RESUMEN
The genetic basis for susceptibility to malaria has been studied widely in African populations but less is known of the contribution of specific genetic variants in Asian populations. We genotyped 67 single-nucleotide polymorphisms (SNPs) in 1030 severe malaria cases and 2840 controls from Vietnam. After data quality control, genotyping data of 956 cases and 2350 controls were analysed for 65 SNPs (3 gender confirmation, 62 positioned in/near 42 malarial candidate genes). A total of 14 SNPs were monomorphic and 2 (rs8078340 and rs33950507) were not in Hardy-Weinberg equilibrium in controls (P<0.01). In all, 7/46 SNPs in 6 genes (ICAM1, IL1A, IL17RC, IL13, LTA and TNF) were associated with severe malaria, with 3/7 SNPs in the TNF/LTA region. Genotype-phenotype correlations between SNPs and clinical parameters revealed that genotypes of rs708567 (IL17RC) correlate with parasitemia (P=0.028, r(2)=0.0086), with GG homozygotes having the lowest parasite burden. Additionally, rs708567 GG homozygotes had a decreased risk of severe malaria (P=0.007, OR=0.78 (95% CI; 0.65-0.93)) and death (P=0.028, OR=0.58 (95% CI; 0.37-0.93)) than those with AA and AG genotypes. In summary, variants in six genes encoding adhesion and proinflammatory molecules are associated with severe malaria in the Vietnamese. Further replicative studies in independent populations will be necessary to confirm these findings.
Asunto(s)
Moléculas de Adhesión Celular/genética , Mediadores de Inflamación/inmunología , Malaria Falciparum/genética , Malaria Falciparum/inmunología , Adulto , Pueblo Asiatico/genética , Estudios de Casos y Controles , Estudios de Cohortes , Femenino , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Variación Genética , Humanos , Molécula 1 de Adhesión Intercelular/genética , Interleucina-13/genética , Interleucina-1alfa/genética , Masculino , Parasitemia/genética , Parasitemia/inmunología , Polimorfismo de Nucleótido Simple , Receptores de Interleucina/genética , Factor de Necrosis Tumoral alfa/genética , Vietnam , Adulto JovenRESUMEN
Infection of mice with the intestinal bacterial pathogen Citrobacter rodentium results in colonic mucosal hyperplasia and a local Th1 inflammatory response similar to that seen in mouse models of inflammatory bowel disease. In these latter models, and in patients with Crohn's disease, neutralization of tumor necrosis factor alpha (TNF-alpha) is of therapeutic benefit. Since there is no information on the role of TNF-alpha in either immunity to noninvasive bacterial pathogens or on the role of TNF-alpha in the immunopathology of infectious colitis, we investigated C. rodentium infection in TNFRp55(-/-) mice. In TNFRp55(-/-) mice, there were higher colonic bacterial burdens, but the organisms were cleared at the same rate as C57BL/6 mice, showing that TNF-alpha is not needed for protective antibacterial immunity. The most striking feature of infection in TNFRp55(-/-) mice, however, was the markedly enhanced pathology, with increased mucosal weight and thickness, increased T-cell infiltrate, and a markedly greater mucosal Th1 response. Interleukin-12 p40 transcripts were markedly elevated in C. rodentium-infected TNFRp55(-/-) mice, and this was associated with enhanced mucosal STAT4 phosphorylation. TNF-alpha is not obligatory for protective immunity to C. rodentium in mice; however, it appears to play some role in downregulating mucosal pathology and Th1 immune responses.
