Comparison of the transcriptome, lipidome, and c-di-GMP production between BCGΔBCG1419c and BCG, with Mincle- and Myd88-dependent induction of proinflammatory cytokines in murine macrophages.

We have previously reported the transcriptomic and lipidomic profile of the first-generation, hygromycin-resistant (HygR) version of the BCGΔBCG1419c vaccine candidate, under biofilm conditions. We recently constructed and characterized the efficacy, safety, whole genome sequence, and proteomic profile of a second-generation version of BCGΔBCG1419c, a strain lacking the BCG1419c gene and devoid of antibiotic markers. Here, we compared the antibiotic-less BCGΔBCG1419c with BCG. We assessed their colonial and ultrastructural morphology, biofilm, c-di-GMP production in vitro, as well as their transcriptomic and lipidomic profiles, including their capacity to activate macrophages via Mincle and Myd88. Our results show that BCGΔBCG1419c colonial and ultrastructural morphology, c-di-GMP, and biofilm production differed from parental BCG, whereas we found no significant changes in its lipidomic profile either in biofilm or planktonic growth conditions. Transcriptomic profiling suggests changes in BCGΔBCG1419c cell wall and showed reduced transcription of some members of the DosR, MtrA, and ArgR regulons. Finally, induction of TNF-α, IL-6 or G-CSF by bone-marrow derived macrophages infected with either BCGΔBCG1419c or BCG required Mincle and Myd88. Our results confirm that some differences already found to occur in HygR BCGΔBCG1419c compared with BCG are maintained in the antibiotic-less version of this vaccine candidate except changes in production of PDIM. Comparison with previous characterizations conducted by OMICs show that some differences observed in BCGΔBCG1419c compared with BCG are maintained whereas others are dependent on the growth condition employed to culture them.

BCGΔBCG1419c increased memory CD8+ T cell-associated immunogenicity and mitigated pulmonary inflammation compared with BCG in a model of chronic tuberculosis.

Previously, we reported that a hygromycin resistant version of the BCGΔBCG1419c vaccine candidate reduced tuberculosis (TB) disease in BALB/c, C57BL/6, and B6D2F1 mice infected with Mycobacterium tuberculosis (Mtb) H37Rv. Here, the second-generation version of BCGΔBCG1419c (based on BCG Pasteur ATCC 35734, without antibiotic resistance markers, and a complete deletion of BCG1419c) was compared to its parental BCG for immunogenicity and protective efficacy against the Mtb clinical isolate M2 in C57BL/6 mice. Both BCG and BCGΔBCG1419c induced production of IFN-γ, TNF-α, and/or IL-2 by effector memory (CD44+CD62L-), PPD-specific, CD4+ T cells, and only BCGΔBCG1419c increased effector memory, PPD-specific CD8+ T cell responses in the lungs and spleens compared with unvaccinated mice before challenge. BCGΔBCG1419c increased levels of central memory (CD62L+CD44+) T CD4+ and CD8+ cells compared to those of BCG-vaccinated mice. Both BCG strains elicited Th1-biased antigen-specific polyfunctional effector memory CD4+/CD8+ T cell responses at 10 weeks post-infection, and both vaccines controlled Mtb M2 growth in the lung and spleen. Only BCGΔBCG1419c significantly ameliorated pulmonary inflammation and decreased neutrophil infiltration into the lung compared to BCG-vaccinated and unvaccinated mice. Both BCG strains reduced pulmonary TNF-α, IFN-γ, and IL-10 levels. Taken together, BCGΔBCG1419c increased memory CD8+T cell-associated immunogenicity and mitigated pulmonary inflammation compared with BCG.

BCG and BCGΔBCG1419c transiently protect hypersusceptible I/St mice and induce different influx of macrophages and neutrophils during pulmonary tuberculosis.

