RESUMEN

The invasion of Pseudomonas aeruginosa and Salmonella enterica serovar Typhi into epithelial cells depends on the cystic fibrosis transmembrane conductance regulator (CFTR) protein as an epithelial receptor. In the case of P. aeruginosa, the bacterial ligand for CFTR is the outer core oligosaccharide portion of the lipopolysaccharide (LPS). To determine whether serovar Typhi LPS is also a bacterial ligand mediating internalization, we used both P. aeruginosa and serovar Typhi LPS as a competitive inhibitor of serovar Typhi invasion into the epithelial cell line T84. P. aeruginosa LPS containing a complete core efficiently inhibited serovar Typhi invasion. However, neither killed wild-type Typhi cells nor purified LPS were effective inhibitors. LPS from mutant Typhi strains defective in O side-chain synthesis, but with an apparently normal core, was capable of inhibiting invasion, but LPS obtained from a deeper rough mutant strain with alterations in fast-migrating core oligosaccharide failed to inhibit invasion. Lastly, exposure of wild-type serovar Typhi to T84 cultures before heat killing resulted in a structural alteration in its LPS that allowed the heat-killed cells to inhibit invasion of wild-type serovar Typhi. These data indicate that the serovar Typhi LPS core, like the P. aeruginosa LPS core, is a ligand mediating internalization of bacteria by epithelial cells, and that exposure of this ligand on wild-type Typhi is induced by the bacteria's interaction with host cells.

Asunto(s)

Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Células Epiteliales/microbiología , Lipopolisacáridos/química , Lipopolisacáridos/farmacología , Salmonella typhi/patogenicidad , Sitios de Unión , Línea Celular , Humanos , Ligandos , Lipopolisacáridos/aislamiento & purificación , Lipopolisacáridos/metabolismo , Mutación , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/patogenicidad , Pseudomonas aeruginosa/fisiología , Salmonella typhi/efectos de los fármacos , Salmonella typhi/fisiología

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/ultraestructura

RESUMEN

Establishment and maintenance of chronic lung infections with mucoid Pseudomonas aeruginosa in patients with cystic fibrosis (CF) require that the bacteria avoid host defenses. Elaboration of the extracellular, O-acetylated mucoid exopolysaccharide, or alginate, is a major microbial factor in resistance to immune effectors. Here we show that O acetylation of alginate maximizes the resistance of mucoid P. aeruginosa to antibody-independent opsonic killing and is the molecular basis for the resistance of mucoid P. aeruginosa to normally nonopsonic but alginate-specific antibodies found in normal human sera and sera of infected CF patients. O acetylation of alginate appears to be critical for P. aeruginosa resistance to host immune effectors in CF patients.

Asunto(s)

Alginatos , Cápsulas Bacterianas , Proteínas Opsoninas , Fagocitosis , Pseudomonas aeruginosa/patogenicidad , Acetilación , Activación de Complemento , Vía Alternativa del Complemento , Fibrosis Quística/complicaciones , Humanos , Enfermedades Pulmonares/complicaciones , Enfermedades Pulmonares/etiología , Infecciones por Pseudomonas/complicaciones , Infecciones por Pseudomonas/etiología

RESUMEN

Pseudomonas aeruginosa is the nosocomial bacterial pathogen most commonly isolated from the respiratory tract. Animal models of this infection are extremely valuable for studies of virulence and immunity. We thus evaluated the utility of a simple model of acute pneumonia for analyzing P. aeruginosa virulence by characterizing the course of bacterial infection in BALB/c mice following application of bacteria to the nares of anesthetized animals. Bacterial aspiration into the lungs was rapid, and 67 to 100% of the inoculum could be recovered within minutes from the lungs, with 0.1 to 1% of the inoculum found intracellularly shortly after infection. At later time points up to 10% of the bacteria were intracellular, as revealed by gentamicin exclusion assays on single-cell suspensions of infected lungs. Expression of exoenzyme U (ExoU) by P. aeruginosa is associated with a cytotoxic effect on epithelial cells in vitro and virulence in animal models. Insertional mutations in the exoU gene confer a noncytotoxic phenotype on mutant strains and decrease virulence for animals. We used the model of acute pneumonia to determine whether introduction of the exoU gene into noncytotoxic strains of P. aeruginosa lacking this gene affected virulence. Seven phenotypically noncytotoxic P. aeruginosa strains were transformed with pUCP19exoUspcU which carries the exoU gene and its associated chaperone. Three of these strains became cytotoxic to cultured epithelial cells in vitro. These strains all secreted ExoU, as confirmed by detection of the ExoU protein with specific antisera. The 50% lethal dose of exoU-expressing strains was significantly lower for all three P. aeruginosa isolates carrying plasmid pUCP19exoUspcU than for the isogenic exoU-negative strains. mRNA specific for ExoU was readily detected in the lungs of animals infected with the transformed P. aeruginosa strains. Introduction of the exoU gene confers a cytotoxic phenotype on some, but not all, otherwise-noncytotoxic P. aeruginosa strains and, for recombinant strains that could express ExoU, there was markedly increased virulence in a murine model of acute pneumonia and systemic spread.