Asunto(s)
Antígenos CD/inmunología , Citrobacter freundii/inmunología , Enfermedades Funcionales del Colon/inmunología , Infecciones por Enterobacteriaceae/inmunología , Receptores del Factor de Necrosis Tumoral/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Animales , Antígenos CD/genética , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Citrobacter freundii/crecimiento & desarrollo , Colon/microbiología , Colon/patología , Enfermedades Funcionales del Colon/patología , Proteínas de Unión al ADN/metabolismo , Infecciones por Enterobacteriaceae/patología , Femenino , Expresión Génica , Hiperplasia/inmunología , Hiperplasia/patología , Interleucina-12/genética , Interleucina-4/genética , Mucosa Intestinal/inmunología , Mucosa Intestinal/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores del Factor de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral , Factor de Transcripción STAT4 , Transactivadores/metabolismoRESUMEN
The formation of attaching and effacing (A/E) lesions on gut enterocytes is central to the pathogenesis of enterohemorrhagic (EHEC) Escherichia coli, enteropathogenic E. coli (EPEC), and the rodent pathogen Citrobacter rodentium. Genes encoding A/E lesion formation map to a chromosomal pathogenicity island termed the locus of enterocyte effacement (LEE). Here we show that the LEE-encoded proteins EspA, EspB, Tir, and intimin are the targets of long-lived humoral immune responses in C. rodentium-infected mice. Mice infected with C. rodentium developed robust acquired immunity and were resistant to reinfection with wild-type C. rodentium or a C. rodentium derivative, DBS255(pCVD438), which expressed intimin derived from EPEC strain E2348/69. The receptor-binding domain of intimin polypeptides is located within the carboxy-terminal 280 amino acids (Int280). Mucosal and systemic vaccination regimens using enterotoxin-based adjuvants were employed to elicit immune responses to recombinant Int280alpha from EPEC strain E2348/69. Mice vaccinated subcutaneously with Int280alpha, in the absence of adjuvant, were significantly more resistant to oral challenge with DBS255(pCVD438) but not with wild-type C. rodentium. This type-specific immunity could not be overcome by employing an exposed, highly conserved domain of intimin (Int388-667) as a vaccine. These results show that anti-intimin immune responses can modulate the outcome of a C. rodentium infection and support the use of intimin as a component of a type-specific EPEC or EHEC vaccine.
Asunto(s)
Adhesinas Bacterianas , Proteínas de la Membrana Bacteriana Externa/inmunología , Vacunas Bacterianas/inmunología , Proteínas Portadoras , Citrobacter freundii/inmunología , Infecciones por Enterobacteriaceae/prevención & control , Proteínas de Escherichia coli , Animales , Anticuerpos Antibacterianos/sangre , Proteínas de la Membrana Bacteriana Externa/administración & dosificación , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/inmunología , Vacunas Bacterianas/administración & dosificación , Vacunas Bacterianas/genética , Citrobacter freundii/crecimiento & desarrollo , Infecciones por Enterobacteriaceae/inmunología , Femenino , Ratones , Ratones Endogámicos C3H , Receptores de Superficie Celular/inmunología , VacunaciónRESUMEN
Mucosal immune responses must discriminate between commensal flora within the lumen and potential pathogens. These responses are highly adapted to induce protection without excessive inflammation. The balances that regulate mucosal immune and inflammatory responses have to be understood if effective mucosal immunity is to be induced through local immunization. This review will summarize some of the lessons learnt from studies of antigens derived from enteric bacterial pathogens and discuss how the gastrointestinal epithelia can 'fight back' when it encounters pathogens.