Background. Host genetic factors influence both susceptibility to Mycobacterium tuberculosis infection and immune responses generated by vaccination. Genetically susceptible mice help to study mechanisms of immune protection which may differ from those operating in more resistant models.Methods. In this work, we compared the efficacy of protection conferred by subcutaneous vaccination of hypersusceptible I/St mice with BCG and the first-generation, hygromycin resistant version of the vaccine candidate BCGΔBCG1419c, against tuberculosis (TB), measured as survival, weight loss and replication in lungs. We further characterized the relative presence of immune cells in lungs.Results. We found that in I/St mice, vaccination with BCG or BCGΔBCG1419c provided similar level of protection against TB-driven weight loss and M. tuberculosis replication in lungs, while prolonging median survival time compared with unvaccinated controls. Despite affording similar protection to parental BCG, BCGΔBCG1419c led to a reduced presence of macrophages in lungs during early TB and to an increased neutrophil recruitment to the lungs during chronic TB.Conclusions. BCGΔBCG1419c protects I/St mice in a different manner than wild-type BCG against pulmonary TB by promoting different influx of macrophages and neutrophils at distinct times post-infection. These findings prompt us to suggest that preclinical evaluation of novel TB vaccine candidates should include evaluation of efficacy not only in commonly used resistant inbred mice, but also in susceptible hosts, to further determine their potential application to populations varying in their genetic. This would likely impact their intended use depending on host resistance or susceptibility to TB.

Macrophage infection with combinations of BCG mutants reduces induction of TNF-α, IL-6, IL-1ß and increases IL-4.

Tuberculosis (TB) is the most prevalent infectious disease worldwide, with no fully effective vaccine yet available. Considering that BCG strains devoid of the BCG1416c or BCG1419c genes afforded protection in mice versus highly virulent M. tuberculosis challenge, or in chronic infection models compared to BCG, respectively, we hypothesized that a synergistic effect of these strains might occur and provide enhanced protection against TB. Herein, we evaluated this hypothesis throughout an experimental design approach, where different combinations of these strains were tested for their capacity to induce cytokines in vitro, compared to individual strains. Our results show that mixed-infection of murine macrophages using these strains significantly decreases induction of TNF-α, IL-1ß, IL-6 but increases IL-4 induction compared with individual strains. These results suggest the existence of interaction effects during infection, which reduce induction of pro-inflammatory cytokines, even though individual intracellular replication is not altered when strains are combined. This is the first report of the evaluation of a potential whole-live combined vaccine against tuberculosis, which paradoxically seems to reduce production of pro-inflammatory cytokines while induces IL-4, leading us to further hypothesize that this combination might contribute as a therapeutic vaccine to reduce inflammation in severe TB cases.

Immune response elicited by two rBCG strains devoid of genes involved in c-di-GMP metabolism affect protection versus challenge with M. tuberculosis strains of different virulence.

Pellicles, a type of biofilm, have gathered a renewed interest in the field of tuberculosis as a structure that mimics some characteristics occurring during M. tuberculosis infection, such as antibiotic recalcitrance and chronicity of infection, and as a source of antigens for humoral response in infected guinea pigs. In other bacteria, it has been well documented that the second messenger c-di-GMP modulates the transition from planktonic cells to biofilm formation. In this work, we used the live vaccine Mycobacterium bovis BCG to determine whether deletion of genes involved in c-di-GMP metabolism would affect interaction with macrophages, capacity to induce immune response in a murine cell line and mice, and how the protein profile was modified when grown as surface pellicles. We found that deletion of the BCG1419c (Delta c-di-GMP phosphodiesterase, ΔPDE) gene, or deletion of the BCG1416c (Delta c-di-GMP diguanylate cyclase, ΔDGC) gene, altered production of TNF-α, IL-6, and IL-1ß, in murine macrophages, and resulted in attenuation in intra-macrophage replication. Moreover, in addition to the improved immunogenicity of the BCGΔBCG1419c mutant already reported, deletion of the BCG1416c gene leads to increased T CD4+ and T CD8+ activation. This correlated with protection versus lethality in mice infected with the highly virulent M. tuberculosis 5186 afforded by vaccination with all the tested BCG strains, and controlled the growth of the mildly virulent M. tuberculosis H37Rv in lungs by vaccination with BCGΔBCG1419c during chronic late infection from 4 to 6 months after challenge. Furthermore, when grown as surface pellicles, a condition used to manufacture BCG vaccine, in comparison to BCG wild type, both rBCGs changed expression of antigenic proteins such as DnaK, HbhA, PstS2, 35KDa antigen, GroEL2, as well as AcpM, a protein involved in synthesis of mycolic acids, molecules relevant to modulate inflammatory responses.

