In deep study of new tuberculosis vaccine candidates based on phoP and fadD26 mutations

El primer objetivo de este trabajo fue caracterizar las interacciones con el hospedador del nuevo candidato a vacuna viva atenuada contra la tuberculosis, MTBVAC. En este estudio se comparó la proteómica de la fracción secretada de MTBVAC y su cepa parental Mt103 a través de las técnicas de western-blot y electroforesis fluorescente diferencial en dos dimensiones (DiGE). Se obtuvieron dos conjuntos de datos de proteínas secretadas mayoritariamente por MTBVAC o Mt103 respectivamente. Gracias a estos resultados se observó el alto porcentaje de sustratos del sistema de secreción TAT (Twin Arginine Translocation) entre la fracción secretada por MTBVAC, y se pudo confirmar la ausencia de los factores de virulencia ESAT-6 y CFP-10 en dicho secretoma. El tráfico intracelular de MTBVAC fue otro aspecto a estudiar de la interacción del candidato a vacuna con su hospedador. MTBVAC es un mutante doble en los genes phoP y fadD26, por lo que se quiso estudiar también la contribución individual de cada mutación al fenotipo final. Se llevaron a cabo infecciones con las correspondientes cepas GFP+ en dos modelos celulares diferentes: la línea celular de macrófagos alveolares de ratón (MHS) y macrófagos primarios humanos derivados de monocitos (hMDM); los resultados se analizaron mediante técnicas de microscopía. Los resultados obtenidos sugieren que MTBVAC, como cepa atenuada que es, no es capaz de detener la maduración del fagosoma en etapas tempranas del proceso, sin embargo la detiene antes de llegar a la fase lisosomal. Mediante esta estrategia MTBVAC evita ser degradada rápidamente en el fagolisosoma. Sin embargo, debido a haber perdido la habilidad de secretar ESAT-6 y CFP-10, MTBVAC no es capaz de escapar al citosol de la célula hospedadora, presentando un fenotipo de persistencia en el cual MTBVAC está “atrapada” en un estadio endosomal tardío. Las mutaciones en los genes phoP y fadD26 de MTBVAC parecen tener un efecto sinérgico sobre el fenotipo del tráfico intracelular del candidato a vacuna. El segundo objetivo fue el estudio exhaustivo del regulon PhoP para tratar de determinar la señal que activa y modula su expresión. Para ello se utilizaron dos estrategias: i) estudiar la expresión de diferentes genes regulados por PhoP a través de un sistema reportero basado en fusiones genéticas de promotores de dichos genes con la proteína GFP, ii) medida directa de la expresión de varios genes regulados por PhoP a través de la extracción de RNA y la técnica de qRT-PCR. En ambos casos, la cepa parental H37Rv y su mutante phoP fueron expuestos a un ambiente intracelular, bien sometiéndolas a condiciones in vitro que trataban de mimetizar las condiciones intrafagosomales (pH ácido, iones, temperatura, hipoxia, etc.) o bien infectando con dichas cepas células fagocíticas y no fagocíticas. Se eligió el promotor de pks2 como reportero más representativo de la actividad del regulón PhoP, demostrando el importante rol que tiene el pH acido en la activación de dicho regulón. Ninguna de las condiciones probadas muestra una señal única que active el regulón PhoP, sugiriendo que es un proceso multifactorial que tiene lugar durante la entrada de la bacteria al interior del macrófago. Sin embargo, la respuesta del regulón tras la exposición a pH ácido demuestra que el pH es un factor clave en la inducción de los genes regulados por PhoP. Basándonos en los resultados obtenidos, se propone una dinámica de expresión de los genes regulados por PhoP dependiente del tiempo post-infección. Los primeros genes activados por PhoP son aquellos relacionados con el metabolismo lipídico y el estrés oxidativo, probablemente para proteger a la bacteria frente al ambiente hostil del interior del fagosoma. Tras 3-5 días, los genes relacionados con el sistema de secreción ESX-1 son activados y en consecuencia, la