Asunto(s)
Mucosa Gástrica/inmunología , Inmunidad Mucosa , Mucosa Intestinal/inmunología , Animales , Antígenos Bacterianos/inmunología , Toxinas Bacterianas/inmunología , Enterotoxinas , Escherichia coli , Mucosa Gástrica/microbiología , Humanos , Sistema Inmunológico/inmunología , Inmunidad Mucosa/inmunología , Mucosa Intestinal/microbiologíaRESUMEN
The hallmark of enteropathogenic (EPEC) and enterohaemorrhagic (EHEC) Escherchia coli adhesion to host cells is intimate attachment leading to the formation of distinctive 'attaching and effacing' lesions. This event is mediated, in part, by binding of the bacterial adhesion molecule intimin to a second bacterial protein, Tir, delivered by a type III secretion system into the host cell plasma membrane. The receptor-binding activity of intimin is localized to the C-terminal 280 amino acids (Int280) and at least five distinct intimin types (alpha, beta, gamma, delta and epsilon) have been identified thus far. In addition to binding to Tir, intimin can also bind to a component encoded by the host. The consequence of latter intimin-binding activity may determine tissue tropism and host specificity. In this study we selected three amino acids in intimin, which are implicated in Tir binding, for site-directed mutagenesis. We used the yeast two-hybrid system and gel overlays to study intimin-Tir protein interaction. In addition, the biological consequences of the mutagenesis was tested using a number of infection models (cultured epithelial cells, human intestinal explants and a mouse model). We report that while an I237/897A substitution (positions numbered according to Int280alpha/whole intimin alpha) in intimin alpha did not have any affect on its biological activity, a T255/914A substitution attenuated intimin activity in vivo. In contrast, the mutation V252/911A affected tissue targeting in the human intestinal explant model and attenuated the biological activity of intimin in the mouse model. This study provides the first clues of the molecular basis of how intimin mediates tissue tropism and host specificity.
Asunto(s)
Adhesinas Bacterianas , Proteínas de la Membrana Bacteriana Externa/fisiología , Proteínas Portadoras , Proteínas de Escherichia coli , Tropismo , Animales , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Secuencia de Bases , Cartilla de ADN , Femenino , Humanos , Mucosa Intestinal/metabolismo , Ratones , Ratones Endogámicos C3H , Mutagénesis Sitio-Dirigida , Técnicas de Cultivo de Órganos , Conformación Proteica , Técnicas del Sistema de Dos HíbridosRESUMEN
Immunologic unresponsiveness (tolerance) is a key feature of the mucosal immune system, and deliberate vaccination by a mucosal route can effectively induce immune suppression. However, some bacterial-derived proteins, e.g. cholera toxin and the heat labile toxin of Escherichia coli, are immunogenic and immunomodulatory at mucosal surfaces and can effectively adjuvant immune responses to codelivered bystander antigens. This review summarizes some of the structural and biological characteristics of these toxins and provides examples of how these properties have been exploited for tolerance induction and mucosal vaccine development.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Toxinas Bacterianas/farmacología , Toxina del Cólera/farmacología , Enterotoxinas/farmacología , Proteínas de Escherichia coli , Adyuvantes Inmunológicos/química , Adyuvantes Inmunológicos/genética , Animales , Células Presentadoras de Antígenos/efectos de los fármacos , Células Presentadoras de Antígenos/enzimología , Células Presentadoras de Antígenos/inmunología , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Toxina del Cólera/química , Toxina del Cólera/genética , Enterotoxinas/química , Enterotoxinas/genética , Humanos , Inmunidad Mucosa/efectos de los fármacos , Ratones , Modelos Moleculares , Poli(ADP-Ribosa) Polimerasas/metabolismo , Subunidades de Proteína , Relación Estructura-Actividad , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Virosis/inmunologíaRESUMEN
