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

<p>Abstract</p> <p>Background</p> <p>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 <it>M. bovis </it>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.</p> <p>Results</p> <p>The 2DE proteomic map of <it>M. bovis </it>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.</p> <p>Conclusions</p> <p>Here we report the detailed 2DE profile of CFPs from <it>M. bovis </it>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.</p

Adenosine A2A receptor as a potential regulator of Mycobacterium leprae survival mechanisms: new insights into leprosy neural damage

BackgroundLeprosy is a chronic infectious disease caused by Mycobacterium leprae, which can lead to a disabling neurodegenerative condition. M. leprae preferentially infects skin macrophages and Schwann cells–glial cells of the peripheral nervous system. The infection modifies the host cell lipid metabolism, subverting it in favor of the formation of cholesterol-rich lipid droplets (LD) that are essential for bacterial survival. Although researchers have made progress in understanding leprosy pathogenesis, many aspects of the molecular and cellular mechanisms of host–pathogen interaction still require clarification. The purinergic system utilizes extracellular ATP and adenosine as critical signaling molecules and plays several roles in pathophysiological processes. Furthermore, nucleoside surface receptors such as the adenosine receptor A2AR involved in neuroimmune response, lipid metabolism, and neuron–glia interaction are targets for the treatment of different diseases. Despite the importance of this system, nothing has been described about its role in leprosy, particularly adenosinergic signaling (AdoS) during M. leprae–Schwann cell interaction.MethodsM. leprae was purified from the hind footpad of athymic nu/nu mice. ST88-14 human cells were infected with M. leprae in the presence or absence of specific agonists or antagonists of AdoS. Enzymatic activity assays, fluorescence microscopy, Western blotting, and RT-qPCR analysis were performed. M. leprae viability was investigated by RT-qPCR, and cytokines were evaluated by enzyme-linked immunosorbent assay.ResultsWe demonstrated that M. leprae-infected Schwann cells upregulated CD73 and ADA and downregulated A2AR expression and the phosphorylation of the transcription factor CREB (p-CREB). On the other hand, activation of A2AR with its selective agonist, CGS21680, resulted in: 1) reduced lipid droplets accumulation and pro-lipogenic gene expression; 2) reduced production of IL-6 and IL-8; 3) reduced intracellular M. leprae viability; 4) increased levels of p-CREB.ConclusionThese findings suggest the involvement of the AdoS in leprosy neuropathogenesis and support the idea that M. leprae, by downmodulating the expression and activity of A2AR in Schwann cells, decreases A2AR downstream signaling, contributing to the maintenance of LD accumulation and intracellular viability of the bacillus

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

Submitted by Sandra Infurna ([email protected]) on 2019-09-03T12:10:25Z No. of bitstreams: 1 TalitaPagani_LeilaMLima_etal_IOC_2019.pdf: 2490553 bytes, checksum: bec0e4e17098191a9db8299c1d950da1 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2019-09-03T12:32:35Z (GMT) No. of bitstreams: 1 TalitaPagani_LeilaMLima_etal_IOC_2019.pdf: 2490553 bytes, checksum: bec0e4e17098191a9db8299c1d950da1 (MD5)Made available in DSpace on 2019-09-03T12:32:35Z (GMT). No. of bitstreams: 1 TalitaPagani_LeilaMLima_etal_IOC_2019.pdf: 2490553 bytes, checksum: bec0e4e17098191a9db8299c1d950da1 (MD5) Previous issue date: 2019Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Genômica Funcional e Bioinformática. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Genômica Funcional e Bioinformática. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Genômica Funcional e Bioinformática. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Genômica Funcional e Bioinformática. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório Interdisciplinar de Pesquisas Médicas. Rio de Janeiro, RJ, Brasil / Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica Leopoldo de Meis. Unidade de Espectometria de Massas e Proteômica. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Microbiologia Celular. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Genômica Funcional e Bioinformática. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Genômica Funcional e Bioinformática. Rio de Janeiro, RJ, Brasil.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

Exploring the "Latin American Mediterranean" family and the RDRio lineage in Mycobacterium tuberculosis isolates from Paraguay, Argentina and Venezuela