bacteria secretará ESAT-6 y CFP-10 induciendo con ello la ruptura del fagosoma y su escape al citosol. A partir de este punto, la actividad del RNA no codificante Mcr7 es altamente inducida por PhoP, lo cual conlleva la inhibición del sistema de secreción TAT y consecuentemente se ve restringida la secreción de los componentes del complejo Antígeno 85 con el objetivo de “esconderse” de la respuesta de la célula hospedadora ahora que la bacteria se encuentra en el citosol. Este mecanismo permite a las bacterias virulentas evadir el control inmunológico del hospedador y en el organismo. El tercer objetivo de la presente tesis fue construir una vacuna hiper atenuada basada en MTBVAC para ser usada en la población con riesgo de inmunosupresión, para la que BCG está contraindicada. Se introdujo una tercera mutación en el gen zmp1 (Rv0198c) de MTBVAC (vacuna viva atenuada con mutaciones en los genes phoP y fadD26) con la intención de hiper atenuar la cepa manteniendo su eficacia protectora frente a tuberculosis. Se usó una estrategia de doble recombinación para generar el triple mutante en los genes phoP, fadD26 y zmp1. Resultados preliminares muestran que el triple mutante no mejora la atenuación, la capacidad de generar respuesta inmunológica o la eficacia protectora de MTBVAC; estudios más exhaustivos en otros modelos animales (cobayas) son necesarios para llegar a alguna conclusión. REFERENCIAS 1. WHO, Global Tuberculosis Control: WHO report 2011. 2011. 2. Russell, D.G., Who puts the tubercle in tuberculosis? Nat Rev Microbiol, 2007. 5(1): p. 39-47. 3. Walters, S.B., et al., The Mycobacterium tuberculosis PhoPR two-component system regulates genes essential for virulence and complex lipid biosynthesis. Mol Microbiol, 2006. 60(2): p. 312-30. 4. Perez, E., et al., An essential role for phoP in Mycobacterium tuberculosis virulence. Mol Microbiol, 2001. 41(1): p. 179-87. 5. Gonzalo Asensio, J., et al., The virulence-associated two-component PhoP-PhoR system controls the biosynthesis of polyketide-derived lipids in Mycobacterium tuberculosis. J Biol Chem, 2006. 281(3): p. 1313-6. 6. Ferrer, N.L., et al., Interactions of attenuated Mycobacterium tuberculosis phoP mutant with human macrophages. PLoS One. 5(9): p. e12978. 7. Sirakova, T.D., et al., The Mycobacterium tuberculosis pks2 gene encodes the synthase for the hepta- and octamethyl-branched fatty acids required for sulfolipid synthesis. J Biol Chem, 2001. 276(20): p. 16833-9. 8. Richter, L., et al., Determination of the minimal acid-inducible promoter region of the lipF gene from Mycobacterium tuberculosis. Gene, 2007. 395(1-2): p. 22-8. 9. Kendall, S.L., et al., The Mycobacterium tuberculosis dosRS two-component system is induced by multiple stresses. Tuberculosis (Edinb), 2004. 84(3-4): p. 247-55. 10. Munoz-Elias, E.J. and J.D. McKinney, Mycobacterium tuberculosis isocitrate lyases 1 and 2 are jointly required for in vivo growth and virulence. Nat Med, 2005. 11(6): p. 638-44. 11. Gonzalo-Asensio, J., et al., PhoP: a missing piece in the intricate puzzle of Mycobacterium tuberculosis virulence. PLoS One, 2008. 3(10): p. e3496. 12. Cardona, P.J., et al., Extended safety studies of the attenuated live tuberculosis vaccine SO2 based on phoP mutant. Vaccine, 2009. 27(18): p. 2499-505. 13. Kamath, A.T., et al., New live mycobacterial vaccines: the Geneva consensus on essential steps towards clinical development. Vaccine, 2005. 23(29): p. 3753-61. 14. Arbues, A., et al., Construction, characterization and preclinical evaluation of MTBVAC, the first live-attenuated M. tuberculosis-based vaccine to enter clinical trials. Vaccine. 31(42): p. 4867-73. 15. Walker, K.B., et al., The second Geneva Consensus: Recommendations for novel live TB vaccines. Vaccine. 28(11): p. 2259-70. 16. Master, S.S., et al., Mycobacterium tuberculosis prevents inflammasome activation. Cell Host Microbe, 2008. 3(4): p. 224-32

