Contenido de glicopeptidolípidos, motilidad y formación de biopelículas en aislados clínicos de Mycobacterium colombiense

Mycobacterium colombiense es una micobacteria perteneciente al Complejo Mycobacterium avium (MAC), aislada a partir de pacientes infectados con el virus de inmunodeficiencia humana (VIH) de la ciudad de Bogotá. M. colombiense ha sido relacionada con la producción de linfadenitis e infecciones pulmonares en pacientes inmunodeprimidos y VIH positivos en Colombia, España y Francia [1][2]. Hasta la fecha, las bases genéticas y morfológicas involucradas en el comportamiento fenotípico de las distintas cepas de M. colombiense son completamente desconocidas. En el presente trabajo se ha demostrado por primera vez que M. colombiense es una micobacteria motil y con capacidad de formar biopelículas (BP) restringida a cepas con morfología de colonia lisa. Hemos observado que las cepas de M. colombiense de morfología lisa contienen en su pared celular glicolípidos con un comportamiento cromatográfico similar a los glicopeptidolípidos (GPLs) de M. avium. Una variante natural rugosa de M. colombiense (57B) mostró ausencia de motilidad, una reducida capacidad de formar BP y sintetizar GPLs en las condiciones experimentales utilizadas en el presente estudio. Utilizando diferentes análisis bioinformáticos, se ubicó una agrupación de genes que podrían estar involucrados en la biosíntesis de GPLs en la cepa de referencia M. colombiense CECT 3035, cuya secuencia genómica se reportó recientemente. Por último, experimentos de qPCR, sugieren que la transcripción de genes posiblemente involucrados en actividades O-metiltransferasa y glicosiltransferasa podrían estar relacionados en la producción de GPLs de las distintas cepas de M. colombiense cultivadas en condiciones planctónicas y de motilidad.Abstract. Mycobacterium colombiense is a novel member of Mycobacterium avium Complex (MAC) that produces respiratory and disseminated infections especially in immunosuppressed patients [1] [2]. At present, the morphologic and genetic bases involved in the phenotypic features of M. colombiense strains are completely unknown. In this study, we show for the first time that M. colombiense strains have the ability to biofilm formation and displayed motility restricted to strains with smooth morphology. In addition, Thin-layer chromatography analysis showed that strains displaying smooth morphology contain glycolipids that migrated as glycopeptidolipids (GPLs). A natural rough variant, M. colombiense 57B, displayed absence of motility and impaired capacity of both, biofilm formation and production of lipids with chromatographic behavior similar to GPLs. According to bioinformatic analyses, a gene cluster of GPLs biosynthesis for the recently sequenced M. colombiense CECT 3035 strain is proposed. Real-time PCR experiments suggested that transcription of genes possibly involved in methyl and glycosyl transfer activity could be correlated to diversity of glycolipids associated to GPLs in M. colombiense strains.Maestrí

P2-type ATPases as targets for the attenuation of Mycobacterium tuberculosis.

