Genomic analysis of Mycobacterium brumae sustains its nonpathogenic and immunogenic phenotype

Mycobacterium brumae is a rapid-growing, non-pathogenic Mycobacterium species, originally isolated from environmental and human samples in Barcelona, Spain. Mycobacterium brumae is not pathogenic and it's in vitro phenotype and immunogenic properties have been well characterized. However, the knowledge of its underlying genetic composition is still incomplete. In this study, we first describe the 4 Mb genome of the M. brumae type strain ATCC 51384T assembling PacBio reads, and second, we assess the low intraspecies variability by comparing the type strain with Illumina reads from three additional strains. Mycobacterium brumae genome is composed of a circular chromosome with a high GC content of 69.2% and containing 3,791 CDSs, 97 pseudogenes, one prophage and no CRISPR loci. Mycobacterium brumae has shown no pathogenic potential in in vivo experiments, and our genomic analysis confirms its phylogenetic position with other non-pathogenic and rapid growing mycobacteria. Accordingly, we determined the absence of virulence-related genes, such as ESX-1 locus and most PE/PPE genes, among others. Although the immunogenic potential of M. brumae was proved to be as high as Mycobacterium bovis BCG, the only mycobacteria licensed to treat cancer, the genomic content of M. tuberculosis T cell and B cell antigens in M. brumae genome is considerably lower than those antigens present in M. bovis BCG genome. Overall, this work provides relevant genomic data on one of the species of the mycobacterial genus with high therapeutic potential

Mycolicibacterium brumae is a safe and non-toxic immunomodulatory agent for cancer treatment

Intravesical Mycobacterium bovis Bacillus Calmette-Guérin (BCG) immunotherapy remains the gold-standard treatment for non-muscle-invasive bladder cancer patients, even though half of the patients develop adverse events to this therapy. On exploring BCG-alternative therapies, Mycolicibacterium brumae, a nontuberculous mycobacterium, has shown outstanding anti-tumor and immunomodulatory capabilities. As no infections due to M. brumae in humans, animals, or plants have been described, the safety and/or toxicity of this mycobacterium have not been previously addressed. In the present study, an analysis was made of M. brumae- and BCG-intravenously-infected severe combined immunodeficient (SCID) mice, M. brumae-intravesically-treated BALB/c mice, and intrahemacoelic-infected-Galleria mellonella larvae. Organs from infected mice and the hemolymph from larvae were processed to count bacterial burden. Blood samples from mice were also taken, and a wide range of hematological and biochemical parameters were analyzed. Finally, histopathological alterations in mouse tissues were evaluated. Our results demonstrate the safety and non-toxic profile of M. brumae. Di erences were observed in the biochemical, hematological and histopathological analysis between M. brumae and BCG-infected mice, as well as survival curves rates and colony forming units (CFU) counts in both animal models. M. brumae constitutes a safe therapeutic biological agent, overcoming the safety and toxicity disadvantages presented by BCG in both mice and G. mellonella animal models

75 años como referente de la investigación agraria y medioambiental española en condiciones de clima mediterráneo [Sitio Web]

1 .pdf con imagen de acceso al “website”, su url y los créditos relacionados con su creación y diseño.-- Créditos: Organización, Estación Experimental de Aula Dei (EEAD-CSIC); Dirección, Jesús Val Falcón; Coordinación, Ana Álvarez-Fernandez, Jorge Álvaro-Fuentes, Ernesto Igartua; Contenido, Anunciación Abadía, Javier Abadía, Carlos Albiñana, Miguel Alfonso, Arancha Arbeloa, Raúl Arbués, Isabel Armillas, Manuel Becana, Santiago Beguería, Carmen Castañeda, Ana Castillo, José Cavero, Bruno Contreras, Azahara Díaz, Edgar García, Elena García, Juan Manuel Gascuñana, Leticia Gaspar, Yolanda Gogorcena, Juan Herrero, Victoria Lafuente, María Victoria López, Juan Antonio Marín, José Martínez, José Carlos Martínez-Giménez, Ana Pilar Mata, Manuel Matamoros, Pierre Mignard, María Ángeles Moreno, Paula Murillo, Ana Navas, Antonio Pérez, Rafael Picorel, María Pilar Vallés, Irene Villar, Inmaculada Yruela, Nery Zapata, Isabel Zarazaga; Diseño y programación: DigitalWorks (Juanjo Ascaso y Asun Dieste); Vídeo, Delegación del CSIC en Aragón (Sara Gutiérrez y Yolanda Hernáiz); Fotografía, Archivo EEAD-CSIC, Anunciación Abadía, Jorge Álvaro-Fuentes, Arancha Arbeloa, Juanjo Ascaso, Santiago Beguería, Elena García, Ernesto Igartua, Ignasi Iglesias, José Manuel Lasa, José Carlos Martínez-Giménez, Pierre Mignard, María Ángeles Moreno, Rubén Sancho, Kosana Suvocarev, María Pilar Vallés, Nery Zapata."Sitio web" de nueva creación y conmemorativo del 75 Aniversario de la EEAD-CSIC que contiene: 1) Foto esférica de su personal en activo; 2) Recopilación de sus hitos históricos más destacados, en orden cronológico; 3) Un vídeo con participación de su personal y muestra de algunas de sus instalaciones; 4) Un mapa con la distribución geográfica de los egresado del Instituto; 5) Algunas fotos, destacando las tomadas a su personal en las celebraciones del 25 y 50 Aniversarios de la EEAD-CSIC.Presentado durante la "Jornada. 75 Aniversario EEAD-CSIC (Zaragoza, Patio de la Infanta. 30 octubre 2019)".Financiación: CSIC, Vicepresidencia Adjunta de Organización y Cultura Científica.N