In an effort to develop a safe and effective vaccine against respiratory syncytial virus (RSV), we used Escherichia coli heat-labile toxin (LT), and LTK63 (an LT mutant devoid of ADP-ribosyltransferase activity) to elicit murine CD8(+) CTL responses to an intranasally codelivered CTL peptide from the second matrix protein (M2) of RSV. M2(82-90)-specific CD8(+) T cells were detected by IFN-gamma enzyme-linked immunospot and (51)Cr release assay in local and systemic lymph nodes, and their induction was dependent on the use of a mucosal adjuvant. CTL elicited by peptide immunization afforded protection against RSV challenge, but also enhanced weight loss. CTL-mediated viral clearance was not dependent on IFN-gamma since depletion using specific mAb during RSV challenge did not affect cellular recruitment or viral clearance. Depletion of IFN-gamma did, however, reduce the concentration of TNF detected in lung homogenates of challenged mice and largely prevented the weight loss associated with CTL-mediated viral clearance. Mice primed with the attachment glycoprotein (G) develop lung eosinophilia after intranasal RSV challenge. Mucosal peptide vaccination reduced pulmonary eosinophilia in mice subsequently immunized with G and challenged with RSV. These studies emphasize that protective and immunoregulatory CD8(+) CTL responses can be mucosally elicited using enterotoxin-based mucosal adjuvants but that resistance against viral infection may be accompanied by enhanced disease.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Toxinas Bacterianas/inmunología , Enterotoxinas/inmunología , Proteínas de Escherichia coli , Mucosa Nasal/inmunología , Fragmentos de Péptidos/inmunología , Infecciones por Virus Sincitial Respiratorio/inmunología , Virus Sincitial Respiratorio Humano/inmunología , Linfocitos T Citotóxicos/inmunología , Proteínas Virales/inmunología , Administración Intranasal , Animales , Toxinas Bacterianas/administración & dosificación , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/virología , Enterotoxinas/administración & dosificación , Eosinofilia/inmunología , Eosinofilia/prevención & control , Epítopos de Linfocito T/inmunología , Inmunidad Innata , Interferón gamma/fisiología , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Fragmentos de Péptidos/administración & dosificación , Infecciones por Virus Sincitial Respiratorio/prevención & control , Porcinos , Linfocitos T Citotóxicos/virología , Células Th2/inmunología , Células Th2/metabolismo , Células Th2/virología , Proteínas Virales/administración & dosificación , Vacunas Virales/administración & dosificación , Vacunas Virales/inmunologíaRESUMEN
Salmonella spp. are regarded as facultative intracellular bacterial pathogens which are found inside macrophages (Mphi) after i. v. infection. It is generally assumed that Mphi restrict the replication of the bacteria during infection. In this study we examined the in vivo activities of Mphi during experimental S. typhimurium infections, using a selective liposome-based Mphi elimination technique. Unexpectedly, elimination of Mphi prior to infection with virulent S. typhimurium decreased morbidity and mortality, suggesting that Mphi mediate the pathology caused by S. typhimurium. Removal of Mphi) during vaccination with attenuated S. typhimurium did not affect protection against challenge with virulent S. typhimurium, suggesting that Mphi are not required for the induction of protective immunity and that other cells must function as antigen-presenting cell to elicit T cell-mediated protection. However, Mphi appeared to be important effectors of protection against challenge infection since elimination of Mphi from vaccinated mice prior to challenge infection with virulent S. typhimurium significantly decreased protection. These results enhance our understanding of the control of S. typhimurium growth in vivo, and moreover suggest that Mphi play a major role in the pathology of virulent S. typhimurium infections. As such, these cells may present a novel target for therapeutic intervention.
