RESUMO
BACKGROUND: Control of Tuberculosis (TB) infection is mainly the result of productive teamwork between T-cell populations and antigen presenting cells (APCs). However, APCs activation at the site of initiating cellular immune response during BCG early infection is not completely understood. METHODS: In this study, we injected C57BL/6 mice in intravenous (i.v) or subcutaneous (s.c) route, then splenic or inguinal lymph node (LN) DCs and MΦs were sorted, and mycobacteria uptake, cytokine production, antigen presentation activity, and cell phenotype were investigated and compared, respectively. RESULTS: Ag85A-specific T-cell immune response began at 6 days post BCG infection, when BCG was delivered in s.c route, Th17 immune response could be induced in inguinal LN. BCG could induce high level of activation phenotype in inguinal LN MΦs, while the MHC II presentation of mycobacteria-derived peptides by DCs was more efficient than MΦs. CONCLUSIONS: The results showed that BCG immunized route can decide the main tissue of T-cell immune response. Compared with s.c injected route, APCs undergo more rapid cell activation in spleen post BCG i.v infection.
Assuntos
Mycobacterium bovis , Tuberculose , Camundongos , Animais , Camundongos Endogâmicos C57BL , Células Apresentadoras de Antígenos , Linfócitos T , Vacina BCGRESUMO
BACKGROUND: Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), and its pathogenicity is associated with its ability to evade the host defense system. The secretory form of the chorismate mutase of M. tuberculosis (TBCM, encoded by Rv1885c) is assumed to play a key role in the pathogenesis of TB; however, the mechanism remains unknown. METHODS: A tbcm deletion mutant (B∆tbcm) was generated by targeted gene knockout in BCG to investigate the pathogenic role of TBCM in mice or macrophages. We compared the pathogenesis of B∆tbcm and wild-type BCG in vivo by measuring the bacterial clearance rate and the degree of apoptosis. Promotion of the intrinsic apoptotic pathway was evaluated in infected bone marrow-derived macrophages (BMDMs) by measuring apoptotic cell death, loss of mitochondrial membrane potential and translocation of pore-forming proteins. Immunocytochemistry, western blotting and real-time PCR were also performed to assess the related protein expression levels after infection. Furthermore, these findings were validated by complementation of tbcm in BCG. RESULTS: Deletion of the tbcm gene in BCG leads to reduced pathogenesis in a mouse model, compared to wild type BCG, by promoting apoptotic cell death and bacterial clearance. Based on these findings, we found that intrinsic apoptosis and mitochondrial impairment were promoted in B∆tbcm-infected BMDMs. B∆tbcm down-regulates the expression of Bcl-2, which leads to mitochondrial outer membrane permeabilization (MOMP), culminating in cytochrome c release from mitochondria. Consistent with this, transcriptome profiling also indicated that B∆tbcm infection is more closely related to altered mitochondrial-related gene expression than wild-type BCG infection, suggesting an inhibitory role of TBCM in mitochondrial dysfunction. Moreover, genetic complementation of B∆tbcm (C∆tbcm) restored its capacity to inhibit mitochondria-mediated apoptotic cell death. CONCLUSIONS: Our findings demonstrate the contribution of TBCM to bacterial survival, inhibiting intrinsic apoptotic cell death of macrophages as a virulence factor of M. tuberculosis complex (MTBC) strains, which could be a potential target for the development of TB therapy.
Assuntos
Corismato Mutase , Mycobacterium bovis , Mycobacterium tuberculosis , Tuberculose , Animais , Camundongos , Apoptose/genética , Corismato Mutase/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mycobacterium bovis/genética , Mycobacterium tuberculosis/genética , Tuberculose/genética , Tuberculose/microbiologiaRESUMO
BACKGROUND: The only licensed live Bacille Calmette-Guérin (BCG) vaccine used to prevent severe childhood tuberculosis comprises genetically divergent strains with variable protective efficacy and rates of BCG-induced adverse events. The whole-genome sequencing (WGS) allowed evaluating the genome stability of BCG strains and the impact of spontaneous heterogeneity in seed and commercial lots on the efficacy of BCG-vaccines in different countries. Our study aimed to assess sequence variations and their putative effects on genes and protein functions in the BCG-1 (Russia) seed lots compared to their progeny isolates available from immunocompetent children with BCG-induced disease (mainly, osteitis). RESULTS: Based on the WGS data, we analyzed the links between seed lots 361, 367, and 368 used for vaccine manufacture in Russia in different periods, and their nine progeny isolates recovered from immunocompetent children with BCG-induced disease. The complete catalog of variants in genes relative to the reference genome (GenBank: CP013741) included 4 synonymous and 8 nonsynonymous single nucleotide polymorphisms, and 3 frameshift deletions. Seed lot 361 shared variants with 2 of 6 descendant isolates that had higher proportions of such polymorphisms in several genes, including ppsC, eccD5, and eccA5 involved in metabolism and cell wall processes and reportedly associated with virulence in mycobacteria. One isolate preserved variants of its parent seed lot 361 without gain of further changes in the sequence profile within 14 years. CONCLUSIONS: The background genomic information allowed us for the first time to follow the BCG diversity starting from the freeze-dried seed lots to descendant clinical isolates. Sequence variations in several genes of seed lot 361 did not alter the genomic stability and viability of the vaccine and appeared accumulated in isolates during the survival in the human organism. The impact of the observed variations in the context of association with the development of BCG-induced disease should be evaluated in parallel with the immune status and host genetics. Comparative genomic studies of BCG seed lots and their descendant clinical isolates represent a beneficial approach to better understand the molecular bases of efficacy and adverse events during the long-term survival of BCG in the host organism.