Estudio in silico, in vitro y ex vivo de la actividad antitumoral de diferentes cepas de Mycobacterium Brumae y su papel en la activación y modulación del sistema inmune

Les instil·lacions intravesicals del Mycobacterium bovis bacillus Calmette-Guerin (BCG) és el tractament d'elecció en pacients de càncer de bufeta no múscul-invasiu (CBNMI) després de la resecció del tumor. El M. bovis BCG presenta una demostrada eficàcia en disminuir la recurrència i la progressió tumoral gràcies a l'activació del sistema immune del pacient, fet que millora la taxa de supervivència dels pacients de CBNMI. No obstant, el tractament intravesical amb aquest agent immunoterapeutic s'associa a efectes adversos en més del 50% dels pacients tractats, que poden desenvolupar complicacions greus fins en un 5% de casos, incloent infeccions pulmonars o disseminades. A més, fins a un 30-40% dels pacients no responen favorablement a la teràpia amb el M. bovis BCG, pel que es veuen obligats a interrompre el tractament. Una de les alternatives proposades per a substituir el tractament amb el M. bovis BCG és l'ús del Mycobacterium brumae, un micobacteri ambiental no patogen amb un efecte antitumoral i immunomodulador demostrat tant in vitro, com ex vivo i in vivo en un model murí de càncer vesical. Amb l'objectiu de desenvolupar noves alternatives terapèutiques que siguin eficaces per a tots els pacients de CBNMI, l' objectiu d' aquesta tesi és aprofundir en les característiques fenotípiques i genotípiques de diferents soques del M. brumae i comparar-les amb el M. bovis BCG; avaluar la seva interacció amb diversos tipus de cèl·lules tumorals; estudiar l'efecte immunomodulador d'ambdós micobacteris en el context específic de persones grans; i caracteritzar-ne la potencial capacitat d' induir immunitat entrenada. Inicialment, es va seqüenciar i analitzar el genoma de la soca de referència del M. brumae i de tres aïllats ambientals del M. brumae. La comparació amb el genoma del M. bovis i del M. tuberculosis va demostrar la no patogenicitat del M. brumae, així com la presència de gens involucrats en l'activació del sistema immune de l'hoste demostrant la seva immunogenicitat. No es van observar diferències a nivell fenotípic entre les diferents soques del M. brumae i molt poques a nivell genotípic. No obstant això, es va observar que la soca CR-103 era capaç d'inhibir la proliferació tumoral d'una manera més eficaç en algunes línies tumorals i que la soca CR-142 induïa una major producció de citocines en macròfags infectats, comparades amb la resta de soques. A més, es va demostrar la capacitat d'inhibir la proliferació tumoral del M. brumae a línies tumorals de localitzacions diferents a la bufeta, com a còlon, fetge o pàncrees. D' altra banda, es va estudiar la capacitat d'activar el sistema immune per part del M. brumae i del M. bovis BCG en cèl·lules mononuclears de sang perifèrica (PBMC) de poblacions sanes joves i grans, així com de pacients amb neoplàsia vesical, observant-se diferències significatives entre les poblacions avaluades en quant a l'inducció de la producció de citocines pro-inflamatòries i la capacitat antitumoral de les PBMC estimulades. Finalment, es va avaluar si el M. brumae era capaç d' induir immunitat entrenada de la mateixa manera que el M. bovis BCG en diferents sistemes de cultius in vitro i ex vivo, utilitzant un model amb monòcits humans THP-1 i monòcits derivats de PBMCs, respectivament. Els resultats van indicar que l'estimulació inicial dels monòcits amb el M. brumae era capaç d' induir un increment a la producció de IL-6, TNF-[Alfa] i IL-1[Beta], així com un increment en la producció de lactat després d'un estímul secundari inespecífic, indicant un canvi al perfil metabòlic de les cèl·lules i revelant el potencial del M. brumae per induir immunitat entrenada.La instilación por vía intravesical de Mycobacterium bovis bacillus Calmette-Guerin (BCG) es el tratamiento de elección en pacientes con cáncer de vejiga no músculo-invasivo (CVNMI) tras la resección del tumor. M. bovis BCG presenta una eficacia demostrada disminuyendo la recurrencia y la progresión tumoral gracias a la activación del sistema inmune del paciente, lo cual mejora la tasa de supervivencia de los individuos con CVNMI. Sin embargo, el tratamiento intravesical con este agente inmunoterapéutico se asocia a efectos adversos en más del 50% de los pacientes tratados, pudiendo desarrollar complicaciones graves en un 5% de casos, incluyendo infecciones pulmonares o diseminadas. Además, hasta un 30-40% de los pacientes no responden favorablemente a la terapia con M. bovis BCG, por lo que se ven obligados a interrumpir el tratamiento. Una de las alternativas propuestas para sustituir el tratamiento con M. bovis BCG es el uso de Mycobacterium brumae, una micobacteria ambiental no patógena con un efecto antitumoral e inmunomodulador demostrado tanto in vitro, como ex vivo e in vivo en modelo murino de cáncer vesical. Con el objetivo de desarrollar nuevas alternativas terapéuticas que sean eficaces para todos los pacientes, el objetivo de esta tesis es profundizar en las características fenotípicas y genotípicas de diferentes cepas de M. brumae y compararlas con M. bovis BCG; evaluar su interacción con diversos tipos de células tumorales; estudiar el efecto inmunomodulador de ambas micobacterias en el contexto específico de personas mayores; y caracterizar su potencial capacidad de inducir