ilustraciones, fotografías, graficas, tablasLa tuberculosis (TB) es una enfermedad infecciosa causada por el bacilo ácido alcohol resistente Mycobacterium tuberculosis (Mtb). La TB es una amenaza para la salud pública, debido a su alta incidencia, la aparición de cepas multi-fármaco-resistentes (MDR) y extremadamente-fármaco-resistentes (XDR), la coinfección con VIH y la eficacia limitada de la vacuna BCG. El diseño de nuevas estrategias de control requiere una mejor comprensión de los mecanismos moleculares utilizados por Mtb para ser un patógeno intracelular tan exitoso. En este sentido, algunos estudios han sugerido la importancia de las ATPasas tipo P en la fisiología y la supervivencia intracelular de las micobacterias. Un meta-análisis del perfil transcripcional de las ATPasas tipo P de Mtb bajo condiciones de estrés como hipoxia, estrés oxidativo, inanición, intoxicación por agentes químicos y procesos de infección in vitro e in vivo, evidenció la expresión diferencial de estos transportadores frente a estas condiciones. De las 12 ATPasas tipo P presentes en el genoma de Mtb, CtpF, que codifica para un transportador de Ca2+, es la ATPasa que muestra mayores niveles de transcripción en las diferentes condiciones de estrés. Particularmente, varias ATPasas tipo P (ctpF, ctpG, ctpC, ctpH y ctpV) exhibieron un aumento en los niveles de expresión durante la infección de macrófagos humanos, sugiriendo la importancia de dichas proteínas en los procesos de infección. Considerando la relevancia funcional de las ATPasas tipo P, el objetivo principal de este trabajo fue evaluar el efecto de la deleción de ATPasas tipo P transportadoras de metales alcalino/alcalinotérreos en la viabilidad y virulencia de Mtb. Para lograr este objetivo, mediante técnicas de recombinería se construyeron los mutantes defectivos en los genes ctpF y ctpH de Mtb, y a partir de distintos análisis funcionales con las cepas mutantes, se demostró que ambos transportadores están implicados en el eflujo de Ca2+. Adicionalmente, las cepas mutantes (MtbΔctpF y MtbΔctpH) mostraron hipersensibilidad frente agentes oxidantes en comparación con la cepa tipo silvestre (MtbWT), indicando un vinculo entre el transporte de Ca2+ y los mecanismos que utiliza el bacilo para neutralizar especies reactivas del ambiente intrafagosomal. Por otro lado, se evaluó en un modelo celular y animal el efecto de la deleción del gen ctpF en la virulencia Mtb. Así, se evidenció que dicha mutación genera una disminución significativa en la capacidad replicativa de Mtb en macrófagos alveolares murinos de la línea celular MH-S. Asimismo, se comparó la virulencia de las cepas MtbΔctpF y MtbWT en un ensayo de sobrevida en ratones BALB/c, encontrando que los ratones infectados con la cepa mutante mostraban mayor tiempo medio de supervivencia, sugiriendo la atenuación de la cepa mutante. Finalmente, se comprobó la existencia de estrategias complementarias que permiten contrarrestar deficiencias en el transporte iónico mediado por las ATPasa tipo P en Mtb. En efecto, se encontró que la cepa MtbΔctpF sobreexpresa el gen ctpH frente a concentraciones tóxicas de Ca2+ y durante los procesos de infección in vitro. De manera similar, el mutante MtbΔctpH sobreexpresó el gen ctpF bajo condiciones tóxicas de Ca2+, sugiriendo una posible actividad compensatoria entre CtpF y CtpH en Mtb. En general, los resultados obtenidos en este trabajo demuestran que las Ca+2-ATPasas están involucradas en la respuesta frente sustancias tóxicas, siendo fundamentales para la supervivencia celular de Mtb. Además, CtpF es relevante para la proliferación intracelular, y su deleción genera atenuación del bacilo tuberculoso en un modelo experimental de TB pulmonar. De esta manera, CtpF es fundamental para la virulencia de Mtb, por lo que podría considerarse un interesante blanco de atenuación. (texto tomado de la fuente)Tuberculosis (TB) is an infectious disease caused by the acid and alcohol-resistant bacillus Mycobacterium tuberculosis (Mtb). TB is considered a public health threat due to its high incidence, the emergence of drug-resistant strains (MDR and XDR), coinfection with HIV, and the limited efficacy of the BCG vaccine. Consequently, the design of new TB control strategies relies on a better comprehension of the molecular mechanisms used by Mtb to be a successful intracellular pathogen. In this sense, some studies have suggested that P-type ATPases are relevant to the physiology and intracellular survival of mycobacteria. Specifically, a meta-analysis of the transcriptional levels of Mtb P-type ATPases under conditions of hypoxia, oxidative stress, starvation, intoxication by chemical agents, and in vitro and in vivo infection processes indicated that these transporters are differentially expressed in these situations. Among the 12 P-type ATPases encoded in the Mtb genome, CtpF encodes a Ca2+ transporter and is the most activated against the conditions studied. Furthermore, several P-type ATPases (ctpF, ctpG, ctpC, ctpH, and ctpV) show over-expression during infection of human macrophages, suggesting the relevance of these proteins in the infection process. Therefore, the main objective of this study was to evaluate the effect of the deletion of alkali/alkaline earth metal-transporting P-type ATPases on the viability and virulence of Mtb. For this, recombination techniques were applied to construct mutants defective in the Mtb ctpF and ctpH genes that encode alkali/alkaline earth metal-transporting ATPases. Diverse functional analyses of these mutants demonstrated that both transporters are involved in Ca2+ efflux. Additionally, the mutant strains (MtbΔctpF and MtbΔctpH) showed hypersensitivity to oxidizing agents compared to the wild type strain (MtbWT), indicating a link between Ca2+ transport and the mechanism used by the bacillus to neutralize reactive species in the intraphagosomal environment. Moreover, the effect ctpF gene deletion on Mtb virulence was evaluated in cellular and animal infection models. Accordingly, this deletion generated a significant decrease in the replicative capacity of Mtb in murine alveolar macrophages of the MH-S cell line. Furthermore, a comparison of the virulence of MtbΔctpF and MtbWT strains through survival tests in BALB/c mice demonstrated that mice infected with the mutant strain showed a longer mean survival time, suggesting the attenuation of the deleted strain. Finally, the existence of a possible compensatory mechanism to counteract deficiencies in ion transport mediated by P-type ATPases in Mtb was evaluated. Indeed, the MtbΔctpF strain over-expresses the ctpH gene against toxic concentrations of Ca2+ and during in vitro infection processes. Similarly, the MtbΔctpH mutant over-expresses the ctpF gene under toxic concentrations of Ca2+, suggesting a possible compensatory activity between ctpF and ctpH in Mtb. This study demonstrates that Ca+2-ATPases are involved in the response to toxic substances, being essential for Mtb survival. Furthermore, CtpF is required for the intracellular proliferation of the mycobacteria, whereas its deletion attenuates the bacillus in an experimental model of pulmonary TB. Overall, CtpF is critical for Mtb virulence and can be an interesting attenuation target.Convocatoria nacional para el apoyo a proyectos de investigación y creación artística de la Universidad Nacional de Colombia 2017-2018 (Código 2010100-29088).Convocatoria nacional para el fomento de alianzas interdisciplinarias que articulen investigación, creación, extensión y formación en la Universidad Nacional de Colombia 2019-2021 (Código 2010100-29665).DoctoradoDoctor en Ciencias - BioquímicaPara el desarrollo de los objetivos propuestos en el presente trabajo, se estableció una estrategia experimental que incluyó herramientas bioquímicas, microbiológicas y de biología molecular.Hospedero-Patógen