Asunto(s)

Proteínas Bacterianas/genética , Citotoxinas/genética , Neumonía Bacteriana/etiología , Infecciones por Pseudomonas/etiología , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidad , Animales , Animales Recién Nacidos , Secuencia de Bases , Cartilla de ADN/genética , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Humanos , Ratones , Ratones Endogámicos BALB C , Pseudomonas aeruginosa/aislamiento & purificación , Transformación Genética , Virulencia/genética

RESUMEN

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 Cultivadas

RESUMEN

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride ion channel, but its relationship to the primary clinical manifestation of CF, chronic Pseudomonas aeruginosa pulmonary infection, is unclear. We report that CFTR is a cellular receptor for binding, endocytosing, and clearing P. aeruginosa from the normal lung. Murine cells expressing recombinant human wild-type CFTR ingested 30-100 times as many P. aeruginosa as cells lacking CFTR or expressing mutant DeltaF508 CFTR protein. Purified CFTR inhibited ingestion of P. aeruginosa by human airway epithelial cells. The first extracellular domain of CFTR specifically bound to P. aeruginosa and a synthetic peptide of this region inhibited P. aeruginosa internalization in vivo, leading to increased bacterial lung burdens. CFTR clears P. aeruginosa from the lung, indicating a direct connection between mutations in CFTR and the clinical consequences of CF.

Asunto(s)

Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Endocitosis , Enfermedades Pulmonares/microbiología , Infecciones por Pseudomonas/microbiología , Receptores de Superficie Celular/metabolismo , Animales , Células Cultivadas , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Endocitosis/efectos de los fármacos , Células Epiteliales/microbiología , Humanos , Pulmón/citología , Ratones , Fragmentos de Péptidos/farmacología , Unión Proteica , Receptores de Superficie Celular/genética

RESUMEN

Patients with cystic fibrosis (CF) have a pronounced hypersusceptibility (80 to 90%) to Pseudomonas aeruginosa infection. We hypothesized that airway epithelial cell ingestion of bacteria followed by cellular desquamation may protect the lung from infection, and epithelial cells expressing mutant forms of the cystic fibrosis transmembrane conductance regulator (CFTR) may be defective in this function. We found that transformed human airway epithelial cells homozygous for the delta F508 allele of CFTR were significantly defective in uptake of P. aeruginosa compared with the same cell line complemented with the wild-type allele of CFTR. Partial membrane expression of the delta F508 CFTR protein occurs in cells grown at 26 degrees C, and under these conditions uptake of P. aeruginosa occurred at levels comparable to cells with a wild-type allele of CFTR. Epithelial cell ingestion assays using isogenic bacterial strains differing in lipopolysaccharide (LPS) phenotype, along with inhibition studies, identified the LPS-core oligosaccharide as the bacterial ligand for epithelial cell invasion. Inhibition of epithelial cell ingestion of P. aeruginosa in a neonatal mouse lung infection model led to increased levels of bacteria in the lungs 24 and 48 h after infection. Defective epithelial cell internalization of P. aeruginosa may be a critical factor in hypersusceptibility of CF patients to chronic lung infections.