Evaluation of the immunogenic capability of the BCG strains BCGΔBCG1419c and BCGΔBCG1416c in a three-dimensional human lung tissue model.

Tuberculosis (TB) still remains as an unmet global threat. The current vaccine is not fully effective and novel alternatives are needed. Here, two vaccine candidate strains derived from BCG carrying deletions in the BCG1416c or BCG1419c genes were analysed for their capacity to modulate the cytokine/chemokine profile and granuloma formation in a human lung tissue model (LTM). We show that the clustering of monocytes, reminiscent of early granuloma formation, in LTMs infected with BCG strains was similar for all of them. However, BCGΔBCG1419c, like M. tuberculosis, was capable of inducing the production of IL-6 in contrast to the other BCG strains. This work suggests that LTM could be a useful ex vivo assay to evaluate the potential immunogenicity of novel TB vaccine candidates.

The adenylyl cyclase Rv2212 modifies the proteome and infectivity of Mycobacterium bovis BCG.

All organisms have the capacity to sense and respond to environmental changes. These signals often involve the use of second messengers such as cyclic adenosine monophosphate (cAMP). This second messenger is widely distributed among organisms and coordinates gene expression related with pathogenesis, virulence, and environmental adaptation. Genomic analysis in Mycobacterium tuberculosis has identified 16 adenylyl cyclases (AC) and one phosphodiesterase, which produce and degrade cAMP, respectively. To date, ten AC have been biochemically characterized and only one (Rv0386) has been found to be important during murine infection with M. tuberculosis. Here, we investigated the impact of hsp60-driven Rv2212 gene expression in Mycobacterium bovis Bacillus Calmette-Guerin (BCG) during growth in vitro, and during macrophage and mice infection. We found that hsp60-driven expression of Rv2212 resulted in an increased capacity of replication in murine macrophages but an attenuated phenotype in lungs and spleen when administered intravenously in mice. Furthermore, this strain displayed an altered proteome mainly affecting proteins associated with stress conditions (bfrB, groEL-2, DnaK) that could contribute to the attenuated phenotype observed in mice.

The synthetic cathelicidin HHC-10 inhibits Mycobacterium bovis BCG in vitro and in C57BL/6 mice.

Tuberculosis causes close to 1.5 million deaths in the world, with new cases exceeding 9 million in recent years. Coinfection with HIV further worsens the global situation. New molecules that overcome the limitations of currently used drugs are needed. We aimed to determine whether HHC-10 is active against the Mycobacterium tuberculosis complex bacteria Mycobacterium bovis bacille calmette guerin (BCG) in vitro and in vivo. For this, HHC-10 was tested in vitro using different peptide concentrations, and in vivo, in C57BL/6 mice infected intratracheally, at two doses (1.25 and 2.5 mg kg(-1), once a week, 4 weeks). Interferon (IFN)-γ, TNF-α, interleukin (IL)-4, and IL-10 mRNA transcript levels were compared between treated and nontreated mice. In vitro, HHC-10 decreased 69% and 88% the number of colony-forming units (CFU) per millileter recovered after 24-hr treatment at 50 and 100 µg/ml, respectively. In vivo, BCG CFUs in mouse lungs were reduced 77.8% and 95.8% at 1.25 and 2.5 mg kg(-1), respectively. IFN-γ expression was lower in the HHC-10-treated group than that of nontreated animals. Considering genomic conservation between BCG and M. tuberculosis, the in vitro and in vivo activities of HHC-10 observed in this study suggest that the use of this peptide may be useful as therapeutic agent against tuberculosis.

Modulation of cAMP metabolism in Mycobacterium tuberculosis and its effect on host infection.

