RESUMEN

Mycobacterium bovis is an etiological agent of bovine tuberculosis (bTB) that also infects other mammals, including humans. The lack of an effective vaccine for the control of bTB highlights the need for developing new vaccines. In this study, we developed and evaluated an M. bovis strain deleted in the virulence genes phoP, esxA and esxB as a vaccine candidate against bTB in BALBc mice. The evaluated strains were the new live vaccine and BCG, alone or in combination with ncH65vD. The immunogen ncH65vD is a fusion protein H65, encapsulated together with vitamin D3, within the oily body of a nanocapsule composed of an antigen-loading polymeric shell. All vaccines conferred protection against the M. bovis challenge. However, no significant differences were detected among the vaccinated groups regarding bacterial loads in lungs and spleen. Mice vaccinated with the mutant strain plus ncH65vD showed negative Ziehl Neelsen staining of mycobacteria in their lungs, which suggests better control of bacteria replication according to this protection parameter. Consistently, this vaccination scheme showed the highest proportion of CD4 + T cells expressing the protection markers PD-1 and CXCR3 among the vaccinated groups. Correlation studies showed that PD-1 and CXCR3 expression levels in lung-resident CD4 T cells negatively correlated with the number of colony forming units of M. bovis in the lungs of mice. Therefore, the results suggest a link between the presence of PD-1 + and CXCR3 + cells at the site of the immune response against mycobacteria and the level of mycobacterial loads.

Asunto(s)

Enfermedades de los Bovinos , Mycobacterium bovis , Mycobacterium tuberculosis , Enfermedades de los Roedores , Vacunas contra la Tuberculosis , Tuberculosis Bovina , Humanos , Bovinos , Animales , Ratones , Tuberculosis Bovina/prevención & control , Vacuna BCG , Receptor de Muerte Celular Programada 1 , Vacunación/veterinaria , Mamíferos

RESUMEN

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-1ß 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.

RESUMEN

Bovine tuberculosis is an important animal and zoonotic disease caused by Mycobacterium bovis. The innate immune response is the first line of defense against pathogens and is also crucial for the development of an efficient adaptive immune response. In this study we used an in vitro co-culture model of antigen presenting cells (APC) and autologous lymphocytes derived from peripheral blood mononuclear cells to identify the cell populations and immune mediators that participate in the development of an efficient innate response capable of controlling the intracellular replication of M. bovis. After M. bovis infection, bovine immune cell cultures displayed upregulated levels of iNOS, IL-22 and IFN-γ and the induction of the innate immune response was dependent on the presence of differentiated APC. Among the analyzed M. bovis isolates, only a live virulent M. bovis isolate induced an efficient innate immune response, which was increased upon stimulation of cell co-cultures with the M. bovis culture supernatant. Moreover, we demonstrated that an allelic variation of the early secreted protein ESAT-6 (ESAT6 T63A) expressed in the virulent strain is involved in this increased innate immune response. These results highlight the relevance of the compounds secreted by live M. bovis as well as the variability among the assessed M. bovis strains to induce an efficient innate immune response.

Asunto(s)

Inmunidad Innata/inmunología , Mycobacterium bovis/inmunología , Tuberculosis Bovina/inmunología , Animales , Antígenos Bacterianos/inmunología , Bovinos , Técnicas de Cocultivo , Citocinas/metabolismo , Interferón gamma/metabolismo , Macrófagos , Cultivo Primario de Células

RESUMEN

Tuberculosis, a lung disease caused by Mycobacterium tuberculosis (Mtb), is one of the ten leading causes of death worldwide affecting mainly developing countries. Mtb can persist and survive inside infected cells through modulation of host antibacterial attack, i.e., by avoiding the maturation of phagosome containing mycobacteria to more acidic endosomal compartment. In addition, bacterial phosphatases play a central role in the interplay between host cells and Mtb. In this study, we characterized the Rv2577 of Mtb as a potential alkaline phosphatase/phosphodiesterase enzyme. By an in vitro kinetic assay, we demonstrated that purified Rv2577 expressed in Mycobacterium smegmatis displays both enzyme activities, as evidenced by using the artificial substrates p-NPP and bis-(p-NPP). In addition, a three-dimensional model of Rv2577 allowed us to define the catalytic amino acid residues of the active site, which were confirmed by site-directed mutagenesis and enzyme activity analysis, being characteristic of a member of the metallophosphatase superfamily. Finally, a mutation introduced in Rv2577 reduced the replication of Mtb in mouse organs and impaired the arrest of phagosomes containing mycobacteria in early endosomes; which indicates Rv2577 plays a role in Mtb virulence.