Asunto(s)

Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/genética , Fibrosis Quística/microbiología , Mutación Puntual , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/fisiología , Infecciones del Sistema Respiratorio/microbiología , Animales , Adhesión Bacteriana , Línea Celular Transformada , Fibrosis Quística/complicaciones , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Susceptibilidad a Enfermedades , Epitelio/metabolismo , Epitelio/microbiología , Humanos , Ratones , Infecciones por Pseudomonas/complicaciones , Pseudomonas aeruginosa/genética , Sistema Respiratorio/metabolismo , Sistema Respiratorio/microbiología , Infecciones del Sistema Respiratorio/complicaciones

RESUMEN

We have reported that some strains of Pseudomonas aeruginosa can enter corneal epithelial cells during experimental murine eye infection and when the cells are cultured in vitro. Following invasion, both the host cell and the intracellular bacteria can remain viable for up to 24 h. Others have reported that toxin-mediated damage of epithelial cells contributes to the pathogenesis of P. aeruginosa keratitis. To clarify the relationship between cell invasion and cytotoxicity, fourteen P. aeruginosa isolates were compared for their capacity to enter epithelial cells and for their ability to induce cytotoxicity. Bacterial invasion was quantified by gentamicin survival assays both in vivo and in vitro. Cytotoxicity was examined qualitatively by trypan blue exclusion assays and quantitatively by chromium release assays in vitro. A significant inverse correlation was found between the ability to induce cytotoxicity and epithelial cell invasion as measured by gentamicin survival assays. Both cytotoxic and noncytotoxic strains were identified among corneal and noncorneal isolates; all isolates that were not cytotoxic were capable of epithelial cell invasion. Efficient host cell invasion could not be demonstrated for cytotoxic strains; however, the gentamicin survival assay relies upon host cells retaining viability in order to yield useful results, and this may limit the effectiveness of this assay for testing epithelial cell invasion by cytotoxic strains. Since all of the corneal isolates that were tested were virulent in vivo, the results show that there are at least two different types of P. aeruginosa-induced disease, one caused by strains that are cytotoxic and the other involving bacteria that can enter epithelial cells and survive intracellularly without killing the host cell.

Asunto(s)

Córnea/microbiología , Pseudomonas aeruginosa/patogenicidad , Animales , Células Cultivadas , Córnea/patología , Epitelio/microbiología , Gentamicinas/farmacología , Humanos , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica , Conejos

RESUMEN

The fate of alpha-melanocyte-stimulating hormone (alpha-MSH) subsequent to binding to the melanoma melanocortin MC1 receptor is of interest with regard to its potential use in targeting cytotoxic drugs or imaging to melanoma. Tools such as iodinated, photoaffinity-labelled, biotinylated and fluorescent melanocortins are required to study the fate of the ligand during its interaction with the receptor. In this study, a series of probes for the receptor based on the potent analogue, [Nle4,Dphe7]alpha-MSH, have been developed and tested for their potential usefulness. All probes contain the core melanocortin motif His-Phe-Arg-Trp. They bind the receptor readily and appear to have similar intrinsic efficacies to the endogenous peptide, so that biological activity is regulated by their receptor binding affinity. Functional photoaffinity-labelled, biotinylated and fluorescent probes are described. The biotinylated probe binds the receptor when coupled to streptavidin, although with reduced affinity.

Asunto(s)

Melanoma Experimental/ultraestructura , Receptores de la Hormona Hipofisaria/metabolismo , alfa-MSH/análogos & derivados , alfa-MSH/metabolismo , Animales , Unión Competitiva , Cinética , Melanoma Experimental/metabolismo , Ratones , Monofenol Monooxigenasa/análisis , Células Tumorales Cultivadas , alfa-MSH/síntesis química

RESUMEN

Cystic fibrosis (CF) patients are hypersusceptible to chronic Pseudomonas aeruginosa lung infections. Cultured human airway epithelial cells expressing the delta F508 allele of the cystic fibrosis transmembrane conductance regulator (CFTR) were defective in uptake of P. aeruginosa compared with cells expressing the wild-type allele. Pseudomonas aeruginosa lipopolysaccharide (LPS)-core oligosaccharide was identified as the bacterial ligand for epithelial cell ingestion; exogenous oligosaccharide inhibited bacterial ingestion in a neonatal mouse model, resulting in increased amounts of bacteria in the lungs. CFTR may contribute to a host-defense mechanism that is important for clearance of P. aeruginosa from the respiratory tract.

