Differences in responses to the intracellular macrophage environment between Mycobacterium bovis BCG vaccine strains Moreau and Pasteur.

BACKGROUND: The Bacille Calmette-Guérin (BCG) vaccine comprises a family of strains with variable protective efficacy against pulmonary tuberculosis (TB) and leprosy, partly due to genetic differences between strains. OBJECTIVES: Previous data highlighting differences between the genomes and proteomic profiles of BCG strains Moreau and Pasteur led us to evaluate their behaviour in the macrophage microenvironment, capable of stimulating molecular responses that can impact the protective effect of the vaccine. METHODS: Strain infectivity, viability, co-localisation with acidified vesicles, macrophage secretion of IL-1 and MCP-1 and lipid droplet biogenesis were evaluated after infection. FINDINGS: We found that BCG Moreau is internalised more efficiently, with significantly better intracellular survival up to 96 h p.i., whereas more BCG Pasteur bacilli were found co-localised in acidified vesicles up to 6 h p.i. IL-1ß and MCP-1 secretion and lipid droplet biogenesis by infected macrophages were more prominent in response to BCG Pasteur. MAIN CONCLUSION: Overall, our results show that, compared to Pasteur, BCG Moreau has increased fitness and better endurance in the harsh intracellular environment, also regulating anti-microbial responses (lower IL-1b and MCP-1). These findings contribute to the understanding of the physiology of BCG Moreau and Pasteur in response to the intraphagosomal environment in a THP-1 macrophage model.

Transcriptional Profiling of Homologous Recombination Pathway Genes in Mycobacterium bovis BCG Moreau.

Mycobacterium bovis BCG Moreau is the main Brazilian strain for vaccination against tuberculosis. It is considered an early strain, more like the original BCG, whereas BCG Pasteur, largely used as a reference, belongs to the late strain clade. BCG Moreau, contrary to Pasteur, is naturally deficient in homologous recombination (HR). In this work, using a UV exposure test, we aimed to detect differences in the survival of various BCG strains after DNA damage. Transcription of core and regulatory HR genes was further analyzed using RT-qPCR, aiming to identify the molecular agent responsible for this phenotype. We show that early strains share the Moreau low survival rate after UV exposure, whereas late strains mimic the Pasteur phenotype, indicating that this increase in HR efficiency is linked to the evolutionary clade history. Additionally, RT-qPCR shows that BCG Moreau has an overall lower level of these transcripts than Pasteur, indicating a correlation between this gene expression profile and HR efficiency. Further assays should be performed to fully identify the molecular mechanism that may explain this differential phenotype between early and late BCG strains.

Mycobacterium bovis BCG dodecin gene codes a functional protein despite of a start codon mutation.

Dodecin is a dodecamer involved in flavin homeostasis, with interesting temperature and osmolarity endurance features in Mycobacterium tuberculosis. A single nucleotide polymorphism in the gene's start codon in BCG, converting ATG to ACG, is predicted to generate a N-terminal shorter isoform, lacking the first 7 amino acids. We previously reported that the shortened recombinant protein has reduced extremophilic features. Here we investigate if within the mycobacterial context dodecin can be produced from both alleles, carrying ATG and ACG start codons. Reporter gene assays using mcherry cloned downstream and in phase to both M.tb and BCG "upstream" regions confirms production of functional proteins. Complementation with both dod alleles similarly enhances M. smegmatis growth after entry into logarithmic phase and exposure to hydrogen peroxide, possibly implicating this protein in oxidative stress response mechanisms. Altogether these data indicate that BCG dodecin is indeed produced, notwithstanding in lower levels compared to M.tb, conferring similar phenotypes, even with the SNP altering the M.tb ATG start codon to the BCG ACG. This protein might be an interesting drug target for the development of new therapeutics against tuberculosis.

Impact of Genomic Deletion RD16 on the Expression of the Mycobacterium bovis BCG Moreau VapBC47 Toxin-Antitoxin System.

Mycobacterium bovis BCG is the only vaccine against tuberculosis. The variable forms of cultivation throughout the years, before seed-lots were developed, allowed in vitro evolution of the original strain, generating a family of vaccines with different phenotypic and genotypic characteristics. Molecular studies revealed regions of difference (RDs) in the genomes of the various BCG strains. This work aims to characterize the gene pair rv3407-rv3408 (vapB47-vapC47), coding for a toxin-antitoxin system of the VapBC family, and to evaluate possible transcriptional effects due to the adjacent BCG Moreau-specific genomic deletion RD16. We show that these genes are co-transcribed in BCG strains Moreau and Pasteur, and that the inactivation of an upstream transcriptional repressor (Rv3405c) due to RD16 has a polar effect, leading to increased vapBC47 expression. Furthermore, we detect VapB47 DNA binding in vitro, dependent on a 5' vapB47 sequence that contributes to a palindrome, spanning the promoter and coding region. Our data shed light on the regulation of VapBC systems and on the impact of the BCG Moreau RD16 deletion in the expression of adjacent genes, contributing to a better understanding of BCG Moreau physiology.