Mycobacterium tuberculosis remains the single most relevant bacterial infectious agent as Tuberculosis is estimated to affect one-third of the world population. Like other microorganisms, M. tuberculosis needs to sense and adapt to changes in the several niches where it is found, ranging from the environment to a number of host-adapted programs, including infection of cell types such as macrophages, dendritic cells, epithelial cells and adipocytes. A strategy commonly used by cells to respond to such changes consists of producing small molecules known as second messengers. 3',5'-cyclic adenosine monophosphate (cAMP) is one of the best-studied second messengers in many organisms, and in recent years its participation during the M. tuberculosis infection cycle has just begun to be thoroughly considered. In this work, we aimed to provide a perspective of how cAMP metabolism proceeds in M. tuberculosis, which genes are activated in response to cAMP signaling in this organism, and discuss the evidence for bacterially produced cAMP use during infection. Furthermore, key issues needing to be addressed for better understanding cAMP physiology in slow-growing pathogenic mycobacteria are presented.

Aspectos biológicos, clínicos y epidemiológicos de la tuberculosis latente: [revision] / Biological, clinical and epidemiological aspects of latent tuberculosis: [review]

Mycobacterium tuberculosis afecta a la humanidad desde hace más de 20 000 años. Su morbimortalidad es elevada, por lo que repercute económicamente en los países en desarrollo. La infección latente, caracterizada por la presencia de bacilos vivos en tejidos del huésped, con ausencia de signos y síntomas clínicos, es una característica de esta enfermedad, ya que la micobacteria puede adaptar su metabolismo para mantenerse viva con baja o nula replicación, dificultando su eliminación de los tejidos por los fármacos antituberculosos y permaneciendo inadvertida al reconocimiento y eliminación por el sistema inmunológico. Varias son las interrogantes de esta forma de tuberculosis (TB): la falta de conocimiento del metabolismo del bacilo en estado durmiente, su relación con la inmunidad del hospedero y la identificación de antígenos como marcadores diagnósticos de infección subclínica durante la latencia. Este artículo resume los aspectos biológicos, clínicos y epidemiológicos más importantes de esta forma de tuberculosis.

Mycobacterium tuberculosis, the causal agent of tuberculosis, has affected humankind for approximately 20 000 years. Tuberculosis is a devastating disease, particularly in developing countries. One of its most notable characteristics is latent infection, in which live bacilli persist in the host tissues without clinical manifestations. Thus, the tuberculous bacilli adapt their metabolism to remain viable with low or no replication, avoiding their elimination by the immune system or conventional chemotherapy. Among the several problems that are particularly important to the understanding of this form of tuberculosis, and are not well-known, are the key metabolic steps that allow mycobacteria to remain in a dormant state and its interaction with host immunity. This article reviews some of the most significant biological, clinical and epidemiological aspects of this form of tuberculosis.

Identificación de una proteasa neutra en intestino de Boophilus microplus por electroforesis en geles de poliacrilamida copolimerizados con gelatina / Identification of a neutral protease in the intestine of Boophilus microplus with electrophoresis in polyacrylamide gels copolymerized with gelatin

A neutral activity of Boophilus microplus in the intestine was identified by electrophoresis in polyacrilamide gel copolymerized with gelatin. The maximum of activity was attained at pH 6.0. The highest specific activity at that pH was obtained with casein substrate. The disappearance of this activity was observed in both substrates after the addition of phenylmethylsulphonyl fluoride in the reaction mixtures. It was very interesting to find out an endopeptidase activity with these characteristics in the intestine, in spite of the fact that the digestive activity in ticks is intracellular at very acid pH, which does not occur in other insects.

Pólipo adenomatoso de vesicula biliar (reporte de un caso) / Polypoid lesion of the gallbladder (case report)

Los pólipos de la vesícula biliar son lesiones raras consideradas como tumores. Su frecuencia llega hasta 13.8, y su benignidad ocurre en 92. El cuadro clínico de presentación es similar a la colelitiasis crónica. Se describe el caso de una mujer joven a quien se le documentó un pólipo adenomatoso vesicular y se le efectuó colecistectomía. El criterio ultrasonográfico es importante. Se efectúa revisión de la entidad y se realizan algunas consideraciones con respecto a esta patología vesicular. Los pólipos de la vesícula biliar son infrecuentes, es importante para su detección una alta sospecha clínica, además deberán ser considerados como tumores, más aún cuando no se detecte litiasis vesicular