Asunto(s)

Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Fibrosis Quística/complicaciones , Infecciones por Pseudomonas/etiología , Pseudomonas aeruginosa/fisiología , Sistema Respiratorio/microbiología , Infecciones del Sistema Respiratorio/etiología , Animales , Animales Recién Nacidos , Línea Celular Transformada , Fibrosis Quística/genética , Fibrosis Quística/microbiología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Susceptibilidad a Enfermedades , Epitelio/microbiología , Humanos , Lipopolisacáridos/farmacología , Pulmón/microbiología , Ratones , Ratones Endogámicos BALB C , Infecciones por Pseudomonas/microbiología , Infecciones del Sistema Respiratorio/microbiología

RESUMEN

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 , Humanos

RESUMEN

Chronic lung infection with mucoid Pseudomonas aeruginosa is the major pathologic feature of cystic fibrosis. Previous studies suggested that a failure to produce opsonic antibody to the mucoid exopolysaccharide (MEP; also called alginate) capsule is associated with the maintenance of chronic bacterial infection. Provision of MEP-specific opsonic antibodies has therapeutic potential. To evaluate the ability of MEP to elicit opsonic antibodies, humans were immunized with two lots of MEP vaccine that differed principally in molecular size. Lot 2 had a larger average MEP polymer size. Both vaccines were well tolerated, but lot 1 was poorly immunogenic, inducing long-lived opsonic antibodies in only 2 of 28 vaccinates given doses of 10 to 150 micrograms. In contrast, at the optimal dose of 100 micrograms, lot 2 elicited long-lived opsonic antibodies in 80 to 90% of the vaccinates. The antibodies elicited by both lots enhanced deposition of C3 onto mucoid P. aeruginosa cells and mediated opsonic killing of heterologous mucoid strains expressing distinct MEP antigens. These results indicate that the polymers of MEP with the largest molecular sizes safely elicit opsonic antibodies in a sufficiently large proportion of vaccinates to permit studies of active and passive immunization of cystic fibrosis patients against infection with mucoid P. aeruginosa.

Asunto(s)

Anticuerpos Antibacterianos/sangre , Vacunas Bacterianas/inmunología , Glicosaminoglicanos/inmunología , Polisacáridos Bacterianos/inmunología , Pseudomonas aeruginosa/inmunología , Adulto , Fibrosis Quística/inmunología , Femenino , Humanos , Masculino , Vacunación

RESUMEN

Virulence comparisons were made in a rabbit model of endocarditis between wild-type and transposon mutants of Staphylococcus epidermidis deficient in elaboration of the capsular polysaccharide/adhesin (PS/A) and slime. The parental phenotype grew from 36 (61%) of 59 cultures of blood. The PS/A-negative phenotype grew in 1 (1%) of 98 cultures of blood (P < .001). No animals infected with PS/A-negative strains developed endocarditis compared with 75% of rabbits infected with PS/A-positive strains. PS/A-producing strains survived better than did PS/A-deficient strains in intact, absorbed rabbit or human serum plus human leukocytes. There was also greater deposition of C3 onto the PS/A-deficient strains than with the PS/A-producing isogenic strains. PS/A functions as an antiphagocytic bacterial capsule preventing C3 deposition and phagocytosis; loss of this structure increases the strain's susceptibility to opsonic killing and decreases its virulence.

Asunto(s)

Elementos Transponibles de ADN , Endocarditis/microbiología , Polisacáridos Bacterianos/metabolismo , Staphylococcus epidermidis/genética , Staphylococcus epidermidis/patogenicidad , Análisis de Varianza , Animales , Adhesión Bacteriana , Activación de Complemento , Endocarditis/inmunología , Humanos , Mutagénesis Insercional , Fenotipo , Conejos , Virulencia/genética

RESUMEN

We examined the basis for the absence in cystic fibrosis (CF) patients of opsonic antibodies to the mucoid exopolysaccharide (MEP) antigen surrounding Pseudomonas aeruginosa that infect these patients. Opsonic antibodies to MEP are found in sera of the minority of CF patients that remain noncolonized into the second to fourth decades of life and protect rodents from chronic P. aeruginosa endobronchial infections. High titers of nonopsonic antibodies to MEP are found in P. aeruginosa-infected CF patients. Immunization of mice with doses of MEP that provoke only nonopsonic antibodies elicited CD3+, CD8+, T cell receptor alpha beta receptor+, major histocompatibility complex-unrestricted cytotoxic lymphocytes specific for hybridoma cells producing opsonic but not nonopsonic antibodies. Cytotoxicity was dependent on immune complexes on the surface of the T cells. Normal murine T cells could be activated by concanavalin A and sensitized with immune complexes for cytotoxic killing of hybridoma targets. CF patients infected with P. aeruginosa had serum immune complexes that sensitized concanavalin A-activated human T cells to kill murine hybridoma cells producing opsonic but not nonopsonic antibody. These results could explain the absence in infected CF patients of MEP-specific opsonins, an occurrence that accompanies the persistence of this infectious state.