Differences in responses to the intracellular macrophage environment between Mycobacterium bovis BCG vaccine strains Moreau and Pasteur

BACKGROUND The Bacille Calmette-Guérin (BCG) vaccine comprises a family of strains with variable protective efficacy against pulmonary tuberculosis (TB) and leprosy, partly due to genetic differences between strains. OBJECTIVES Previous data highlighting differences between the genomes and proteomic profiles of BCG strains Moreau and Pasteur led us to evaluate their behaviour in the macrophage microenvironment, capable of stimulating molecular responses that can impact the protective effect of the vaccine. METHODS Strain infectivity, viability, co-localisation with acidified vesicles, macrophage secretion of IL-1 and MCP-1 and lipid droplet biogenesis were evaluated after infection. FINDINGS We found that BCG Moreau is internalised more efficiently, with significantly better intracellular survival up to 96 h p.i., whereas more BCG Pasteur bacilli were found co-localised in acidified vesicles up to 6 h p.i. IL-1β and MCP-1 secretion and lipid droplet biogenesis by infected macrophages were more prominent in response to BCG Pasteur. MAIN CONCLUSION Overall, our results show that, compared to Pasteur, BCG Moreau has increased fitness and better endurance in the harsh intracellular environment, also regulating anti-microbial responses (lower IL-1b and MCP-1). These findings contribute to the understanding of the physiology of BCG Moreau and Pasteur in response to the intraphagosomal environment in a THP-1 macrophage model.

¿Qué es la Medicina del Estilo de Vida y por qué la necesitamos? / What is Lifestyle Medicine and why do we need it?

Medicina del Estilo de Vida (MEV), se define como la práctica basada en la evidencia, de asistir a individuos y familias en la adopción y mantención de conductas que mejoran la salud y calidad de vida, tales como alimentación saludable, realización de actividad física periódica, sueño reparador, manejo del estrés, cese del uso de sustancias tóxicas y una sólida red de apoyo social. Esta disciplina de la medicina, ha demostrado ser efectiva en la prevención, manejo y a veces reversión de las patologías que conllevan la mayor morbimortalidad global, tales como hipertensión arterial, diabetes mellitus tipo 2, enfermedad coronaria y obesidad. Es más, se estima que el 80% de las enfermedades crónicas no transmisibles podrían prevenirse llevando un estilo de vida más saludable. Ciertas barreras estructurales han hecho que la incorporación de la MEV en las mallas curriculares universitarias y establecimientos de salud sea más lenta de lo esperado, sin embargo, cada vez son más las instituciones académicas y prestadoras de salud que adoptan los principios de la MEV, y la aparición de sociedades médicas relacionadas a esta disciplina en casi todos los continentes, están acelerando el paso hacia una medicina más focalizada en tratar las causas de la enfermedad, en lugar de centrarse en lo sintomático

Lifestyle Medicine (LM) is the evidence based practice of assisting individuals and families to adopt and sustain behaviors that can improve health and quality of life. These include healthy diet, participating in regular physical activity, having good quality sleep, managing stress, avoiding risky substance abuse and building strong social connections. LM has demonstrated its effectiveness at preventing, managing and sometimes reversing the diseases that globally carry the biggest morbidity and mortality burden, such as hypertension, type 2 diabetes mellitus, coronary artery disease and obesity. More so, it is estimated that 80% of non-communicable chronic diseases could be avoided by living a healthier lifestyle. Certain structural barriers have made LM's incorporation into the medical curriculum and clinical practice slower than expected, however, more and more academic institutions and healthcare providers are adopting LM's principles. The appearance of medical associations related to this discipline in almost every continent is accelerating the pace towards a medicine that is more centered on the root-causes of disease, rather than focusing on symptoms

