Rv2617c and P36 are virulence factors of pathogenic mycobacteria involved in resistance to oxidative stress

In this study, we characterized the role of Rv2617c in the virulence of Mycobacterium tuberculosis. Rv2617c is a protein of unknown function unique to M. tuberculosis complex (MTC) and Mycobacterium leprae. In vitro, this protein interacts with the virulence factor P36 (also named Erp) and KdpF, a protein linked to nitrosative stress. Here, we showed that knockout of the Rv2617c gene in M. tuberculosis CDC1551 reduced the replication of the pathogen in a mouse model of infection and favored the trafficking of mycobacteria to phagolysosomes. We also demonstrated that Rv2617c and P36 are required for resistance to in vitro hydrogen peroxide treatment in M. tuberculosis and Mycobacterium bovis, respectively. These findings indicate Rv2617c and P36 act in concert to prevent bacterial damage upon oxidative stress.Instituto de BiotecnologíaFil: Forrellad, Marina Andrea. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Vazquez, Cristina Lourdes. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Blanco, Federico Carlos. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Klepp, Laura Ines. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Garcia, Elizabeth Andrea. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Rocha, Rosana Valeria. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Villafañe, Luciana Maria. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bigi, María Mercedes. Fil: Bigi, María Mercedes. Universidad de Buenos Aires. Facultad de Agronomía; ArgentinaFil: Gutierrez, Maximiliano Gabriel. The Francis Crick Institute, Host-Pathogen Interactions in Tuberculosis Laboratory; Reino UnidoFil: Bigi, Fabiana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de investigaciones Científicas y Tecnológicas; Argentin

A Transcriptional Analysis of Cattle Immune Cells Reveals a Central Role of Type 1 Interferon in the In Vitro Innate Immune Response against <i>Mycobacterium bovis</i>

Bovine tuberculosis is a chronic infectious disease primarily caused by Mycobacterium bovis, a bacterium that affects cattle and other mammals, including humans. Despite the availability of vast research about the immune response mechanisms of human tuberculosis caused by Mycobacterium tuberculosis, the knowledge of bovine tuberculosis’s immunology, particularly regarding the innate immune response, still remains scarce. In this study, we compared the transcriptome of cell cultures containing lymphocytes and M. bovis infected-macrophages with two strains of variable virulence, the virulent Mb04-303 strain and the attenuated Mb534. To that end, we infected bovine macrophages at a multiplicity of infection of one, and co-cultured the infections with autologous lymphocytes. RNA obtained from the co-cultures was sequenced to identify differentially expressed gene pathways by using the database Reactome. The RNA-seq analysis showed that the Mb04-303 infection upregulated the type 1 interferon signalling pathway, while it downregulated the KEAP1-NFE2L2 pathway. According to the literature, this last pathway is involved in the activation of antioxidant genes and inflammasome. In addition, the macrophages infected with Mb04-303 recruited more Galectin 8 than those infected with Mb534. This result indicates that Mb04-303 induced higher phagosome membrane damage, with the possible concomitant release of bacterial compounds into the cytoplasm that activates the type I signalling pathway. Altogether, Mb04-303 repressed the antioxidant and anti-inflammatory responses, likely impairing interleukin-1β activation, and trigged the canonical type 1 interferon signalling. Although these responses led to the control of bacterial replication during early infection, the virulent strain eventually managed to establish a successful infection

Polymorphisms of 20 regulatory proteins between Mycobacterium tuberculosis and Mycobacterium bovis

Mycobacterium tuberculosis and Mycobacterium bovis are responsible for tuberculosis in humans and animals, respectively. Both species are closely related and belong to the Mycobacterium tuberculosis complex (MTC). M. tuberculosis is the most ancient species from which M. bovis and other members of the MTC evolved. The genome of M. bovis is over >99.95% identical to that of M. tuberculosis but with seven deletions ranging in size from 1 to 12.7 kb. In addition, 1200 single nucleotide mutations in coding regions distinguish M. bovis from M. tuberculosis. In the present study, we assessed 75 M. tuberculosis genomes and 23 M. bovis genomes to identify non-synonymous mutations in 202 coding sequences of regulatory genes between both species. We identified species-specific variants in 20 regulatory proteins and confirmed differential expression of hypoxia-related genes between M. bovis and M. tuberculosisInst. de BiotecnologíaFil: Bigi, María Mercedes. Universidad de Buenos Aires; ArgentinaFil: Blanco, Federico Carlos. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Araujo, Flabio R. EMBRAPA Mato Grosso do Sul; BrasilFil: Thacker, Tyler C. United States Department of Agriculture. Agricultural Research Service. National Animal Disease Center; Estados UnidosFil: Zumarraga, Martin Jose. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Cataldi, Angel Adrian. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Soria, Marcelo Abel. Universidad de Buenos Aires. Facultad de Agronomía. Departamento de Biología Aplicada y Alimentos. Cátedra de Microbiología Agrícola; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales; ArgentinaFil: Bigi, Fabiana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentin

Characterization of the two component regulatory system PhoPR in Mycobacterium bovis