inmunidad entrenada. Inicialmente se secuenció y analizó el genoma de la cepa de referencia de M. brumae y de tres aislados ambientales de M. brumae. La comparación con el genoma de M. bovis y M. tuberculosis demostró la no patogenicidad de M. brumae, así como la presencia de genes involucrados en la activación del sistema inmune del huésped demostrando su inmunogenicidad. No se observaron diferencias a nivel fenotípico de las diferentes cepas de M. brumae y muy pocas a nivel genotípico. Sin embargo, se observó que la cepa CR-103 era capaz de inhibir la proliferación tumoral de una forma más eficaz en algunas líneas tumorales y que la cepa CR-142 inducía una mayor producción de citocinas en macrófagos infectados, comparando con el resto de las cepas de M. brumae. Se demostró la capacidad de inhibir la proliferación tumoral de M. brumae en líneas tumorales de localizaciones diferentes a vejiga, como colon, hígado o páncreas. Por otro lado, se estudió la capacidad de activar el sistema inmune por parte de M. brumae y M. bovis BCG en células mononucleares de sangre periférica (PBMC) de individuos sanos jóvenes y mayores, así como de pacientes con neoplasia vesical, obteniendo una diferencia significativa entre las poblaciones evaluadas en cuando a la inducción de la producción de citocinas proinflamatorias y la capacidad antitumoral de las PBMC estimuladas. Finalmente, se evaluó si M. brumae era capaz de inducir inmunidad entrenada como M. bovis BCG en diferentes cultivos in vitro y ex vivo, utilizando un modelo in vitro con monocitos humanos THP-1 y monocitos derivados de PBMC, respectivamente. Los resultados indicaron que la estimulación inicial de monocitos con M. brumae era capaz de inducir un incremento en la producción de IL-6, TNF-[Alfa] e IL-1[Beta], así como un incremento en la producción de lactato tras un estímulo secundario inespecífico, indicando un cambio en el perfil metabólico y revelando el potencial de M. brumae para inducir inmunidad entrenada.Intravesical instillations of Mycobacterium bovis bacillus Calmette-Guerin (BCG) is the preferred treatment in patients with non-muscle-invasive bladder cancer (NMIBC) after tumour resection. M. bovis BCG has demonstrated efficacy in reducing the recurrence and tumor progression due to the activation of the patient immune system, which enhances the survival rate of individuals with NMIBC. However, intravesical treatment with this immunotherapeutic agent is associated with adverse events in more than 50% of treated patients, being able to develop serious complications in 5% of cases, including lung or disseminated M. bovis BCG infections. Furthermore, up to 30-40% of patients do not respond to M. bovis BCG therapy and, consequently, they have to finish the treatment. One of the alternatives proposed to replace the M. bovis BCG treatment is the use of Mycobacterium brumae, an environmental non-pathogenic mycobacterium with an antitumor and immunomodulatory effect demonstrated in vitro, ex vivo and in vivo in a murine model of bladder cancer. With the aim of developing new therapeutic alternatives that are effective for all patients, the objective of this thesis is to decipher into the phenotypic characteristics and genotypes of different strains of M. brumae and compare them with M. bovis BCG; to evaluate their interaction with different tumour cells; to study the immunemodulatory effect of both mycobacteria in the specific context of older people; and to characterize its potential ability to induce trained immunity. Initially, the genome of the reference strain and three environmental isolates of M. brumae were sequenced and analysed. The comparison with the genome of M. bovis BCG and M. tuberculosis demonstrated the non-pathogenicity of M. brumae, as well as the presence of genes involved in the activation of the host immune system demonstrating its immunogenicity. No differences were observed at the phenotypic level of the different strains of M. brumae and very few at the genotypic level. However, it was observed that the CR-103 strain could inhibit some tumour cell proliferation more efficiently. Besides, the CR-142 strain induced an increase in cytokine production in infected macrophages, compared with the rest of the strains. The ability of M. brumae to inhibit the tumour proliferation was demonstrated in tumour cell lines from locations other than the bladder, such as colon, liver, or pancreas. On the other hand, the ability of M. brumae and M. bovis BCG to activate the immune system from young and old healthy populations, as well as from patients with bladder neoplasia was evaluated. Using peripheral blood mononuclear cells (PBMC) a significant difference between the evaluated populations in terms of triggering the production of proinflammatory cytokines and the antitumor capacity of the activated PBMCs. Finally, the capacity of M. brumae to induce trained immunity, compared to M. bovis BCG, was evaluated in different in vitro and ex vivo cultures using an in vitro model with THP-1 human monocytes and PBMC-derived monocytes, respectively. The results indicated that the initial stimulation of monocytes with M. brumae followed by a secondary non-specific stimulus induce an elevated production of IL-1[Beta], IL-6, and TNF-[Alfa], and an enhanced lactate production. This indicates a change in the metabolic profile of the cells and shows the potential of M. brumae to induce trained immunity

