RESUMEN
Development of multigenic constructs expressing Mycobacterium tuberculosis (Mtb) antigens may be a strategy to obtain improved DNA vaccines against tuberculosis (TB). Several multigenic constructs expressing two or three Mtb antigens as fusion proteins were developed, both as tPA- and ubiquitin-fusion proteins. To demonstrate proper protein expression and intracellular turnover all multiantigens were tagged with the HA epitope and constructs were used to transfect rhabdomyosarcoma (RD) cells. Antigen expression was demonstrated by immunofluorescence using anti-HA antibodies. C57Bl/6 mice were immunized with selected constructs and protective activity was assessed following aerogenic challenge with Mtb. Several of these constructs induced a significant level of protection in the lung and in the spleen. Immunization with the construct expressing tPA85B-ES6 induced a level of protection that approached that provided by BCG. Immunization with a combination of these constructs induced levels of protection that were not superior to those elicited by a single combination, and immunization with a construct expressing five Mtb antigens could not provide an improved level of protection compared to tPA85B-ES6. We conclude that the activity of a DNA vaccine based on tPA85B-ES6 cannot be enhanced by broadening the antigen repertoire with other highly immunogenic secreted Mtb proteins.
Asunto(s)
Antígenos Bacterianos/inmunología , Vacunas contra la Tuberculosis , Tuberculosis/prevención & control , Vacunas de ADN/inmunología , Animales , Antígenos Bacterianos/genética , Ratones , Ratones Endogámicos C57BL , Mycobacterium tuberculosis/inmunología , Proteínas Recombinantes de Fusión/inmunología , Células Tumorales Cultivadas , Vacunas de ADN/genética , Vacunas SintéticasRESUMEN
Characterization of PE_PGRS gene expression will help define the role of this protein family in the biology of Mycobacterium tuberculosis. In this report, quantitative real-time RT-PCR (QRT-PCR) was implemented to assess expression of three PE_PGRS genes (rv0746, rv1651c and rv1818c) under different experimental conditions. The three PE_PGRS genes showed a similar expression profile in axenic cultures, with a significant up-regulation occurring at late log and early stationary phases. rv1651c gene expression increased following intracellular growth in bone marrow-derived macrophages but not in type-II human pneumocytes, while rv0746 was induced in both in vitro systems. Following the infection of mice with M. tuberculosis, expression levels of rv1651c and rv0746 normalized to ftsZ and 16S rRNA were highest in the spleen tissue during the chronic stages of murine tuberculosis, with a >20- and >30-fold up-regulation, respectively. Levels of expression remained lower in the lung over the same time period. Expression of the rv1818c gene did not change significantly under different experimental conditions tested. The results of this study indicate that M. tuberculosis can differentially regulate expression of PE_PGRS genes and that genes such as rv0746 and rv1651c are significantly induced while M. tuberculosis persists in host cells and tissues.
Asunto(s)
Adaptación Fisiológica , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Proteínas de la Membrana/genética , Mycobacterium tuberculosis/genética , Animales , Antígenos Bacterianos/biosíntesis , Proteínas Bacterianas/biosíntesis , Línea Celular , Modelos Animales de Enfermedad , Humanos , Pulmón/microbiología , Macrófagos/microbiología , Proteínas de la Membrana/biosíntesis , Ratones , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/fisiología , ARN Bacteriano/análisis , ARN Bacteriano/genética , ARN Mensajero/análisis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Bazo/microbiología , Tuberculosis/microbiologíaRESUMEN
Reactivities of human sera against selected recombinant Mycobacterium tuberculosis antigens were assessed by enzyme-linked immunosorbent assay. The results obtained indicate that patients with tuberculosis (TB) do not develop a strong humoral response against PE_PGRS and PPE proteins or against the Ag85B and heparin-binding hemagglutinin (HBHA) recombinant antigens. Conversely, purified methylated HBHA was strongly recognized by sera obtained from TB patients compared to controls.
Asunto(s)
Formación de Anticuerpos/inmunología , Hemaglutininas/inmunología , Mycobacterium tuberculosis/inmunología , Tuberculosis Pulmonar/diagnóstico , Tuberculosis Pulmonar/inmunología , Anticuerpos Antibacterianos/inmunología , Especificidad de Anticuerpos , Antígenos Bacterianos/inmunología , Ensayo de Inmunoadsorción Enzimática , Hemaglutininas/metabolismo , Humanos , Lectinas , Metilación , Proteínas Recombinantes/inmunologíaRESUMEN
The Heparin-Binding Haemagglutinin (HBHA) is a mycobacterial adhesin involved in the dissemination of Mycobacterium tuberculosis from the site of primary infection and a potential candidate for the development of a new vaccine against tuberculosis. Methylation of HBHA is a novel post-translational event that imparts important immunological properties to the protein. Since recombinant HBHA expressed in Escherichia coli is not methylated, we investigated the possibility of producing recombinant methylated HBHA in fast growing mycobacteria for use in immunological and biochemical studies. The complete coding sequence of HBHA was cloned in the plasmid pMV206, under the control of a strong promoter (hsp60) or its own promoter. The constructs generated were electroporated into Mycobacterium smegmatis and the recombinant strains obtained were analyzed for the presence of the HBHA protein using the anti-HBHA monoclonal antibodies D2 and E4. Our results indicate that expression of high amounts of intact protein can be toxic for the mycobacteria, that methylated HBHA can be obtained in M. smegmatis only when using a promoter sequence weaker than hsp60 and that the expression of the complete structural gene is required in order to obtain methylated HBHA. We constructed a recombinant M. smegmatis strain (pMV3-38) that expresses a histidine-tagged methylated HBHA that can be easily purified. The use of fast-growing strains of M. smegmatis to obtain significant amounts of purified HBHA protein within a short timeframe, should be an effective strategy for the evaluation of a new HBHA-based vaccine candidate for tuberculosis.
Asunto(s)
Hemaglutininas/genética , Hemaglutininas/metabolismo , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/metabolismo , Proteínas Recombinantes/metabolismo , Hemaglutininas/aislamiento & purificación , Lectinas , Metilación , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/crecimiento & desarrollo , Mycobacterium tuberculosis/genética , Proteínas Recombinantes/aislamiento & purificación , Transformación GenéticaRESUMEN
Identification of the novel PE multigene family was an unexpected finding of the genomic sequencing of Mycobacterium tuberculosis. Presently, the biological role of the PE and PE_PGRS proteins encoded by this unique family of mycobacterial genes remains unknown. In this report, a representative PE_PGRS gene (Rv1818c/PE_PGRS33) was selected to investigate the role of these proteins. Cell fractionation studies and fluorescence analysis of recombinant strains of Mycobacterium smegmatis and M. tuberculosis expressing green fluorescent protein (GFP)-tagged proteins indicated that the Rv1818c gene product localized in the mycobacterial cell wall, mostly at the bacterial cell poles, where it is exposed to the extracellular milieu. Further analysis of this PE_PGRS protein showed that the PE domain is necessary for subcellular localization. In addition, the PGRS domain, but not PE, affects bacterial shape and colony morphology when Rv1818c is overexpressed in M. smegmatis and M. tuberculosis. Taken together, the results indicate that PE_PGRS and PE proteins can be associated with the mycobacterial cell wall and influence cellular structure as well as the formation of mycobacterial colonies. Regulated expression of PE genes could have implications for the survival and pathogenesis of mycobacteria within the human host and in other environmental niches.