RESUMEN
The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) has been proposed to be an epithelial cell receptor for Pseudomonas aeruginosa involved in bacterial internalization and clearance from the lung. We evaluated the role of CFTR in clearing P. aeruginosa from the respiratory tract using transgenic CF mice that carried either the DeltaF508 Cftr allele or an allele with a Cftr stop codon (S489X). Intranasal application achieved P. aeruginosa lung infection in inbred C57BL/6 DeltaF508 Cftr mice, whereas DeltaF508 Cftr and S489X Cftr outbred mice required tracheal application of the inoculum to establish lung infection. CF mice showed significantly less ingestion of LPS-smooth P. aeruginosa by lung cells and significantly greater bacterial lung burdens 4.5 h postinfection than C57BL/6 wild-type mice. Microscopy of infected mouse and rhesus monkey tracheas clearly demonstrated ingestion of P. aeruginosa by epithelial cells in wild-type animals, mostly around injured areas of the epithelium. Desquamating cells loaded with P. aeruginosa could also be seen in these tissues. No difference was found between CF and wild-type mice challenged with an LPS-rough mucoid isolate of P. aeruginosa lacking the CFTR ligand. Thus, transgenic CF mice exhibit decreased clearance of P. aeruginosa and increased bacterial burdens in the lung, substantiating a key role for CFTR-mediated bacterial ingestion in lung clearance of P. aeruginosa.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/genética , Fibrosis Quística/microbiología , Infecciones por Pseudomonas/genética , Infecciones por Pseudomonas/microbiología , Infecciones del Sistema Respiratorio/genética , Infecciones del Sistema Respiratorio/microbiología , Animales , Adhesión Bacteriana/genética , Línea Celular Transformada , Fibrosis Quística/patología , Fibrosis Quística/prevención & control , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Células Epiteliales/microbiología , Células Epiteliales/ultraestructura , Femenino , Pulmón/microbiología , Macaca mulatta , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Confocal , Microscopía Electrónica de Rastreo , Infecciones por Pseudomonas/patología , Infecciones por Pseudomonas/prevención & control , Pseudomonas aeruginosa/fisiología , Pseudomonas aeruginosa/ultraestructura , Infecciones del Sistema Respiratorio/patología , Infecciones del Sistema Respiratorio/prevención & control , Tráquea/microbiología , Tráquea/ultraestructuraRESUMEN
Homozygous mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis (CF). In the heterozygous state, increased resistance to infectious diseases may maintain mutant CFTR alleles at high levels in selected populations. Here we investigate whether typhoid fever could be one such disease. The disease is initiated when Salmonella typhi enters gastrointestinal epithelial cells for submucosal translocation. We found that S. typhi, but not the related murine pathogen S. typhimurium, uses CFTR for entry into epithelial cells. Cells expressing wild-type CFTR internalized more S. typhi than isogenic cells expressing the most common CFTR mutation, a phenylalanine deleted at residue 508 (delta508). Monoclonal antibodies and synthetic peptides containing a sequence corresponding to the first predicted extracellular domain of CFTR inhibited uptake of S. typhi. Heterozygous deltaF508 Cftr mice translocated 86% fewer S. typhi into the gastrointestinal submucosa than wild-type Cftr mice; no translocation occurred in deltaF508 Cftr homozygous mice. The Cftr genotype had no effect on the translocation of S. typhimurium. Immunoelectron microscopy revealed that more CFTR bound to S. typhi in the submucosa of Cftr wild-type mice than in deltaF508 heterozygous mice. We conclude that diminished levels of CFTR in heterozygotes may decrease susceptibility to typhoid fever.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Intestinos/microbiología , Receptores de Superficie Celular/metabolismo , Salmonella typhi/fisiología , Animales , Línea Celular , Colon/microbiología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Epitelio/microbiología , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Intestinos/ultraestructura , Yeyuno/microbiología , Yeyuno/ultraestructura , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Membrana Mucosa/microbiología , Mutación , Receptores de Superficie Celular/genética , Proteínas Recombinantes , Salmonella typhi/patogenicidad , Salmonella typhi/ultraestructura , Salmonella typhimurium/fisiología , Células Tumorales CultivadasRESUMEN
BACKGROUND: The capsular polysaccharide/adhesin (PS/A) antigen of Staphylococcus epidermidis was required to produce endocarditis in a rabbit model in which infection resulted from hematogenous spread of bacteria from a contaminated catheter in the jugular vein. However, many prosthetic valve endocarditis (PVE) infections probably result from direct contamination of the valve with small numbers of bacteria during surgery. The role of PS/A in this situation was evaluated by modifying a rabbit model of endocarditis to partially mimic PVE. METHODS AND RESULTS: A Teflon catheter was contaminated with graded inocula of either PS/A-positive S epidermidis strain M187sp11 or the PS/A-negative, isogenic strain M187sn3 and inserted into the left ventricle through the aortic valve. The PS/A-positive strain had a 50% infectious dose of 1.1 x 10(2) cfu (95% CI, 3.3 to 3.7 x 10(3)) compared with 8.5 x 10(4) cfu of the PS/A-negative strain (95% CI, 8.6 x 10(3) to 8.5 x 10(5)). The odds for developing endocarditis were estimated to be 42 times higher for any given inoculum level of the PS/A-positive strain (P = .1). When the PS/A-positive strain was adherent to a catheter surface it survived in rabbit blood, whereas under the same conditions the PS/A-negative strain was killed approximately 90% in 1 hour. CONCLUSIONS: Direct contamination of an intraventricular foreign body by low levels of PS/A-positive S epidermidis results in endocarditis in rabbits, but at suitably high doses PS/A-negative strains have sufficient virulence to infect cardiac vegetations. PS/A enhances but is not absolutely required for bacterial virulence in a rabbit model of PVE.