Asunto(s)

Anticuerpos Antibacterianos/inmunología , Complejo Antígeno-Anticuerpo/inmunología , Fibrosis Quística/inmunología , Citotoxicidad Inmunológica , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/inmunología , Linfocitos T Citotóxicos/inmunología , Linfocitos T/inmunología , Animales , Complejo Antígeno-Anticuerpo/sangre , Antígenos CD/análisis , Linfocitos B/inmunología , Fibrosis Quística/sangre , Fibrosis Quística/complicaciones , Humanos , Hibridomas/inmunología , Ratones , Infecciones por Pseudomonas/sangre , Infecciones por Pseudomonas/complicaciones , Receptores de Antígenos de Linfocitos T/análisis

RESUMEN

The failure of cystic fibrosis patients to limit chronic infection due to mucoid Pseudomonas aeruginosa might be due to ineffective opsonins produced against this bacterium. Nonopsonizing antibody to the bacterial capsule, mucoid exopolysaccharide (MEP), appears at elevated titers during chronic colonization of cystic fibrosis patients, as do opsonins not specific for MEP. Nonopsonic antibodies to MEP occur naturally in most adults and can be induced in animals by immunization. A limited number of humans produce MEP-specific opsonic antibodies after immunization. The purpose of this study was to compare the activation and deposition of C components onto the bacterial surface in the presence of these different antibodies. Opsonic killing uses the classical C pathway. MEP-specific opsonic and nonopsonic antibodies bound to whole bacteria and activated C to a comparable degree, but opsonic antibody deposited 3 to 40 times more C3 onto bacteria, mostly as C3bi, compared to nonopsonic antibody. In addition, two to three times as much nonopsonic mAb as opsonic mAb (both IgG2b) bound to the bacteria at comparable input concentrations, indicating the difference in C deposition was not due to differences in antibody binding. Non-MEP-specific opsonins also bound C3 to the bacteria, but only a mean of 27 +/- 14% was ester linked, compared with 81 +/- 11% of C3 deposited by MEP-specific opsonins. Immunoprecipitation experiments indicated that two-thirds of the C3 bound in the presence of MEP-specific opsonins was linked to MEP, whereas non-MEP-specific opsonins obtained from infected patients deposited the C3 onto LPS and other unidentified Ag. These data show that MEP-specific opsonins function by depositing C3 onto the outer bacterial surface that differentiates them from non-MEP-specific opsonins produced in response to chronic infection.

Asunto(s)

Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/inmunología , Proteínas del Sistema Complemento/metabolismo , Glicosaminoglicanos/inmunología , Polisacáridos Bacterianos/inmunología , Pseudomonas aeruginosa/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Actividad Bactericida de la Sangre , Activación de Complemento , Complemento C3/metabolismo , Relación Dosis-Respuesta Inmunológica , Humanos , Ratones , Proteínas Opsoninas

RESUMEN

Mucoid strains of Pseudomonas aeruginosa are the major pulmonary pathogens for cystic fibrosis patients. Opsonizing antibodies to the mucoid exopolysaccharide (MEP) antigen may protect animals and some cystic fibrosis patients from infection. However, MEP does not readily elicit opsonic antibodies either during chronic infection or after vaccination. To evaluate alternative means to induce opsonic antibodies, a murine monoclonal anti-idiotypic antibody directed to an opsonic monoclonal antibody specific to MEP was produced. The anti-idiotypic antibody bound to F(ab')2 fragments of the opsonic antibody, blocked binding to MEP, bound to cross-reactive idiotopes on human opsonic antibodies to MEP, and elicited MEP-specific antibodies in syngeneic and allogeneic mice. These anti-idiotype-induced, MEP-specific antibodies fixed complement to mucoid P. aeruginosa cells and opsonized them for phagocytic killing by human leukocytes. These studies demonstrate the potential utility of anti-idiotypic monoclonal antibody for generating protective immunity against bacterial polysaccharides.

Asunto(s)

Anticuerpos Antiidiotipos/inmunología , Anticuerpos Antibacterianos/inmunología , Anticuerpos Monoclonales/inmunología , Glicosaminoglicanos/inmunología , Proteínas Opsoninas , Pseudomonas aeruginosa/inmunología , Animales , Anticuerpos Antiidiotipos/biosíntesis , Anticuerpos Monoclonales/biosíntesis , Proteínas del Sistema Complemento/inmunología , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Fagocitosis