Project description:Tuberculosis (TB) is one of major causes of death worldwide. Bacillus Calmette-Guerin (BCG) is the only licensed TB vaccine and its inability to protect against adult pulmonary TB can be due to genetic differences among strains described since the 1940s. In this work, we compared the proteomic profile of the surface-associated proteins from M. bovis BCG Moreau, the Brazilian vaccine strain, and the BCG Pasteur reference strain. The methodology used was 2D-gel electrophoresis combined with mass spectrometry techniques (MALDI-TOF/TOF). We identified 115 proteins. Of these, 24 proteins showed differential expression between the two BCG strains. Furthermore, 27 proteins previously described as displaying moonlighting function were identified, 8 of these proteins showed variation in abundance comparing BCG Moreau to Pasteur and 2 of them presented two different domain hits.
Project description:The century-old Mycobacterium bovis Bacillus Calmette-Guerin (BCG) remains the only licensed vaccine against tuberculosis (TB). Despite this, there is still a lot to learn about the immune response induced by BCG, both in terms of phenotype and specificity. Here, we investigated the BCG-specific gene expression changes induced in PBMCs and CD4 memory T cells by BCG in individuals pre- and 8m post vaccination. We also determined whether reactivity against a peptide pool defined in individuals with controlled latent TB infection (MTB300), and with peptides homologous to peptides found in BCG, was boosted following BCG vaccination.
Project description:In the present study, we applied microarray technology to define a biosignature from the whole genome expression in lung and spleen samples after BCG vaccination and M. bovis infection of BALB/c mice. The aims were two-fold, namely to define biosignatures that could predict vaccine success before challenge, and biomarker patterns that correlated with anamnestic protective responses following exposure to virulent M. bovis. Further, Our aim was to define these markers to be detectable without in vitro antigenic challenge. After BCG vaccination, we defined a specific pulmonary gene expression signature related to the connective tissue development and function network that predicted vaccine success before M. bovis challenge. In addition, a Th17-related cytokine profile was found that correlated with vaccine-induced protective immunity following infection with virulent M. bovis in the lung as well as additional genes that were up-regulated in the spleens of vaccinated animals post-infection that was related to neutrophil biology and inflammation. This study has therefore prioritized both biomarkers predicting vaccination success before challenge and bio-signatures that are associated with protective immune responses that will be useful to evaluate future vaccine candidates. Two groups of 20 mice each were immunised by a single intradermal injection of 2 x 10 to 5 CFU of M. bovis BCG (Vaccinated), or Hanks buffered salt solution (HBSS) (Unvaccinated). Six weeks later 5 mice from each group were euthanized for immunological analyses and the remaining mice from each group were challenged with approx 600 CFU M. bovis via the intranasal route. At days 3 and 14 post challenge five mice per group were euthanized and spleens and lungs harvested.
Project description:Mycobacterium tuberculosis is the causative agent of tuberculosis, a disease that affects one-third of the world’s population. The sole extant vaccine for tuberculosis is the live attenuated Mycobacterium bovis bacille Calmette-Guerin (BCG). We examined 13 representative BCG strains from around the world to ascertain their ability to express DosR-regulated dormancy antigens. These are known to be recognized by T-cells of M. tuberculosis infected individuals, especially those harboring latent infections. Differences in expression of these antigens could be valuable for use as diagnostic markers to distinguish BCG vaccination from latent tuberculosis. We determined that all BCG strains were defective for induction of two dormancy genes, narK2 (Rv1737c) and narX (Rv1736c). NarK2 is known to be necessary for nitrate respiration during anaerobic dormancy. Analysis of the narK2/X promoter region revealed a base substitution mutation in all tested BCG strains and M. bovis in comparison to the M. tuberculosis sequence. We also show that nitrate reduction by BCG strains during dormancy was greatly reduced compared to M. tuberculosis and varied between tested strains. Several dormancy regulon transcriptional differences were also identified among the strains, as well as variation in their growth and survival. These findings demonstrate defects in DosR regulon expression during dormancy and phenotypic variation between commonly used BCG vaccine strains. 12 different BCG strains were examined as well as M. tuberculosis H37Rv and M. bovis. Two arrays per strain were analyzed, one with the addition of nitric oxide and the other utilizing hypoxia treatment, both conditions shown to induce expression of the dormancy regulon. The reference sample for each array was log phase M. tuberculosis H37Rv.
Project description:In the present study, we applied microarray technology to define a biosignature from the whole genome expression in lung and spleen samples after BCG vaccination and M. bovis infection of BALB/c mice. The aims were two-fold, namely to define biosignatures that could predict vaccine success before challenge, and biomarker patterns that correlated with anamnestic protective responses following exposure to virulent M. bovis. Further, Our aim was to define these markers to be detectable without in vitro antigenic challenge. After BCG vaccination, we defined a specific pulmonary gene expression signature related to the connective tissue development and function network that predicted vaccine success before M. bovis challenge. In addition, a Th17-related cytokine profile was found that correlated with vaccine-induced protective immunity following infection with virulent M. bovis in the lung as well as additional genes that were up-regulated in the spleens of vaccinated animals post-infection that was related to neutrophil biology and inflammation. This study has therefore prioritized both biomarkers predicting vaccination success before challenge and bio-signatures that are associated with protective immune responses that will be useful to evaluate future vaccine candidates.
Project description:Mycobacterial Ser/Thr kinases play a critical role in bacterial physiology and pathogenesis. Linking kinases to the substrates they phosphorylate in vivo, thereby elucidating their exact functions, is still a challenge. The aim of this work was to associate protein phosphorylation in mycobacteria with important subsequent macro cellular events by identifying the physiological substrates of PknG in Mycobacterium bovis BCG. The study compared the phosphoproteome dynamics during the batch growth of M. bovis BGC versus the respective PknG knock-out mutant (ΔPknG-BCG) strains.
Project description:Intralesional therapy using Mycobacterium bovis Bacillus Calmette-Guérin (BCG) for cutaneous metastatic melanoma induces regression of injected, but also non-injected lesions. Tumor-associated macrophages (also known as M2) infiltrate solid tumors and impair antitumor immunity. Since macrophages play a pivotal role in both tumors and mycobacterial infections, we hypothesized BCG alters M2 to promote antitumor immunity. BCG-treated, in vitro-polarized M2 (M2-BCG) show dramatic transcriptional changes involving inflammation, immune cell recruitment, cross-talk and activation pathways. Mechanistic network analysis indicates potential for M2-BCG to improve IFN-γ responses, frequently used as a marker for successful antitumor immunity. Accordingly, we found an increased frequency of IFN-γ-producing CD4+ T cells responding to M2-BCG vs mock-treated M2 (p<0.05). Moreover, supernatant from M2-BCG increased the frequency of granzyme B-producing CD8+ tumor-infiltrating lymphocytes (TIL) responding to autologous melanoma cell lines (p<0.01). Comparing the transcriptome of injected vs uninjected lesions biopsied from IL-BCG patients showed immune function prevailing in injected lesions, with the most enriched pathways representing T cell activation mechanisms. In vitro BCG-infected tumor cells also stimulated IFN-γ production from HLA-A2-restricted syngeneic TIL from the same melanoma patient (p<0.05). Together, our data indicates BCG can promote an antitumor microenvironment in cutaneous metastatic melanoma.
Project description:Global gene expression analysis of Mycobacterium bovis BCG following Triclosan treatment using Affymetrix GeneChip arrays. Results from this study provide insight into the molecular mechanisms underlying the cellular response of Mycobacterium bovis BCG to Triclosan