Submitted by Sandra Infurna ([email protected]) on 2020-03-28T17:15:58Z No. of bitstreams: 1 HarrrisonGomes_SidraVasconcelos_etal_IOC_2019.pd.pdf: 1746618 bytes, checksum: 5564ed65983e850073abda10c0053dea (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2020-03-28T17:43:14Z (GMT) No. of bitstreams: 1 HarrrisonGomes_SidraVasconcelos_etal_IOC_2019.pd.pdf: 1746618 bytes, checksum: 5564ed65983e850073abda10c0053dea (MD5)Made available in DSpace on 2020-03-28T17:43:14Z (GMT). No. of bitstreams: 1 HarrrisonGomes_SidraVasconcelos_etal_IOC_2019.pd.pdf: 1746618 bytes, checksum: 5564ed65983e850073abda10c0053dea (MD5) Previous issue date: 2019Universidad Nacional de Asunción. Instituto de Investigaciones en Ciencias de la Salud. Departamento de Biología Molecular y Biotecnología. Asunción, Paraguay / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular aplicada às Micobactérias. Rio de Janeiro, RJ, Brasil.Universidad Nacional de Asunción. Instituto de Investigaciones en Ciencias de la Salud. Departamento de Biología Molecular y Biotecnología. Asunción, Paraguay.Universidad Nacional de Asunción. Instituto de Investigaciones en Ciencias de la Salud. Departamento de Biología Molecular y Biotecnología. Asunción, Paraguay.Instituto Nacional de Enfermedades Infecciosas, ANLIS “Carlos G. Malbran”. Servicio de Micobacterias. Buenos Aires. Argentina.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular aplicada às Micobactérias. Rio de Janeiro, RJ, Brasil..Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Microbiologia Celular, Rio de Janeiro, RJ, Brasil.Laboratorio Central de Salud Pública, MSP y BS. Asunción, Paraguay.Instituto Nacional de Enfermedades Respiratorias Emilio Coni. Buenos Aires, Argentina.Instituto de Biomedicina. Laboratorio de Tuberculosis. Caracas, Venezuela / Universidad de Las Américas Facultad de Ciencias de la Salud. One Health Research Group. Quito, Ecuador.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular aplicada às Micobactérias. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular aplicada às Micobactérias. Rio de Janeiro, RJ, Brasil.The Latin American & Mediterranean (LAM) spoligotype family is one of the most successful genotype of Mycobacterium tuberculosis worldwide and particularly prevalent in South-America. Within this family, a sublineage named Region of Difference Rio (RDRio) was reported initially in Brazil and is characterized by a genomic deletion of about 26.3 kb. This lineage seems to show a specific adaptation to the Euro-Latin American population. In this context, we sought to evaluate the LAM family and the presence of the RDRio genotype in samples from three Latin American countries including Paraguay, Venezuela and Argentina. To detect LAM strains reliably we applied a typing scheme using spoligotyping, 12 loci MIRU-VNTR, the Ag85C103 SNP and the regions of difference RDRio and RD174. IS6110-RFLP results were also used when available

Kinetoplastid membrane protein-11 exacerbates infection with Leishmania amazonensis in murine macrophages

In Leishmania amazonensis, kinetoplastid membrane protein-11 (KMP-11) expression increases during metacyclogenesis and is higher in amastigotes than in promastigotes, suggesting a role for this protein in the infection of the mammalian host. We show that the addition of KMP-11 exacerbates L. amazonensis infection in peritoneal macrophages from BALB/c mice by increasing interleukin (IL)-10 secretion and arginase activity while reducing nitric oxide (NO) production. The doses of KMP-11, the IL-10 levels and the intracellular amastigote loads were strongly, positively and significantly correlated. The increase in parasite load induced by KMP-11 was inhibited by anti-KMP-11 or anti-IL-10 neutralising antibodies, but not by isotype controls. The neutralising antibodies, but not the isotype controls, were also able to significantly decrease the parasite load in macrophages cultured without the addition of KMP-11, demonstrating that KMP-11-induced exacerbation of the infection is not dependent on the addition of exogenous KMP-11 and that the protein naturally expressed by the parasite is able to promote it. In this study, the exacerbating effect of KMP-11 on macrophage infection with Leishmania is for the first time demonstrated, implicating it as a virulence factor in L. amazonensis. The stimulation of IL-10 production and arginase activity and the inhibition of NO synthesis are likely involved in this effect