Assuntos
Mycobacterium bovis , Tuberculose , Vacina BCG/efeitos adversos , Criança , Genoma , Humanos , Mycobacterium bovis/genética , Federação Russa , Tuberculose/prevenção & controleRESUMO
BACKGROUND: Tuberculosis is an important health problem worldwide. The only available vaccine is M. bovis/BCG, an attenuated mycobacterium that activates the innate and the acquired immune system after being phagocytosed by macrophages and dendritic cells. Vaccination fails to prevent adult pulmonary tuberculosis although it may have a protective effect in childhood infection. Understanding how BCG interacts with macrophages and other immunocompetent cells is crucial to develop new vaccines. RESULTS: In this study we showed that macrophages phagocytose M. bovis/BCG bacilli with higher efficiency when they are cultured without phosphate. We isolated mycobacterial membranes to search for mycobacterial molecules that could be involved in these processes; by immunoblot, it was found that the plasma membranes of phosphate-deprived bacilli express the adhesins PstS-1, LpqH, LprG, and the APA antigen. These proteins are not detected in membranes of bacilli grown with usual amounts of phosphate. CONCLUSIONS: The interest of our observations is to show that under the metabolic stress implied in phosphate deprivation, mycobacteria respond upregulating adhesins that could improve their capacity to infect macrophages. These observations are relevant to understand how M. bovis/BCG induces protective immunity.
Assuntos
Vacina BCG/imunologia , Macrófagos/imunologia , Mycobacterium bovis/imunologia , Fagocitose/imunologia , Fosfatos/imunologia , Tuberculose Pulmonar/imunologia , Imunidade Adaptativa/imunologia , Animais , Antígenos/imunologia , Linhagem Celular Tumoral , Membrana Celular/imunologia , Imunidade Inata/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Vacinação/métodosRESUMO
Mycobacterium tuberculosis is the main etiological agent of tuberculosis. The Bacillus Calmette-Guérin (BCG) microbes that are primarily used as a vaccine against tuberculosis also constitute the dominant infection model for studying the interaction of mycobacteria with the host cell types. The majority of interaction experiments have been conducted using macrophages and monocytes as prototype phagocyte cell types. Here, we report that M. bovis BCG infects mouse primary B cells as well as human B cell line. The complement receptors, along with B cell receptors, are engaged in the process of bacterial entry into the host B cells. Once inside the B cells, the intracellular trafficking of BCG follows the complete endocytic pathway of the ingested particles, which is in contrast to the events taking place during ingestion of BCG by macrophages. In vivo infection of mice with M. bovis BCG activated peritoneal as well as splenic B cells to produce proinflammatory cytokines. This paper further supports the evidence that B cells are involved in a host's early interactions with intracellular bacterial pathogens and participate in the induction of innate defense responses.