M. bovis BCG Moreau N-Terminal Loss Leads to a Less Stable Dodecin With Lower Flavin Binding Capacity.

Tuberculosis still remains a concerning health problem worldwide. Its etiologic agent, Mycobacterium tuberculosis, continues to be the focus of research to unravel new prophylactic and therapeutic strategies against this disease. The only vaccine in use against tuberculosis is based on the in vitro attenuated strain, M. bovis BCG. Dodecin is a dodecameric complex important for flavin homeostasis in Archea and Eubacteria, and the M. tuberculosis protein is described as thermo- and halostable. M. bovis BCG Moreau, the Brazilian vaccine strain, has a single nucleotide polymorphism in the dodecin start codon, leading to a predicted loss of seven amino acids at the protein N-terminal end. In this work we aimed to characterize the effect of this mutation in the BCG Moreau protein features. Our recombinant protein assays show that the predicted BCG homolog is less thermostable than M.tb's but maintains its dodecamerization ability, although with a lower riboflavin-binding capacity. These data are corroborated by structural analysis after comparative modeling, showing that the predicted BCG dodecin complex has a lower interaction energy among its monomers and also a distinct electrostatic surface near the flavin binding pocket. However, western blotting assays with the native proteins were unable to detect significant differences between the BCG Moreau and M.tb orthologs, indicating that other factors may be modulating protein structure/function in the bacterial context.

Insights into the proteomic profile and gene expression of Lutzomyia longipalpis-derived Lulo cell line.

BACKGROUND: Lutzomyia longipalpis-derived cell line (Lulo) has been suggested as a model for studies of interaction between sandflies and Leishmania. OBJECTIVES: Here, we present data of proteomic and gene expression analyses of Lulo cell related to interactions with Leishmania (Viannia) braziliensis. METHODS: Lulo cell protein extracts were analysed through a combination of two-dimensional gel electrophoresis and mass spectrometry and resulting spots were further investigated in silico to identify proteins using Mascot search and, afterwards, resulting sequences were applied for analysis with VectorBase. RESULTS: Sixty-four spots were identified showing similarities to other proteins registered in the databases and could be classified according to their biological function, such as ion-binding proteins (23%), proteases (14%), cytoskeletal proteins (11%) and interactive membrane proteins (9.5%). Effects of interaction with L. (V.) braziliensis with the expression of three genes (enolase, tubulin and vacuolar transport protein) were observed after an eight-hour timeframe and compared to culture without parasites, and demonstrated the impact of parasite interaction with the expression of the following genes: LLOJ000219 (1.69-fold), LLOJ000326 (1.43-fold) and LLOJ006663 (2.41-fold). CONCLUSIONS: This set of results adds relevant information regarding the usefulness of the Lulo cell line for studies with Leishmania parasites that indicate variations of protein expression.

Mycobacterium bovis BCG moreau is naturally deficient in homologous recombination.

The ability to perform genetic manipulation of mycobacteria is important for characterization of gene function. Homologous recombination-based protocols are frequently used for reverse genetics studies with mycobacteria. It is known that Mycobacteriumbovis BCG Russia, closely related to M. bovis BCG Moreau, is a natural recA deficient strain and is non-permissive to homologous recombination assays. In this work we show that M. bovis BCG Moreau is also deficient in homologous recombination, shown by a specialized transduction assay, but this phenotype can be reverted by complementation with heterologous recombinases, using a recombineering protocol. Sequence analysis of the genes known to be involved in homologous recombination annotated in the genome of BCG Moreau detected no differences compared to the genome of BCG Pasteur. Further studies are needed in order to determine the exact mechanism underlying this deficiency in BCG Moreau.

Mycobacterium tuberculosis and M. bovis BCG Moreau Fumarate Reductase Operons Produce Different Polypeptides That May Be Related to Non-canonical Functions.

Tuberculosis is a world widespread disease, caused by Mycobacterium tuberculosis (M.tb). Although considered an obligate aerobe, this organism can resist life-limiting conditions such as microaerophily mainly due to its set of enzymes responsible for energy production and coenzyme restoration under these conditions. One of these enzymes is fumarate reductase, an heterotetrameric complex composed of a catalytic (FrdA), an iron-sulfur cluster (FrdB) and two transmembrane (FrdC and FrdD) subunits involved in anaerobic respiration and important for the maintenance of membrane potential. In this work, aiming to further characterize this enzyme function in mycobacteria, we analyzed the expression of FrdB-containing proteins in M.tb and Mycobacterium bovis Bacillus Calmette-Guérin (BCG) Moreau, the Brazilian vaccine strain against tuberculosis. We identified three isoforms in both mycobacteria, two of them corresponding to the predicted encoded polypeptides of M.tb (27 kDa) and BCG Moreau (40 kDa) frd sequences, as due to an insertion on the latter's operon a fused FrdBC protein is expected. The third 52 kDa band can be explained by a transcriptional slippage event, typically occurring when mutation arises in a repetitive region within a coding sequence, thought to reduce its impact allowing the production of both native and variant forms. Comparative modeling of the M.tb and BCG Moreau predicted protein complexes allowed the detection of subtle overall differences, showing a high degree of structure and maybe functional resemblance among them. Axenic growth and macrophage infection assays show that the frd locus is important for proper bacterial development in both scenarios, and that both M.tb's and BCG Moreau's alleles can partially revert the hampered phenotype of the knockout strain. Altogether, our results show that the frdABCD operon of Mycobacteria may have evolved to possess other yet non-described functions, such as those necessary during aerobic logarithmic growth and early stage steps of infection.