Asunto(s)
Endocarditis Bacteriana/microbiología , Prótesis Valvulares Cardíacas/efectos adversos , Polisacáridos Bacterianos/inmunología , Infecciones Estafilocócicas/microbiología , Staphylococcus epidermidis/inmunología , Animales , Contaminación de Equipos , Prótesis Valvulares Cardíacas/microbiología , ConejosRESUMEN
The colonization of mucosal surfaces by Pseudomonas aeruginosa can lead to local or disseminated disease. Secretory immunoglobulin A (IgA) has been assumed to be responsible for preventing mucosal colonization by interfering with the binding of bacterial ligands to epithelial surface receptors. However, the efficacy of this mechanism of immunity derives little actual support from in vivo experiments. In an investigation of the role of local and systemic immunization strategies in reducing colonization of the gastrointestinal tract of mice by P. aeruginosa, the bacterial antigens that were potential targets for immune effectors promoting mucosal clearance were identified. Levels of gastrointestinal colonization were reduced when immunity to homologous O antigens, but not that to pili or flagella, was elicited. Oral vaccination with attenuated Salmonella typhimurium expressing P. aeruginosa serogroup O11 antigen elicited mucosal and serum IgA antibodies and serum IgG antibodies specific for the recombinant antigen. Oral challenge of immunized mice with P. aeruginosa serogroup O11 demonstrated protection against gastrointestinal colonization. Intraperitoneal immunization with a serogroup O11 high-molecular-weight O-polysaccharide antigen elicited only serum IgG and IgM antibodies yet was as effective as oral vaccination in protecting mice against gastrointestinal colonization. This finding was confirmed by the demonstration that intraperitoneal immunization with purified lipopolysaccharide was also protective against mucosal surface colonization. These results call into question the need for local immune effectors, particularly secretory IgA, directed at bacterial ligands for epithelial surface components, in protecting a mucosal surface from bacterial challenge.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/inmunología , Sistema Digestivo/inmunología , Polisacáridos Bacterianos/inmunología , Pseudomonas aeruginosa/inmunología , Administración Oral , Animales , Vacunas Bacterianas/administración & dosificación , Femenino , Lipopolisacáridos/inmunología , Ratones , Ratones Endogámicos BALB C , Antígenos O , Proteínas Recombinantes/inmunología , Salmonella typhimuriumRESUMEN
Serum opsonophagocytic-killing titers often indicate the level of immune resistance to bacterial pathogens, yet in almost all cystic fibrosis (CF) patients that have chronic lung infections with mucoid Pseudomonas aeruginosa, high titers of opsonic-killing Abs can be measured and the infectious pathology still progresses through pulmonary failure and death. This anomalous finding may be due to the use of suspended cells of P. aeruginosa to evaluate phagocytic killing, whereas in the lungs of CF patients the organisms grow in a microcolony or biofilm, encased in mucoid exopolysaccharide (MEP, also called alginate). To determine whether the microcolony mode of growth contributes to bacterial resistance to host defenses, we evaluated opsonophagocytic killing of mucoid P. aeruginosa growing in a biofilm. Abs from infected CF patients were poorly able to mediate opsonic killing of biofilm, but not suspended, mucoid P. aeruginosa cells. Bacterial resistance to killing could be overcome by disruption of the biofilm layer with an enzyme that degrades MEP. Chronically infected CF patients also fail to produce opsonic-killing Abs specific to MEP, and when these Abs were evaluated in sera of older, noninfected CF patients and humans vaccinated with MEP, comparable killing of P. aeruginosa in biofilms and suspensions was obtained. In this case, C3 was deposited onto the MEP layer and could be visualized by fluorescence microscopy deposited throughout the biofilm. We conclude that opsonic Abs made by CF patients in response to chronic infection are ineffective at mediating phagocytic killing and elimination of bacterial cells growing as microcolonies in their lungs.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Cápsulas Bacterianas/inmunología , Biopelículas , Fibrosis Quística/inmunología , Fagocitosis , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/inmunología , Enfermedad Crónica , Complemento C3/análisis , HumanosRESUMEN