Asunto(s)
Macrófagos/inmunología , Salmonelosis Animal/etiología , Salmonelosis Animal/inmunología , Salmonella typhimurium/inmunología , Salmonella typhimurium/patogenicidad , Animales , Anticuerpos Antibacterianos/sangre , Vacunas Bacterianas/farmacología , Ácido Clodrónico/administración & dosificación , Recuento de Colonia Microbiana , Femenino , Liposomas , Activación de Linfocitos , Macrófagos/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Salmonelosis Animal/prevención & control , Salmonella typhimurium/aislamiento & purificación , Linfocitos T/inmunología , VirulenciaRESUMEN
The ability of enterotoxin-based mucosal adjuvants to induce CD8+ MHC class I-restricted CTL responses to a codelivered bystander Ag was examined. Escherichia coli heat-labile toxin (LT), or derivatives of LT carrying mutations in the A subunit (LTR72, LTK63), were tested in parallel with cholera toxin (CT) or a fusion protein consisting of the A1 subunit of CT fused to the Ig binding domain of Staphylococcus aureus protein A (called CTA1-DD). Intranasal (i.n.) immunization of C57BL/6 mice with CT, CTA1-DD, LT, LTR72, LTK63, but not rLT-B, elicited MHC class I-restricted CD8+ T cell responses to coadministered OVA or the OVA CTL peptide SIINFEKL (OVA257-264). CT, LT, and LTR72 also induced CTL responses to OVA after s.c. or oral coimmunization whereas LTK63 only activated responses after s.c. coimmunization. rLT-B was unable to adjuvant CTL responses to OVA or OVA257-264 administered by any route. Mice treated with an anti-CD4 mAb to deplete CD4+ T cells mounted significant OVA-specific CTL responses after i.n. coadministration of LT with OVA or OVA257-264. Both 51Cr release assays and IFN-gamma enzyme-linked immunospot assays indicated that IFN-gamma-/- and IL-12 p40-/- gene knockout mice developed CTL responses equivalent to those detected in normal C57BL/6 mice. The results highlight the versatility of toxin-based adjuvants and suggest that LT potentiates CTL responses independently of IL-12 and IFN-gamma and probably by a mechanism unrelated to cross-priming.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Toxinas Bacterianas/farmacología , Toxina del Cólera/farmacología , Citotoxicidad Inmunológica , Enterotoxinas/farmacología , Proteínas de Escherichia coli , Antígenos de Histocompatibilidad Clase I/inmunología , Activación de Linfocitos , Mucosa Nasal/inmunología , Linfocitos T Citotóxicos/inmunología , Adenosina Difosfato Ribosa/metabolismo , Adyuvantes Inmunológicos/administración & dosificación , Administración Intranasal , Administración Oral , Sustitución de Aminoácidos , Animales , Arginina/metabolismo , Toxinas Bacterianas/administración & dosificación , Toxinas Bacterianas/genética , Toxina del Cólera/administración & dosificación , Enterotoxinas/administración & dosificación , Enterotoxinas/genética , Epítopos de Linfocito T/inmunología , Inyecciones Subcutáneas , Interferón gamma/fisiología , Interleucina-12/fisiología , Lisina/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ovalbúmina/administración & dosificación , Ovalbúmina/inmunología , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/inmunología , Linfocitos T Citotóxicos/metabolismo , Linfocitos T Colaboradores-Inductores/inmunología , Células Tumorales CultivadasRESUMEN
This study describes the construction and analysis of three in vivo-inducible promoter expression plasmids, containing pnirB, ppagC, and pkatG, for the delivery of foreign antigens in the DeltaaroAD mutant of Salmonella enterica var. Typhimurium (hereafter referred to as S. typhimurium). The reporter genes encoding beta-galactosidase and firefly luciferase were used to assess the comparative levels of promoter activity in S. typhimurium in vitro in response to different induction stimuli and in vivo in immunized mice. It was determined that the ppagC construct directed the expression of more beta-galactosidase and luciferase in S. typhimurium than the pnirB and pkatG constructs, both in vitro and in vivo. The gene encoding the C fragment of tetanus toxin was expressed in the aroAD mutant of S. typhimurium (BRD509) under the control of the three promoters. Mice orally immunized with attenuated S. typhimurium expressing C fragment under control of the pagC promoter [BRD509(pKK/ppagC/C frag)] mounted the highest tetanus toxoid-specific serum antibody response. Levels of luciferase expression in vivo and C-fragment expression in vitro from the pagC promoter appeared to be equivalent to if not lower than the levels of expression detected with the constitutive trc promoter. However, mice immunized with BRD509(pKK/ppagC/C frag) induced significantly higher levels of tetanus toxoid-specific antibody than BRD509(pKK/C frag)-immunized mice, suggesting that the specific location of foreign antigen expression may be important for immunogenicity. Mutagenesis of the ribosome binding sites (RBS) in the three promoter/C fragment expression plasmids was also performed. Despite optimization of the RBS in the three different promoter elements, the expression levels in vivo and overall immunogenicity of C fragment when delivered to mice by attenuated S. typhimurium were not affected. These studies suggest that in vivo-inducible promoters may give rise to enhanced immunogenicity and increase the efficacy of S. typhimurium as a vaccine vector.
