The PGRS Domain from PE_PGRS33 of Mycobacterium tuberculosis is Target of Humoral Immune Response in Mice and Humans.

The PE_PGRS33 protein is a member of the PE family, which encompasses the PE and the PE_PGRS subfamilies. Among PE_PGRS's, this protein is one of the most studied antigens and its immunomodulatory properties are influence by both PE and PGRS domains. However, the contribution of these domains to the host immune recognition of the PE_PGRS33 protein and their potential role in latent tuberculosis infection in humans is still unknown. In this study, the immunogenic properties of the complete PE_PGRS33 protein and each domain separately were evaluated in BALB/c mice and latent tuberculosis infected (LTBI) humans. In mice, PE_PGRS33 and its domains induced similar antibody production and secretion of IFN-γ. PE_PGRS33 and the PE domain stimulated higher CD4(+) and CD8(+) T-cell proliferation compared to the PGRS domain. This demonstrated that the principal difference in the immune recognition of the domains is the higher activation of T-cell subpopulations involved in the control of tuberculosis. In humans, the secretion of IFN-γ in response to PE_PGRS33 was detected in both LTBI and in non-infected vaccinated individuals. The same was observed for antibody response, which targets epitopes located in the PGRS domain but not in the PE domain. These observations suggest that T and B cell responses to PE_PGRS33 are induced by BCG vaccination and can be maintained for many years in non-infected individuals. This also indicates that the IFN-γ response detected might not be associated with latent tuberculosis infection. These results contribute to the elucidation of the role of the PE_PGRS33 protein and its PE and PGRS domains in the immune response against Mycobacterium tuberculosis.

Specific bacterial genotypes of Mycobacterium tuberculosis cause extensive dissemination and brain infection in an experimental model.

Meningeal tuberculosis is a severe type of extrapulmonary disease, which is thought to begin with respiratory infection, followed by hematogenous dissemination and brain infection. Host genetic susceptibility factors and specific mycobacterial substrains could be involved in its development. From an epidemiological study in Colombia, we selected three Mycobacterium tuberculosis clinical strains isolated from the cerebrospinal fluid (CSF) of patients with meningeal tuberculosis, and used them to infect BALB/c mice through the intratracheal route. These strains showed a distinctive spoligotype pattern. The course of infection in terms of strain virulence (mice survival, bacillary loads in lungs), bacilli dissemination and extrapulmonary infection (bacilli loads in blood, brain, liver, kidney and spleen), and immune responses (cytokine expression determined by real time PCR in brain and lung) was studied and compared with that induced by the laboratory strain H37Rv and other five clinical strains isolated from patients with pulmonary TB. All the clinical isolates from meningeal TB patients disseminated extensively through the hematogenous route infecting the brain, producing inflammation in the cerebral parenchyma and meninges, whereas H37Rv and clinical isolates from pulmonary TB patients showed very limited efficiency to infect the brain. Thus, it seems that mycobacterial strains with a distinctive genotype are able to disseminate extensively after the respiratory infection and infect the brain.

Expression, secretion, and glycosylation of the 45- and 47-kDa glycoprotein of Mycobacterium tuberculosis in Streptomyces lividans.

The gene encoding the 45/47 kDa glycoprotein (Rv1860) of Mycobacterium tuberculosis was expressed in Streptomyces lividans under its own promoter and under the thiostrepton-inducible Streptomyces promoter PtipA. The recombinant protein was released into the culture medium and, like the native protein, migrated as a double band at 45 and 47 kDa in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gels. However, in contrast to the native protein, only the 47-kDa recombinant protein could be labeled with concanavalin A (ConA). Carbohydrate digestion with jack bean alpha-D-mannosidase resulted in a reduction in the molecular mass of the recombinant protein upper band and completely eliminated ConA binding. Two-dimensional gel electrophoresis revealed only one isoelectric point for the recombinant protein. Comparative fingerprinting analysis of the individually purified upper and lower recombinant protein bands, treated under the same conditions with specific proteases, resulted in similar peptide patterns, and the peptides had the same N-terminal sequence, suggesting that migration of the recombinant protein as two bands in SDS-PAGE gels could be due to differences in glycosylation. Mass spectrometry analysis of the recombinant protein indicated that as in native protein, both the N-terminal and C-terminal domains of the recombinant protein are glycosylated. Furthermore, it was determined that antibodies of human tuberculosis patients reacted mainly against the carbohydrate residues of the glycoprotein. Altogether, these observations show that expression of genes for mycobacterial antigens in S. lividans is very useful for elucidation of the functional role and molecular mechanisms of glycosylation in bacteria.

Comparación de métodos moleculares útiles en la detección rápida de Mycobacterium tuberculosis Multirresistente (MTB-MRD) / Comparation of useful molecular methods for fast detection of Multidrug Resistent Mycobacterium tuberculosis (MTB-MDR)

Objetivo: evaluar y comparar dos métodos moleculares rápidos utilizables en la detección de pacientes con MTB-MRD. Material y Métodos: realizamos un estudio experimental para comparar la eficiencia de los métodos: "rifoligotyping" e Inno-LIPA, utilizando como "gold standard" el método de las proporciones múltiples. La población de estudio fueron 41 aislamientos de pacientes con tuberculosis MRD y 43 de pacientes con tuberculosis farmacosensible. Se amplificó por PCR un fragmento del gen rpob utilizando un par de iniciadores diferentes para cada método y posteriormente se detectó mediante hibridización reversa en membranas y tiras, la presencia de mutaciones que confieren resistencia a rifampicina. Se calculó la sensibilidad, la especificidad, el valor predictivo de MRD (VPMRD) y el valor predictivo de fármaco sensibilidad (VPFS) para cada una de las pruebas y se compararon en términos de eficiencia. Resultados: las dos metodologías fueron igualmente eficientes. Los VPMRD fueron del 100 por ciento y los VPFS fueron 81.1 por ciento y 84.3 por ciento La sensibilidad fue de 75.6 por ciento y 80.5 por ciento respectivamente, y la especificidad fue de 100 por ciento para ambos métodos. Conclusiones: se pueden considerar los dos como posibles métodos de elección para determinar rápidamente la presencia de tuberculosis MRD en poblaciones de riesgo, facilitando la prevención de la diseminación de estas cepas y agilizando la prescripción de esquemas de quimioterapias apropiados. La distribución de las mutaciones demostró que se hace necesario diseñar otras membranas o tiras que contengan las mutaciones más comunes, específicamente en las cepas colombianas, porque cerca de 1/3 de los aislamientos colombianos estudiados presentaron mutaciones que no fueron identificadas por ninguno de los dos métodos