The ctpF Gene Encoding a Calcium P-Type ATPase of the Plasma Membrane Contributes to Full Virulence of Mycobacterium tuberculosis

Identification of alternative attenuation targets of Mycobacterium tuberculosis (Mtb) is pivotal for designing new candidates for live attenuated anti-tuberculosis (TB) vaccines. In this context, the CtpF P-type ATPase of Mtb is an interesting target; specifically, this plasma membrane enzyme is involved in calcium transporting and response to oxidative stress. We found that a mutant of MtbH37Rv lacking ctpF expression (MtbΔctpF) displayed impaired proliferation in mouse alveolar macrophages (MH-S) during in vitro infection. Further, the levels of tumor necrosis factor and interferon-gamma in MH-S cells infected with MtbΔctpF were similar to those of cells infected with the parental strain, suggesting preservation of the immunogenic capacity. In addition, BALB/c mice infected with Mtb∆ctpF showed median survival times of 84 days, while mice infected with MtbH37Rv survived 59 days, suggesting reduced virulence of the mutant strain. Interestingly, the expression levels of ctpF in a mouse model of latent TB were significantly higher than in a mouse model of progressive TB, indicating that ctpF is involved in Mtb persistence in the dormancy state. Finally, the possibility of complementary mechanisms that counteract deficiencies in Ca2+ transport mediated by P-type ATPases is suggested. Altogether, our results demonstrate that CtpF could be a potential target for Mtb attenuation