The essential role of cholesterol metabolism in the intracellular survival of Mycobacterium 2 leprae is not coupled to central carbon metabolism and energy production

Submitted by sandra infurna ([email protected]) on 2016-05-17T16:02:04Z No. of bitstreams: 1 mariaangela_marques_etal_IOC_2015.pdf: 1511352 bytes, checksum: 4396e5f1e7be4b619876fe93a31f911a (MD5)Approved for entry into archive by sandra infurna ([email protected]) on 2016-05-17T16:50:23Z (GMT) No. of bitstreams: 1 mariaangela_marques_etal_IOC_2015.pdf: 1511352 bytes, checksum: 4396e5f1e7be4b619876fe93a31f911a (MD5)Made available in DSpace on 2016-05-17T16:50:23Z (GMT). No. of bitstreams: 1 mariaangela_marques_etal_IOC_2015.pdf: 1511352 bytes, checksum: 4396e5f1e7be4b619876fe93a31f911a (MD5) Previous issue date: 2015Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Microbiologia Celular. Rio de Janeiro, RJ, Brasil / Colorado State University. Department of Microbiology, Immunology and Pathology. Fort Collins, CO, USA.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Microbiologia Celular. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Microbiologia Celular. Rio de Janeiro, RJ, BrasilColorado State University. Department of Microbiology, Immunology and Pathology. Fort Collins, CO, USAFundação Oswaldo Cruz.de Microbiologia Celular. Rio de Janeiro, RJ, Brasil Instituto Oswaldo Cruz. LaboratórioFundação Oswaldo Cruz.de Microbiologia Celular. Rio de Janeiro, RJ, Brasil Instituto Oswaldo Cruz. LaboratórioColorado State University. Department of Microbiology, Immunology and Pathology. Fort Collins, CO, USAFundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Genômica Funcional e Bioinformática. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica. Laboratório de Bioquímica de Lipídeos e Lipoproteínas. Rio de Janeiro, RJ, Brasil.Colorado State University. Department of Microbiology, Immunology and Pathology. Fort Collins, CO, USAColorado State University. Department of Microbiology, Immunology and Pathology. Fort Collins, CO, USAColorado State University. Department of Microbiology, Immunology and Pathology. Fort Collins, CO, USAColorado State University. Department of Microbiology, Immunology and Pathology. Fort Collins, CO, USAFundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Microbiologia Celular. Rio de Janeiro, RJ, Brasil.Mycobacterium leprae induces the formation of lipid droplets, which are recruited to pathogen-containing phagosomes in infected macrophages and Schwann cells. Cholesterol is among the lipids with increased abundance in M. leprae-infected cells, and intracellular survival relies on cholesterol accumulation. The present study investigated the capacity of M. leprae to acquire and metabolize cholesterol. In silico analyses showed that oxidation of cholesterol to cholest-4-en-3-one (cholestenone), the first step of cholesterol degradation catalyzed by the enzyme 3β-hydroxysteroid dehydrogenase (3β-HSD), is apparently the only portion of the cholesterol catabolic pathway seen in Mycobacterium tuberculosis preserved by M. leprae. Incubation of bacteria with radiolabeled cholesterol confirmed the in silico predictions. Radiorespirometry and lipid analyses performed after incubating M. leprae with [4-(14)C]cholesterol or [26-(14)C]cholesterol showed the inability of this pathogen to metabolize the sterol rings or the side chain of cholesterol as a source of energy and carbon. However, the bacteria avidly incorporated cholesterol and, as expected, converted it to cholestenone both in vitro and in vivo. Our data indicate that M. leprae has lost the capacity to degrade and utilize cholesterol as a nutritional source but retains the enzyme responsible for its oxidation to cholestenone. Thus, the essential role of cholesterol metabolism in the intracellular survival of M. leprae is uncoupled from central carbon metabolism and energy production. Further elucidation of cholesterol metabolism in the host cell during M. leprae infection will establish the mechanism by which this lipid supports M. leprae intracellular survival and will open new avenues for novel leprosy therapies. Our study focused on the obligate intracellular pathogen Mycobacterium leprae and its capacity to metabolize cholesterol. The data make an important contribution for those interested in understanding the mechanisms of mycobacterial pathogenesis, since they indicate that the essential role of cholesterol for M. leprae intracellular survival does not rely on its utilization as a nutritional source. Our findings reinforce the complexity of cholesterol's role in sustaining M. leprae infection. Further elucidation of cholesterol metabolism in the host cell during M. leprae infection will establish the mechanism by which this lipid supports M. leprae intracellular survival and will open new avenues for novel leprosy therapies

