in the capsule and O-antigen will generate a greater immune response against antigens other than the capsular and O-antigen epitopes than a wild-type strain. Lastly, we hypothesize that mucosal immunization with killed E. coli has the potential to generate a significant immune response. In this study, we demonstrated that nasal immunization with a formalin-killed ExPEC derivative deficient in capsule and O-antigen results in a significantly greater overall humoral response compared to its wild-type derivative (which demonstrates that capsule and/or the O-antigen impede the development of an optimal humoral immune response) and a significantly greater immune response against non-capsular and O-antigen epitopes. These antibodies also bound to a subset of heterologous ExPEC strains and enhanced neutrophil-mediated bactericidal activity against the homologous and a heterologous strain. Taken together, these studies support the concept that formalin-killed genetically engineered ExPEC derivatives are whole cell vaccine candidates to prevent infections due to ExPEC.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Infecciones por Escherichia coli/prevención & control , Vacunas contra Escherichia coli , Ingeniería Genética/métodos , Vacunas de Productos Inactivados , Administración Intranasal , Animales , Cápsulas Bacterianas/genética , Sangre/microbiología , Escherichia coli/genética , Escherichia coli/patogenicidad , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/inmunología , Vacunas contra Escherichia coli/administración & dosificación , Vacunas contra Escherichia coli/genética , Vacunas contra Escherichia coli/inmunología , Femenino , Formaldehído/farmacología , Humanos , Inmunización , Masculino , Ratones , Antígenos O/genética , Conejos , Vacunas de Productos Inactivados/administración & dosificación , Vacunas de Productos Inactivados/genética , Vacunas de Productos Inactivados/inmunologíaRESUMEN
Extracellular pathogenic Escherichia coli (ExPEC) strains are common causes of a variety of clinical syndromes, including urinary tract infections, abdominal infections, nosocomial pneumonia, neonatal meningitis, and sepsis. ExPEC strains are extracellular bacterial pathogens; therefore, the innate immune response (e.g., professional phagocytes) plays a crucial role in the host defense against them. Studies using the model ExPEC strain CP9 demonstrated that it is relatively resistant to neutrophil-mediated bactericidal activity. Although this could be due to resistance to phagocytosis, the ability of CP9 to survive the intracellular killing mechanisms of neutrophils is another possibility. Using a variation of the intracellular invasion assay, we studied the survival of CP9 within peripheral blood-derived human neutrophils. Our results indicated that CP9 did survive within human neutrophils, but we were unable to demonstrate that intracellular replication occurred. This finding was not unique to CP9, since when a conservative assessment of survival was used, four of six additional ExPEC strains, but not an E. coli laboratory strain, were also capable of survival within neutrophils. Initial studies in which we began to decipher the mechanisms by which CP9 is able to successfully survive intracellular neutrophil-mediated bactericidal activity demonstrated that CP9 was at least partially susceptible to the neutrophil oxidative burst. Therefore, absolute resistance to the oxidative burst is not a mechanism by which ExPEC survives within neutrophils. In addition, electron microscopy studies showed that CP9 appeared to be present in phagosomes within neutrophils. Therefore, avoidance of phagosomal uptake or subsequent escape from the phagosome does not appear to be a mechanism that contributes to CP9's survival. These findings suggest that survival of ExPEC within neutrophils may be an important virulence mechanism.
Asunto(s)
Infecciones por Escherichia coli/microbiología , Escherichia coli/fisiología , Neutrófilos/microbiología , Actividad Bactericida de la Sangre , Escherichia coli/patogenicidad , Humanos , Neutrófilos/inmunología , Neutrófilos/ultraestructuraRESUMEN
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.