Mycobacterium bovis is the causative agent of bovine tuberculosis and is a member of Mycobacterium tuberculosis complex, which causes tuberculosis in a number of mammals including humans. Previous studies have shown that the genes encoding the two-component system PhoPR, which regulates several genes involved in the virulence of M. tuberculosis, are polymorphic in M. bovis, when compared to M. tuberculosis, which results in a dysfunctional two-component system. In this study we investigated the role of PhoPR in two M. bovis strains with differing degrees of virulence. We found that the deletion of phoP in an M. bovis isolate reduced its capacity of inducing phagosomal arrest in bovine macrophages. By gene expression analysis, we demonstrated that, in both M. bovis strains, PhoP regulates the expression of a putative lipid desaturase Mb1404-Mb1405, a protein involved in redox stress AhpC, the sulfolipid transporter Mmpl8 and the secreted antigen ESAT-6. Furthermore, the lack of PhoP increased the sensitivity to acidic stress and alteration of the biofilm/pellicle formation of M. bovis. Both these phenotypes are connected to bacterial redox homeostasis. Therefore, the results of this study suggest a role of PhoPR in M. bovis to be linked to the mechanisms that mycobacteria display to maintain their redox balance.Instituto de BiotecnologíaFil: Garcia, Elizabeth Andrea. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Blanco, Federico Carlos. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bigi, María Mercedes. Universidad de Buenos Aires. Facultad de Agronomía, Cátedra de Microbiología Agrícola; Argentina. . Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales; ArgentinaFil: Vazquez, Cristina Lourdes. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Forrellad, Marina Andrea. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Rocha, Rosana Valeria. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Golby, Paul. CN Bio Innovations, Welwyn Garden City; Reino UnidoFil: Soria, Marcelo Abel. Universidad de Buenos Aires. Facultad de Agronomía. Departamento de Biología Aplicada y Alimentos. Cátedra de Microbiología Agrícola; ArgentinaFil: Bigi, Fabiana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentin

Expression of tuberculosis antigen ESAT-6 in Nicotiana tabacum using a potato virus X-based vector

A good candidate antigen to create a therapeutic vaccine against TB is the ESAT-6 protein. Antigens produced in plants have already been successfully used as experimental vaccines, and small single-stranded RNA plant viruses have emerged as promising tools to rapidly express large amounts of foreign proteins in susceptible host plants. Here, we present the expression of ESAT-6 protein in Nicotiana tabacum using a vector based on potato virus X (PVX). The complete ESAT-6 open reading frame is expressed as a fusion protein with the 2A peptide of Foot and Mouth Disease Virus and the amino terminal of the PVX coat protein (CP) (PVXESAT-6). This strategy allows the production of free CP and ESAT-6 as well as fused ESAT-2A-CP to obtain recombinant chimaeric virions expressing ESAT-6 at the surface to be used as particulate antigen in vaccination. ESAT-6 expression was tested in agroinfiltrated tobacco leaves and products of the expected molecular masses corresponding to cleaved CP and ESAT-2A-CP fusion protein were observed, with ESAT-6 yields ranging from 0.5% to 1% of total soluble protein. Our study describes for the first time the expression of the ESAT-6 protein in tobacco plants using a PVX-derived vector. This strategy should serve as a convenient, rapid, low-cost expression system and can also be used for the assessment of ESAT-6 production and function prior to stable plant transformation.Fil: Zelada, Alicia Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Calamante, Gabriela. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Santangelo, María de la Paz. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Bigi, Fabiana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Verna, Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Mentaberry, Alejandro Nestor. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Cataldi, Ángel Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentin

Single nucleotide polymorphisms may explain the contrasting phenotypes of two variants of a multidrug-resistant Mycobacterium tuberculosis strain

Globally, about 4.5% of new tuberculosis (TB) cases are multi-drug-resistant (MDR), i.e. resistant to the two most powerful first-line anti-TB drugs. Indeed, 480,000 people developed MDR-TB in 2015 and 190,000 people died because of MDR-TB. The MDR Mycobacterium tuberculosis M family, which belongs to the Haarlem lineage, is highly prosperous in Argentina and capable of building up further drug resistance without impairing its ability to spread. In this study, we sequenced the whole genomes of a highly prosperous M-family strain (Mp) and its contemporary variant, strain 410, which produced only one recorded tuberculosis case in the last two decades. Previous reports have demonstrated that Mp induced dysfunctional CD8þ cytotoxic T cell activity, suggesting that this strain has the ability to evade the immune response against M. tuberculosis. Comparative analysis of Mp and 410 genomes revealed non-synonymous polymorphisms in eleven genes and five intergenic regions with polymorphisms between both strains. Some of these genes and promoter regions are involved in the metabolism of cell wall components, others in drug resistance and a SNP in Rv1861, a gene encoding a putative transglycosylase that produces a truncated protein in Mp. The mutation in Rv3787c, a putative S-adenosyl-L methioninedependent methyltransferase, is conserved in all of the other prosperous M strains here analysed and absent in non-prosperous M strains. Remarkably, three polymorphic promoter regions displayed differential transcriptional activity between Mp and 410. We speculate that the observed mutations/polymorphisms are associated with the reported higher capacity of Mp for modulating the host's immune response.Inst. de BiotecnologíaFil: Bigi, María Mercedes. Universidad de Buenos Aires. Cátedra de de Microbiología; ArgentinaFil: López, Beatriz. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas “Dr. Carlos G. Malbrán”; ArgentinaFil: Blanco, Federico Carlos. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Sasiain, María del Carmen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: de la Barrera, Silvia Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Marti, Marcelo Adrian. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Sosa, Ezequiel Jorge. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Cálculo. Plataforma de Bioinformática Argentina; ArgentinaFil: Fernandez Do Porto, Darío Augusto. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Cálculo. Plataforma de Bioinformática Argentina; ArgentinaFil: Ritacco, Viviana. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas “Dr. Carlos G. Malbrán”; ArgentinaFil: Bigi, Fabiana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Soria, Marcelo Abel. Universidad de Buenos Aires. Facultad de Agronomía. Departamento de Biología Aplicada y Alimentos. Cátedra de Microbiología Agrícola; Argentin