Asunto(s)
Vacunas Bacterianas/genética , Regiones Promotoras Genéticas , Salmonella typhimurium/genética , Toxina Tetánica/genética , Vacunas Sintéticas/genética , Animales , Anticuerpos Antibacterianos/biosíntesis , Vacunas Bacterianas/inmunología , Femenino , Vectores Genéticos , Inmunización , Luciferasas/genética , Ratones , Ratones Endogámicos BALB C , Reacción en Cadena de la PolimerasaRESUMEN
Corynebacterium pseudotuberculosis, a gram-positive facultative intracellular bacterial pathogen, is the etiological agent of the economically important disease caseous lymphadenitis (CLA) in both sheep and goats. Attenuated mutants of C. pseudotuberculosis have the potential to act as novel vaccines against CLA and as veterinary vaccine vectors. In this report, we have assessed the virulence of both aroQ and pld mutants of C. pseudotuberculosis in sheep and concurrently their capacity to act as vaccines against homologous challenge. The results suggest that aroQ mutants of C. pseudotuberculosis are attenuated with regard to both lymph node persistence and vaccination site reactogenicity. Immunologically, aroQ mutants failed to elicit detectable specific gamma interferon (IFN-gamma)-secreting lymphocytes and induced low levels of antibodies to C. pseudotuberculosis culture supernatant antigens. Following subcutaneous vaccination, the immune responses induced by aroQ mutants did not protect sheep from infection with the wild-type strain but did appear to reduce the clinical severity of disease resulting from challenge. Conversely, an attenuated C. pseudotuberculosis strain expressing an enzymatically inactive phospholipase D exotoxin, when used as a vaccine, elicited a protective immune response. Protection appeared to correlate with in vivo persistence of the vaccine strain, the induction of IFN-gamma-secreting lymphocytes, and relatively high levels of antibodies to culture supernatant antigens. The results suggest that aroQ mutants of C. pseudotuberculosis may be overly attenuated for use as a CLA vaccines or as vaccine vectors.
Asunto(s)
Vacunas Bacterianas/inmunología , Corynebacterium pseudotuberculosis/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Hidroliasas/genética , Inmunoglobulina G/sangre , Mutación , Ovinos , Vacunación , Vacunas Atenuadas/inmunologíaRESUMEN
We compared the abilities of different Salmonella enterica var. Typhimurium (S. typhimurium) strains harboring mutations in the genes aroA, aroAD, purA, ompR, htrA, and cya crp to present the heterologous antigen, C fragment of tetanus toxin, to the mouse immune system. Plasmid pTETtac4, encoding C fragment, was transferred into the various S. typhimurium mutants, and the levels of antigen expression were found to be equivalent. After primary oral immunization of BALB/c mice, all attenuated strains were capable of penetrating the gut epithelium and colonizing the Peyer's patches and spleens of mice. Of all strains compared, the delta purA mutant colonized and persisted in the Peyer's patches at the lowest level, whereas the delta htrA mutant colonized and persisted in the spleen at the lowest level. The level of specific antibody elicited by the different strains against either S. typhimurium lipopolysaccharide or tetanus toxoid was strain dependent and did not directly correlate to the mutants' ability to colonize the spleen. The level of immunoglobulin G1 (IgG1) and IgG2a antibody specific for tetanus toxoid was determined in mice immunized with four S. typhimurium mutants. The level of antigen-specific IgG1 and IgG2a was significantly lower in animals immunized with S. typhimurium delta purA. Antigen-specific T-cell proliferation assays indicated a degree of variability in the capacity of some strains to elicit T cells to the heterologous antigen. Cytokine profiles (gamma interferon and interleukin-5) revealed that the four S. typhimurium mutants tested induced a Th1-type immune response. Mice were challenged with a lethal dose of tetanus toxin 96 days after oral immunization. With the exception of the S. typhimurium delta purA mutant, all strains elicited a protective immune response. These data indicate that the level of total Ig specific for the carried antigen, C fragment, does not correlate with the relative invasiveness of the vector, but it is determined by the carrier mutation and the background of the S. typhimurium strain.