Advances and challenges in recombinant Mycobacterium bovis

Introduction: Human Immunodeficiency Virus/Acquired Immune Deficiency Syndrome, tuberculosis, and malaria are responsible for most human deaths produced by infectious diseases worldwide. Vaccination against HIV requires generation of memory T cells and neutralizing antibodies, mucosal immunity, and stimulation of an innate immune responses. In this context, the use of Mycobacterium bovis bacillus Calmette–Guérin (BCG) as a live vaccine vehicle is a promising approach for T-cell induction. Areas covered: In this review, we provide a comprehensive summary of the literature regarding immunogenicity studies in animal models performed since 2005. Furthermore, we provide expert commentary and 5-year view on how the development of potential recombinant BCG-based HIV vaccines involves careful selection of the HIV antigen, expression vectors, promoters, BCG strain, preclinical animal models, influence of preexisting immunity, and safety issues, for the rational design of recombinant BCG:HIV vaccines to prevent HIV transmission in the general population. Expert commentary: The three critical issues to be considered when developing a rBCG:HIV vaccine are codon optimization, antigen localization, and plasmid stability in vivo. The use of integrative expression vectors are likely to improve the mycobacterial vaccine stability and immunogenicity to develop not only recombinant BCG-based vaccines expressing second generation of HIV-1 immunogens but also other major pediatric pathogens to prime protective responses shortly following birth

Brazilian Flora 2020: Leveraging the power of a collaborative scientific network

International audienceThe shortage of reliable primary taxonomic data limits the description of biological taxa and the understanding of biodiversity patterns and processes, complicating biogeographical, ecological, and evolutionary studies. This deficit creates a significant taxonomic impediment to biodiversity research and conservation planning. The taxonomic impediment and the biodiversity crisis are widely recognized, highlighting the urgent need for reliable taxonomic data. Over the past decade, numerous countries worldwide have devoted considerable effort to Target 1 of the Global Strategy for Plant Conservation (GSPC), which called for the preparation of a working list of all known plant species by 2010 and an online world Flora by 2020. Brazil is a megadiverse country, home to more of the world's known plant species than any other country. Despite that, Flora Brasiliensis, concluded in 1906, was the last comprehensive treatment of the Brazilian flora. The lack of accurate estimates of the number of species of algae, fungi, and plants occurring in Brazil contributes to the prevailing taxonomic impediment and delays progress towards the GSPC targets. Over the past 12 years, a legion of taxonomists motivated to meet Target 1 of the GSPC, worked together to gather and integrate knowledge on the algal, plant, and fungal diversity of Brazil. Overall, a team of about 980 taxonomists joined efforts in a highly collaborative project that used cybertaxonomy to prepare an updated Flora of Brazil, showing the power of scientific collaboration to reach ambitious goals. This paper presents an overview of the Brazilian Flora 2020 and provides taxonomic and spatial updates on the algae, fungi, and plants found in one of the world's most biodiverse countries. We further identify collection gaps and summarize future goals that extend beyond 2020. Our results show that Brazil is home to 46,975 native species of algae, fungi, and plants, of which 19,669 are endemic to the country. The data compiled to date suggests that the Atlantic Rainforest might be the most diverse Brazilian domain for all plant groups except gymnosperms, which are most diverse in the Amazon. However, scientific knowledge of Brazilian diversity is still unequally distributed, with the Atlantic Rainforest and the Cerrado being the most intensively sampled and studied biomes in the country. In times of “scientific reductionism”, with botanical and mycological sciences suffering pervasive depreciation in recent decades, the first online Flora of Brazil 2020 significantly enhanced the quality and quantity of taxonomic data available for algae, fungi, and plants from Brazil. This project also made all the information freely available online, providing a firm foundation for future research and for the management, conservation, and sustainable use of the Brazilian funga and flora