Exploring the Potential Role of Moonlighting Function of the Surface-Associated Proteins From Mycobacterium bovis BCG Moreau and Pasteur by Comparative Proteomic.

Surface-associated proteins from Mycobacterium bovis BCG Moreau RDJ are important components of the live Brazilian vaccine against tuberculosis. They are important targets during initial BCG vaccine stimulation and modulation of the host's immune response, especially in the bacterial-host interaction. These proteins might also be involved in cellular communication, chemical response to the environment, pathogenesis processes through mobility, colonization, and adherence to the host cell, therefore performing multiple functions. In this study, the proteomic profile of the surface-associated proteins from M. bovis BCG Moreau was compared to the BCG Pasteur reference strain. The methodology used was 2DE gel electrophoresis combined with mass spectrometry techniques (MALDI-TOF/TOF), leading to the identification of 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. Moonlighting proteins are multifunctional proteins in which two or more biological functions are fulfilled by a single polypeptide chain. Therefore, the identification of such proteins with moonlighting predicted functions can contribute to a better understanding of the molecular mechanisms unleashed by live BCG Moreau RDJ vaccine components.

Gain of function in Mycobacterium bovis BCG Moreau due to loss of a transcriptional repressor.

The Bacille Calmette-Guérin (BCG) vaccine comprises a family of genetically different strains derived by the loss of genomic regions (RDs) and other mutations. In BCG Moreau, loss of RD16 inactivates rv3405c * , encoding a transcriptional repressor that negatively regulates the expression of Rv3406, an alkyl sulfatase. To evaluate the impact of this loss on the BCG and host cell viability and the cytokine profile, THP-1 cells were infected with BCG Moreau (harbouring the empty vector) and a complemented strain carrying a functional copy of rv3405c. Viability of the host cells and bacteria as well as the pattern of cytokine secretion were evaluated. Our results show that the viability of BCG Moreau is higher than that of the complemented strain in an axenic medium, suggesting a possible functional gain associated with the constitutive expression of Rv3406. Viability of the host cells did not vary significantly between recombinant strains, but differences in the profiles of the cytokine secretion (IL-1ß and IL-6) were observed. Our results suggest an example of a functional gain due to gene loss contributing to the elucidation of the impact of RD16 on the physiology of BCG Moreau.

The BCG Moreau RD16 deletion inactivates a repressor reshaping transcription of an adjacent gene.

The Brazilian anti-tuberculosis vaccine strain Mycobacterium bovis bacillus Calmette-Guérin (BCG) BCG Moreau is unique in having a deletion of 7608 bp (RD16) that results in the truncation of a putative TetR transcriptional regulator, the ortholog of Mycobacterium tuberculosis rv3405c, BCG_M3439c. We investigated the effect of this truncation on the expression of the rv3406 ortholog (BCG_M3440), lying 81 bp downstream in the opposite orientation. RT-PCR and western blot experiments show that rv3406 mRNA and Rv3406 accumulate in BCG Moreau but not in BCG Pasteur (strain that bears an intact rv3405c), suggesting this to be a result of rv3405c truncation. Recombinant Rv3405c forms a complex with the rv3405c-rv3406 intergenic region, which contains a characteristic transcription factor binding site, showing it to have DNA binding activity. Complementation of M. bovis BCG Moreau with an intact copy of rv3405c abolishes Rv3406 accumulation. These results show that Rv3405c is a DNA binding protein that acts as a transcriptional repressor of rv3406.