Reductive Power Generated by Mycobacterium leprae Through Cholesterol Oxidation Contributes to Lipid and ATP Synthesis

&lt;jats:p&gt;Upon infection, &lt;jats:italic&gt;Mycobacterium leprae&lt;/jats:italic&gt;, an obligate intracellular bacillus, induces accumulation of cholesterol-enriched lipid droplets (LDs) in Schwann cells (SCs). LDs are promptly recruited to &lt;jats:italic&gt;M. leprae&lt;/jats:italic&gt;-containing phagosomes, and inhibition of this process decreases bacterial survival, suggesting that LD recruitment constitutes a mechanism by which host-derived lipids are delivered to intracellular &lt;jats:italic&gt;M. leprae&lt;/jats:italic&gt;. We previously demonstrated that &lt;jats:italic&gt;M. leprae&lt;/jats:italic&gt; has preserved only the capacity to oxidize cholesterol to cholestenone, the first step of the normal cholesterol catabolic pathway. In this study we investigated the biochemical relevance of cholesterol oxidation on bacterial pathogenesis in SCs. Firstly, we showed that &lt;jats:italic&gt;M. leprae&lt;/jats:italic&gt; increases the uptake of LDL-cholesterol by infected SCs. Moreover, fluorescence microscopy analysis revealed a close association between &lt;jats:italic&gt;M. leprae&lt;/jats:italic&gt; and the internalized LDL-cholesterol within the host cell. By using &lt;jats:italic&gt;Mycobacterium smegmatis&lt;/jats:italic&gt; mutant strains complemented with &lt;jats:italic&gt;M. leprae&lt;/jats:italic&gt; genes, we demonstrated that &lt;jats:italic&gt;ml1942&lt;/jats:italic&gt; coding for 3β-hydroxysteroid dehydrogenase (3β-HSD), but not &lt;jats:italic&gt;ml0389&lt;/jats:italic&gt; originally annotated as cholesterol oxidase (ChoD), was responsible for the cholesterol oxidation activity detected in &lt;jats:italic&gt;M. leprae&lt;/jats:italic&gt;. The 3β-HSD activity generates the electron donors NADH and NADPH that, respectively, fuel the &lt;jats:italic&gt;M. leprae&lt;/jats:italic&gt; respiratory chain and provide reductive power for the biosynthesis of the dominant bacterial cell wall lipids phthiocerol dimycocerosate (PDIM) and phenolic glycolipid (PGL)-I. Inhibition of &lt;jats:italic&gt;M. leprae&lt;/jats:italic&gt; 3β-HSD activity with the 17β-[N-(2,5-di-t-butylphenyl)carbamoyl]-6-azaandrost-4-en-3one (compound 1), decreased bacterial intracellular survival in SCs. In conclusion, our findings confirm the accumulation of cholesterol in infected SCs and its potential delivery to the intracellular bacterium. Furthermore, we provide strong evidence that cholesterol oxidation is an essential catabolic pathway for &lt;jats:italic&gt;M. leprae&lt;/jats:italic&gt; pathogenicity and point to 3β-HSD as a prime drug target that may be used in combination with current multidrug regimens to shorten leprosy treatment and ameliorate nerve damage.&lt;/jats:p&gt