New insights in the genome content of the bacterium Mycobacterium brumae

Resumen del póster presentado al 41st Annual Congress of the European Society of Mycobacteriology (ESM), celebrado de forma virtual del 28 al 29 de junio de 2019.Mycobacterium brumae is a fast-growing, non-pathogenic Mycobacterium species, originally isolated from environmental and human samples in Barcelona, Spain. Previous studies have shown that the implementation of non-pathogenic mycobacteria, such as Mycobacterium bovis BCG can improve the treatment against high-risk non-muscle invasive bladder cancer (BC) by intravesical administration. M. brumae has been shown to be non-pathogenic and its phenotype and immunogenic effect have been well characterized. However, the knowledge of its underlying genetic composition is still incomplete. In this study we have sequenced the genome by means of PACBIO of the M. brumae strain CR-270 obtaining the most complete assembly to date. We describe its genetic content by showing evolutionary relationships between different mycobacteria and we compare its virulence gene content with other virulent mycobacteria such as H37Rv reference strain. Furthermore, we describe the genetic variability of M. brumae by comparative genomics using obtained Illumina sequences. Our results contribute to increase the knowledge about the genetic bases that explain the non-pathogenic phenotype of this bacterium with therapeutic potential.Peer reviewe

Genomic analysis of Mycobacterium brumae sustains its nonpathogenic and immunogenic phenotype