Asunto(s)
Anticuerpos Antibacterianos/biosíntesis , Vacunas Bacterianas/inmunología , Salmonella typhimurium/genética , Toxoide Tetánico/inmunología , Animales , Femenino , Inmunización , Inmunoglobulina G/clasificación , Interferón gamma/biosíntesis , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Mutación , Salmonella typhimurium/inmunología , Vacunas Atenuadas/inmunologíaRESUMEN
Initiation of attaching-effacing lesions, which characterize infections with rabbit enteropathogenic Escherichia coli (REPEC), requires bacteria to adhere to the intestinal epithelium. This adherence is reflected in vitro by the affinity of these E. coli strains for various types of eukaryotic cells. TnphoA mutants of REPEC 83/39 (O15:H-) which had lost the ability to adhere to HEp-2 epithelial cells, guinea pig ileal brush borders, and mouse erythrocytes were generated. DNA sequencing of the region surrounding the inactivating transposon insertions within a 95-kb plasmid, designated pRAP for REPEC adherence plasmid, revealed extensive homology between that region and the structural genes of enterotoxigenic E. coli operons encoding the K88 and CS31A fimbrial adhesins and the genes for the afr2 adhesin from REPEC B10 (O103:H2). Seven genes of the ral operon (for REPEC adherence locus), including three putative minor fimbrial subunit genes (ralC, ralF, and ralH), a major fimbrial subunit gene (ralG), a gene of unknown function (ralI), and genes for two fimbrial subunit chaperones (ralD and ralE), were sequenced. When inoculated perorally into weanling rabbits, a mutant with a TnphoA insertion in the ralE gene showed a 10-fold reduction in colonizing ability, with only 1 of 10 rabbits excreting bacteria compared to all 5 of those infected with the wild-type parent strain (P = 0.002). The severity of the diarrheal illness caused by the mutant strain was also reduced. Western blotting of surface protein extracts of strain 83/39 with hyperimmune anti-83/39 antiserum, adsorbed with the ralE mutant, revealed a 32-kDa protein which was absent from protein extracts of two nonadherent mutants. The adsorbed antiserum also bound to the surface of strain 83/39 but not to nonadherent mutants, as detected by immunogold labeling. These results indicate that the ral operon of REPEC 83/39 contains genes necessary for the biosynthesis of fine fimbriae which are responsible for in vitro adherence of the bacteria and play a role in their colonization of, and hence virulence for, rabbits. The putative major fimbrial subunit is a protein with an observed molecular size of approximately 32 kDa which, when assembled, appears to form a capsule of fimbriae surrounding the bacterium similar to that described for CS31A.