Caracterização da resposta de mycobacterium bovis BCG Moreau e BCG Pasteur ao estresse oxidativo / Characterization of the response of mycobacterium bovis BCG Moreau and BCG Pasteur to oxidative stress

A vacina BCG (Bacilo de Calmette-Guèrin), única medida profilática contra a tuberculose (TB), foi obtida no início do século XX por Calmette e Guèrin após 231 passagens de um isolado clínico de M. bovis cultivado em meio glicerinado contendo bile bovina. Sua eficácia protetora contra a TB pulmonar em adultos varia de 0-80% e as diferenças genéticas entre as diversas cepas vacinais utilizadas mundialmente contribuem para esta variação. A cepa vacinal brasileira, BCG Moreau, é considerada uma cepa primitiva, mais próxima do BCG original, quando comparada às cepas mais recentes, como BCG Pasteur (cepa de referência). Dados prévios indicaram um perfil proteômico bidimensional diferencial entre a cepa brasileira e a de referência, quando cultivadas em meio Sauton, sem agitação (condição de produção da vacina). Essas proteínas (PepA, DnaK, GroEL2, AhpC, HspX, BCG2993 e GroES) foram identificadas e pertencem ao grupo de proteínas que respondem a diferentes tipos de estresse. A partir desses dados optamos por investigar o estresse oxidativo, com o objetivo de caracterizar o perfil de resposta da cepa BCG Moreau, comparado ao da cepa de referência. Para isso, selecionamos dois agentes oxidantes: peróxido de hidrogênio (H2O2) e diamida. A fração protéica intracelular foi resolvida em eletroforese bidimensional (2D) (pH 4-7) para confecção de mapas protéicos de alta resolução; o perfil transcricional dos genes codificantes para as proteínas selecionadas foi avaliado por PCR em tempo real (qRT-PCR) e a expressão proteica foi quantificada por western-blot empregando anticorpos policlonais produzidos a partir da forma recombinante destas proteínas (HspX, AhpC e BCG2993) expressas em E. coli. Na condição de cultivo para produção da vacina, o proteoma 2D revelou a maior expressão intracelular das proteínas PepA, HspX, AhpC e GroES na cepa BCG Moreau. Enquanto que as proteínas DnaK, GroEL2 e BCG2993 foram mais expressas na cepa BCG Pasteur. A comparação do meio vacinal com o meio 7H9 revelou que as proteínas PepA e DnaK estão sendo diferentemente expressas nesses meios. Os ensaios com diamida e H2O2 revelaram a ausência do transcrito para o gene hspX e consequentemente da proteína (2D e WB). A análise transcricional revelou que o agente oxidativo H2O2 modulou todos os genes em estudo, enquanto que o composto diamida só não alterou a transcrição do gene ahpC. Os resultados da análise proteômica mostram que as proteínas selecionadas foram moduladas de forma diferente pelos agentes oxidantes e que o comportamento diferencial entre a cepa Moreau e Pasteur foi evidenciado na condição com peróxido de hidrogênio. Estes resultados contribuem para a caracterização molecular da resposta ao estresse da cepa BCG Moreau comparada à cepa BCG Pasteur, permitindo uma melhor compreensão da cepa vacinal brasileira contra a TB.

Proteomic profile of culture filtrate from the Brazilian vaccine strain Mycobacterium bovis BCG Moreau compared to M. bovis BCG Pasteur.

BACKGROUND: Bacille Calmette-Guerin (BCG) is currently the only available vaccine against tuberculosis (TB) and comprises a heterogeneous family of sub-strains with genotypic and phenotypic differences. The World Health Organization (WHO) affirms that the characterization of BCG sub-strains, both on genomic and proteomic levels, is crucial for a better comprehension of the vaccine. In addition, these studies can contribute in the development of a more efficient vaccine against TB. Here, we combine two-dimensional electrophoresis (2DE) and mass spectrometry to analyse the proteomic profile of culture filtrate proteins (CFPs) from M. bovis BCG Moreau, the Brazilian vaccine strain, comparing it to that of BCG Pasteur. CFPs are considered of great importance given their dominant immunogenicity and role in pathogenesis, being available for interaction with host cells since early infection. RESULTS: The 2DE proteomic map of M. bovis BCG Moreau CFPs in the pH range 3-8 allowed the identification of 158 spots corresponding to 101 different proteins, identified by MS/MS. Comparison to BCG Pasteur highlights the great similarity between these BCG strains. However, quantitative analysis shows a higher expression of immunogenic proteins such as Rv1860 (BCG1896, Apa), Rv1926c (BCG1965c, Mpb63) and Rv1886c (BCG1923c, Ag85B) in BCG Moreau when compared to BCG Pasteur, while some heat shock proteins, such as Rv0440 (BCG0479, GroEL2) and Rv0350 (BCG0389, DnaK), show the opposite pattern. CONCLUSIONS: Here we report the detailed 2DE profile of CFPs from M. bovis BCG Moreau and its comparison to BCG Pasteur, identifying differences that may provide relevant information on vaccine efficacy. These findings contribute to the detailed characterization of the Brazilian vaccine strain against TB, revealing aspects that may lead to a better understanding of the factors leading to BCG's variable protective efficacy against TB.