Mycobacterium brumae is a rapid-growing, non-pathogenic Mycobacterium species, originally isolated from environmental and human samples in Barcelona, Spain. Mycobacterium brumae is not pathogenic and it's in vitro phenotype and immunogenic properties have been well characterized. However, the knowledge of its underlying genetic composition is still incomplete. In this study, we first describe the 4 Mb genome of the M. brumae type strain ATCC 51384 T assembling PacBio reads, and second, we assess the low intraspecies variability by comparing the type strain with Illumina reads from three additional strains. Mycobacterium brumae genome is composed of a circular chromosome with a high GC content of 69.2% and containing 3,791 CDSs, 97 pseudogenes, one prophage and no CRISPR loci. Mycobacterium brumae has shown no pathogenic potential in in vivo experiments, and our genomic analysis confirms its phylogenetic position with other non-pathogenic and rapid growing mycobacteria. Accordingly, we determined the absence of virulence-related genes, such as ESX-1 locus and most PE/PPE genes, among others. Although the immunogenic potential of M. brumae was proved to be as high as Mycobacterium bovis BCG, the only mycobacteria licensed to treat cancer, the genomic content of M. tuberculosis T cell and B cell antigens in M. brumae genome is considerably lower than those antigens present in M. bovis BCG genome. Overall, this work provides relevant genomic data on one of the species of the mycobacterial genus with high therapeutic potential

Mycolicibacterium brumae is a safe and non-toxic immunomodulatory agent for cancer treatment

Intravesical Mycobacterium bovis Bacillus Calmette-Guérin (BCG) immunotherapy remains the gold-standard treatment for non-muscle-invasive bladder cancer patients, even though half of the patients develop adverse events to this therapy. On exploring BCG-alternative therapies, Mycolicibacterium brumae, a nontuberculous mycobacterium, has shown outstanding anti-tumor and immunomodulatory capabilities. As no infections due to M. brumae in humans, animals, or plants have been described, the safety and/or toxicity of this mycobacterium have not been previously addressed. In the present study, an analysis was made of M. brumae - and BCG-intravenously-infected severe combined immunodeficient (SCID) mice, M. brumae -intravesically-treated BALB/c mice, and intrahemacoelic-infected- Galleria mellonella larvae. Organs from infected mice and the hemolymph from larvae were processed to count bacterial burden. Blood samples from mice were also taken, and a wide range of hematological and biochemical parameters were analyzed. Finally, histopathological alterations in mouse tissues were evaluated. Our results demonstrate the safety and non-toxic profile of M. brumae. Differences were observed in the biochemical, hematological and histopathological analysis between M. brumae and BCG-infected mice, as well as survival curves rates and colony forming units (CFU) counts in both animal models. M. brumae constitutes a safe therapeutic biological agent, overcoming the safety and toxicity disadvantages presented by BCG in both mice and G. mellonella animal models

Biodegradación de la lignocelulosa: aspectos microbiológicos, químicos y enzimáticos del ataque fúngico a la lignina

Wood is the main renewable material on Earth and is largely used as building material and in paper-pulp manufacturing. This review describes the composition of lignocellulosic materials, the different processes by which fungi are able to alter wood, including decay patterns caused by white, brown, and soft-rot fungi, and fungal staining of wood. The chemical, enzymatic, and molecular aspects of the fungal attack of lignin, which represents the key step in wood decay, are also discussed. Modern analytical techniques to investigate fungal degradation and modification of the lignin polymer are reviewed, as are the different oxidative enzymes (oxidoreductases) involved in lignin degradation. These include laccases, high redox potential ligninolytic peroxidases (lignin peroxidase, manganese peroxidase, and versatile peroxidase), and oxidases. Special emphasis is given to the reactions catalyzed, their synergistic action on lignin, and the structural bases for their unique catalytic properties. Broadening our knowledge of lignocellulose biodegradation processes should contribute to better control of wood-decaying fungi, as well as to the development of new biocatalysts of industrial interest based on these organisms and their enzymes.These studies have been partially supported by ENCE (Spain), by Spanish projects AGL2002-393 and BIO2002-1166, by EU projects QLK5-99-1357 and QLK3-99-590, and by an EUFORES(ENCE)-PDT/MEC(Uruguay) grant. Carmen Ascaso (CCMA, CSIC, Madrid) is acknowledged for low-temperature scanning-electron microscopy facilities. Klaus Piontek (ETH, Zurich) is acknowledged for solving the VP crystal structure. Lina Bettucci (Universidad de la República, Montevideo) is acknowledged for an Inocutis jamaicensis strain. M.S acknowledges MEC for a Postdoctoral Fellowship. F.J.R.-D. thanks CSIC for an I3P contract. A.G. and S.C. thank MEC for their “Ramón y Cajal” contracts.Peer reviewe