Structural and antigenic heterogeneity has been noted among lipopolysaccharides (LPS) produced by Pseudomonas aeruginosa within serogroups previously considered to be serologically homogeneous. We characterized murine monoclonal antibodies (MAbs) and immunization-induced human polyclonal antibodies reactive with one or more of five structurally variant LPS subtypes belonging to serogroup 06 of the International Antigenic Typing System. Analyses of five different MAbs employing purified LPS or whole patterns of subtype specificity, ranging from recognition of a single subtype to reactivity with all five. MAb-mediated opsonophagocytic killing and in vivo protection against live challenge in mice correlated, in general, with differential binding to various LPS subtypes. In comparison, sera from human vaccinees immunized with LPS-derived high-molecular-weight polysaccharide from P. aeruginosa Fisher immunotype 1, one of five serogroup 06 subtypes, exhibited LPS binding and opsonic activity against all five subtypes. Antibodies in the human sera effectively inhibited binding to all five LPS subtype antigens of the cross-reactive MAb, LC3-2H2, suggesting the existence of a common serogroup-related epitope. These findings emphasize the importance of defining subtype-associated variations in LPS antigenicity and corresponding differences in antibody specificity and function as a basis for designing immunoprophylactic or therapeutic strategies which target P. aeruginosa LPS.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Anticuerpos Monoclonales/inmunología , Lipopolisacáridos/inmunología , Pseudomonas aeruginosa/inmunología , Animales , Especificidad de Anticuerpos , Vacunas Bacterianas/inmunología , Secuencia de Carbohidratos , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunización , Ratones , Datos de Secuencia Molecular , Peso Molecular , Fagocitosis , Polisacáridos Bacterianos/inmunologíaRESUMEN
Chronic mucosal colonization by Pseudomonas aeruginosa is an integral part of the pathologic process associated with disease due to infection with this organism. We have adapted the streptomycin-treated murine model of chronic mucosal colonization by enteric pathogens to study colonization by P. aeruginosa. Mice first received 1 mg of streptomycin per ml of drinking water for 2 to 5 days and then ingested 10(7) CFU of P. aeruginosa per ml of drinking water for a minimum of 5 days. The result of this regimen was chronic mucosal colonization with P. aeruginosa for up to 10 weeks, which was determined by fecal cultures and confirmed by culture of the intestines after killing of the experimental animals. Bacterial counts were highest in the cecum and colon, with some evidence for extraintestinal bacterial translocation as well. Use of P. aeruginosa mutants deficient in the production of colonization factors such as pili and those dependent on the rpoN gene product resulted in a lower level of chronic colonization. Immune responses to type-specific lipopolysaccharide, pili, and flagellar antigens were measured, and increases in both serum and intestinal antibodies were usually elicited when a strain elaborated a given antigen. This model represents an easy method of routinely achieving chronic mucosal colonization by P. aeruginosa and should prove useful for the study of both bacterial virulence factors and host responses associated with this infectious process.
Asunto(s)
Enfermedades Gastrointestinales/microbiología , Mucosa Intestinal/microbiología , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/patogenicidad , Animales , Anticuerpos Antibacterianos/biosíntesis , Fibrosis Quística/microbiología , Modelos Animales de Enfermedad , Enfermedades Gastrointestinales/patología , Ratones , Infecciones por Pseudomonas/inmunología , Infecciones por Pseudomonas/patologíaRESUMEN
As a step toward developing recombinant oral vaccines, we have explored the feasibility of expression of O polysaccharide antigens from Pseudomonas aeruginosa by Escherichia coli. We cloned in E. coli HB101 a 26.2-kilobase DNA fragment from P. aeruginosa strain PA103 that specifies the production of the O polysaccharide of Fisher immunotype 2 (IT-2) strains. The recombinant organism incorporated the P. aeruginosa IT-2 O polysaccharide onto the core of the E. coli lipopolysaccharide (LPS). Transfer of the recombinant plasmid to three LPS-rough strains of P. aeruginosa resulted in synthesis of IT-2 O antigen, and two of these transconjugant strains also synthesized a second O polysaccharide, presumably representing expression of a repressed, or an incomplete, set of genes for an endogenous O polysaccharide. Rabbits injected with the purified recombinant LPS made antibody specific for P. aeruginosa IT-2 O side chains, as did mice fed the recombinant E. coli strain. Expression of P. aeruginosa O antigens by enteric bacteria makes it possible to study these recombinant strains as oral vaccines to prevent P. aeruginosa infections.