Asunto(s)
Infecciones por Escherichia coli/microbiología , Escherichia coli/genética , Fimbrias Bacterianas/genética , Genes Bacterianos , Intestinos/microbiología , Operón , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Escherichia coli/patogenicidad , Ratones , Datos de Secuencia Molecular , Conejos , Virulencia/genéticaRESUMEN
Corynebacterium pseudotuberculosis, a gram-positive intracellular bacterial pathogen, is the etiological agent of the disease caseous lymphadenitis (CLA) in both sheep and goats. Attenuated mutants of C. pseudotuberculosis have the potential to act as novel live veterinary vaccine vectors. We have cloned and sequenced the aroB and aroQ genes from C. pseudotuberculosis C231. By allelic exchange, aroQ mutants of both C231, designated CS100, and a pld mutant strain TB521, designated CS200, were constructed. Infection of BALB/c mice indicated that introduction of the aroQ mutation into C231 and TB521 attenuated both strains. In sublethally infected BALB/c mice, both CS100 and CS200 were cleared from spleens and livers by day 8 postinfection. The in vivo persistence of these strains was increased when the intact aroQ gene was supplied on a plasmid in trans. Mice infected with TB521 harbored bacteria in organs at least till day 8 postinfection without ill effect. When used as a vaccine, only the maximum tolerated dose of CS100 had the capacity to protect mice from homologous challenge. Vaccination with TB521 also elicited protective immunity, and this was associated with gamma interferon (IFN-gamma) production from splenocytes stimulated 7 days postvaccination. The role of IFN-gamma in controlling primary infections with C. pseudotuberculosis was examined in mice deficient for the IFN-gamma receptor (IFN-gammaR(-/-) mice). IFN-gammaR(-/-) mice cleared an infection with CS100 but were significantly more susceptible than control littermates to infection with C231 or TB521. These studies support an important role for IFN-gamma in control of primary C. pseudotuberculosis infections and indicate that aroQ mutants remain attenuated even in immunocompromised animals. This is the first report of an aroQ mutant of a bacterial pathogen, and the results may have implications for the construction of aromatic mutants of Mycobacterium tuberculosis for use as vaccines.
Asunto(s)
Vacunas Bacterianas/inmunología , Corynebacterium pseudotuberculosis/inmunología , Secuencia de Aminoácidos , Animales , Clonación Molecular , Corynebacterium pseudotuberculosis/crecimiento & desarrollo , Interferón gamma/fisiología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Mutación , Vacunas Atenuadas/inmunologíaRESUMEN
DNA vaccines are an exciting development in vaccine technology which may have a special role in preventing viral infections and as 'theracines' for cancer. Their use in preventing bacterial infections has, by comparison, been less well documented. While it is unlikely that traditional, highly successful and cheap vaccines for diseases such as diphtheria will be replaced by DNA vaccines, naked DNA may be particularly appropriate for preventing bacterial infections where cytotoxic T cells confer protection, or where a Th1 type T cell response mediates resistance. For example, DNA vaccines containing different mycobacterial antigens have been shown to inhibit overt infections by Mycobacterium tuberculosis in rodent models. The use of DNA vaccines in bacterial infections may be complicated by fundamental differences between prokaryotic and eukaryotic genes and gene products, including mRNA stability, codon bias, secondary structures surrounding native start sequences and glycosylation. These problems can be solved by re-synthesis of bacterial genes to produce 'new' sequences which are more highly expressed by eukaryotic cells.
Asunto(s)
Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Infecciones Bacterianas/prevención & control , Vacunación/métodos , Vacunas de ADN/uso terapéutico , Animales , Infecciones por Chlamydia/prevención & control , Chlamydia trachomatis/genética , Chlamydia trachomatis/inmunología , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/inmunología , Tuberculosis/prevención & controlRESUMEN
Corynebacterium pseudotuberculosis is an intracellular bacterial pathogen causing a chronic abscessing disease in sheep and goats called caseous lymphadenitis. We are developing this bacterial species as a live vector system to deliver vaccine antigens to the animal immune system. Foreign genes expressed in bacterial hosts can be unstable so we undertook to delete the C. pseudotuberculosis chromosomal recA gene to determine whether a recA- background would reduce the frequency of recombination in cloned DNA. Homologous DNA recombination within an isogenic recA- C. pseudotuberculosis was 10-12-fold lower than that in the recA+ parental strain. Importantly, the recA mutation had no detectable affect upon the virulence of C. pseudotuberculosis in a mouse model. Taken together these results suggest that a recA- background may be useful in the further development of C. pseudotuberculosis as a vaccine vector.