TNF-α antagonists differentially induce TGF-β1-dependent resuscitation of dormant-like Mycobacterium tuberculosis

TNF-α- as well as non-TNF-α-targeting biologics are prescribed to treat a variety of immune-mediated inflammatory disorders. The well-documented risk of tuberculosis progression associated with anti-TNF-α treatment highlighted the central role of TNF-α for the maintenance of protective immunity, although the rate of tuberculosis detected among patients varies with the nature of the drug. Using a human, in-vitro granuloma model, we reproduce the increased reactivation rate of tuberculosis following exposure to Adalimumab compared to Etanercept, two TNF-α-neutralizing biologics. We show that Adalimumab, because of its bivalence, specifically induces TGF-β1-dependent Mycobacterium tuberculosis (Mtb) resuscitation which can be prevented by concomitant TGF-β1 neutralization. Moreover, our data suggest an additional role of lymphotoxin-α-neutralized by Etanercept but not Adalimumab-in the control of latent tuberculosis infection. Furthermore, we show that, while Secukinumab, an anti-IL-17A antibody, does not revert Mtb dormancy, the anti-IL-12-p40 antibody Ustekinumab and the recombinant IL-1RA Anakinra promote Mtb resuscitation, in line with the importance of these pathways in tuberculosis immunity

Live attenuated TB vaccines representing the three modern Mycobacterium tuberculosis lineages reveal that the Euro–American genetic background confers optimal vaccine potential

Background: Human tuberculosis (TB) is caused by a plethora of Mycobacterium tuberculosis complex (MTBC) strains belonging to seven phylogenetic branches. Lineages 2, 3 and 4 are considered “modern” branches of the MTBC responsible for the majority of worldwide TB. Since the current BCG vaccine confers variable protection against pulmonary TB, new candidates are investigated. MTBVAC is the unique live attenuated vaccine based on M. tuberculosis in human clinical trials. Methods: MTBVAC was originally constructed by unmarked phoP and fadD26 deletions in a clinical isolate belonging to L4. Here we construct new vaccines based on isogenic gene deletions in clinical isolates of the L2 and L3 modern lineages. These three vaccine candidates were characterized at molecular level and also in animal experiments of protection and safety. Findings: Safety studies in immunocompromised mice showed that MTBVAC-L2 was less attenuated than BCG Pasteur, while the original MTBVAC was found even more attenuated than BCG and MTBVAC-L3 showed an intermediate phenotype. The three MTBVAC candidates showed similar or superior protection compared to BCG in immunocompetent mice vaccinated with each MTBVAC candidate and challenged with three representative strains of the modern lineages. Interpretation: MTBVAC vaccines, based on double phoP and fadD26 deletions, protect against TB independently of the phylogenetic linage used as template strain for their construction. Nevertheless, lineage L4 confers the best safety profile

The Transcriptional Regulatory Network of Mycobacterium tuberculosis

Under the perspectives of network science and systems biology, the characterization of transcriptional regulatory (TR) networks beyond the context of model organisms offers a versatile tool whose potential remains yet mainly unexplored. In this work, we present an updated version of the TR network of Mycobacterium tuberculosis (M.tb), which incorporates newly characterized transcriptional regulations coming from 31 recent, different experimental works available in the literature. As a result of the incorporation of these data, the new network doubles the size of previous data collections, incorporating more than a third of the entire genome of the bacterium. We also present an exhaustive topological analysis of the new assembled network, focusing on the statistical characterization of motifs significances and the comparison with other model organisms. The expanded M.tb transcriptional regulatory network, considering its volume and completeness, constitutes an important resource for diverse tasks such as dynamic modeling of gene expression and signaling processes, computational reliability determination or protein function prediction, being the latter of particular relevance, given that the function of only a small percent of the proteins of M.tb is known

Multiple deletions in the polyketide synthase gene repertoire of Mycobacterium tuberculosis reveal functional overlap of cell envelope lipids in host-pathogen interactions

International audienceSeveral specific lipids of the cell envelope are implicated in the pathogenesis of M. tuberculosis (Mtb), including phthiocerol dimycocerosates (DIM) that have clearly been identified as virulence factors. Others, such as trehalose-derived lipids, sulfolipids (SL), diacyltrehaloses (DAT) and polyacyltrehaloses (PAT), are believed to be essential for Mtb virulence, but the details of their role remain unclear. We therefore investigated the respective contribution of DIM, DAT/PAT and SL to tuberculosis by studying a collection of mutants, each with impaired production of one or several lipids. We confirmed that among those with a single lipid deficiency, only strains lacking DIM were affected in their replication in lungs and spleen of mice in comparison to the WT Mtb strain. We found also that the additional loss of DAT/PAT, and to a lesser extent of SL, increased the attenuated phenotype of the DIM-less mutant. Importantly, the loss of DAT/PAT and SL in a DIM-less background also affected Mtb growth in human monocyte-derived macrophages (hMDMs). Fluorescence microscopy revealed that mutants lacking DIM or DAT/PAT were localized in an acid compartment and that bafilomycin A1, an inhibitor of phagosome acidification, rescued the growth defect of these mutants. These findings provide evidence for DIM being dominant virulence factors that mask the functions of lipids of other families, notably DAT/PAT and to a lesser extent of SL, which we showed for the first time to contribute to Mtb virulence