Assuntos
Linfócitos B , Citocinas/metabolismo , Mycobacterium bovis/imunologia , Tuberculose/imunologia , Animais , Linfócitos B/imunologia , Linfócitos B/microbiologia , Vacina BCG , Humanos , Imunidade Inata , Camundongos , Cultura Primária de Células , Tuberculose/microbiologiaRESUMO
Within tuberculous granulomas, a subpopulation of Mycobacterium tuberculosis resides inside foamy macrophages (FM) that contain abundant cytoplasmic lipid bodies (LB) filled with triacylglycerol (TAG). Upon fusion of LB with M. tuberculosis-containing phagosomes, TAG is hydrolyzed and reprocessed by the bacteria into their own lipids, which accumulate as intracytosolic lipid inclusions (ILI). This phenomenon is driven by many mycobacterial lipases, among which LipY participates in the hydrolysis of host and bacterial TAG. However, the functional contribution of LipY's PE domain to TAG hydrolysis remains unclear. Here, enzymatic studies were performed to compare the lipolytic activities of recombinant LipY and its truncated variant lacking the N-terminal PE domain, LipY(ΔPE). Complementarily, an FM model was used where bone marrow-derived mouse macrophages were infected with M. bovis BCG strains either overexpressing LipY or LipY(ΔPE) or carrying a lipY deletion mutation prior to being exposed to TAG-rich very-low-density lipoprotein (VLDL). Results indicate that truncation of the PE domain correlates with increased TAG hydrolase activity. Quantitative electron microscopy analyses showed that (i) in the presence of lipase inhibitors, large ILI (ILI+3) were not formed because of an absence of LB due to inhibition of VLDL-TAG hydrolysis or inhibition of LB-neutral lipid hydrolysis by mycobacterial lipases, (ii) ILI+3 profiles in the strain overexpressing LipY(ΔPE) were reduced, and (iii) the number of ILI+3 profiles in the ΔlipY mutant was reduced by 50%. Overall, these results delineate the role of LipY and its PE domain in host and mycobacterial lipid consumption and show that additional mycobacterial lipases take part in these processes.
Assuntos
Proteínas de Bactérias/química , Hidrolases de Éster Carboxílico/química , Metabolismo dos Lipídeos , Macrófagos/microbiologia , Macrófagos/fisiologia , Triglicerídeos/metabolismo , Fatores de Virulência/química , Animais , Proteínas de Bactérias/genética , Hidrolases de Éster Carboxílico/genética , Domínio Catalítico , Células Cultivadas , Feminino , Lipase/metabolismo , Lipoproteínas VLDL/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica , Mycobacterium bovis , Estrutura Terciária de Proteína , Tuberculose/microbiologia , Fatores de Virulência/genéticaRESUMO
BACKGROUND: Control of Mycobacterium tuberculosis (Mtb) infection requires CD4+ T-cell responses and major histocompatibility complex class II (MHC II) presentation of Mtb antigens (Ags). Dendritic cells (DCs) are the most potent of the Ag-presenting cells and are central to the initiation of T-cell immune responses. Much research has indicated that DCs play an important role in anti-mycobacterial immune responses at early infection time points, but the kinetics of Ag presentation by these cells during these events are incompletely understood. RESULTS: In the present study, we evaluated in vivo dynamics of early Ag presentation by murine lymph-node (LN) DCs in response to Mycobacterium bovis bacillus Calmette-Guérin (BCG) Ag85A protein. Results showed that the early Ag-presenting activity of murine DCs induced by M. bovis BCG Ag85A protein in vivo was transient, appearing at 4 h and being barely detectable at 72 h. The transcription levels of CIITA, MHC II and the expression of MHC II molecule on the cell surface increased following BCG infection. Moreover, BCG was found to survive within the inguinal LN DC pool, representing a continuing source of mycobacterial Ag85A protein, with which LN DCs formed Ag85A peptide-MHCII complexes in vivo. CONCLUSIONS: Our results demonstrate that a decrease in Ag85A peptide production as a result of the inhibition of Ag processing to is largely responsible for the short duration of Ag presentation by LN DCs during BCG infection in vivo.
Assuntos
Aciltransferases/imunologia , Apresentação de Antígeno/imunologia , Antígenos de Bactérias/imunologia , Células Dendríticas/imunologia , Linfonodos/imunologia , Mycobacterium bovis/imunologia , Tuberculose/imunologia , Aciltransferases/metabolismo , Adjuvantes Imunológicos/administração & dosagem , Animais , Antígenos de Bactérias/metabolismo , Vacina BCG/administração & dosagem , Vacina BCG/imunologia , Sobrevivência Celular/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/microbiologia , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Interferon gama/imunologia , Interferon gama/metabolismo , Linfonodos/metabolismo , Linfonodos/microbiologia , Camundongos Endogâmicos C57BL , Mycobacterium bovis/fisiologia , Fatores de Tempo , Tuberculose/prevenção & controle , Tuberculose/veterináriaRESUMO
Environmental mycobacteria, highly prevalent in natural and artificial (including chlorinated municipal water) niches, are emerging as new threat to human health, especially to HIV-infected population. These seemingly harmless non-pathogenic mycobacteria, which are otherwise cleared, establish as opportunistic infections adding to HIV-associated complications. Although immune-evading strategies of pathogenic mycobacteria are known, the mechanisms underlying the early events by which opportunistic mycobacteria establish infection in macrophages and influencing HIV infection are unclear. Proteomics of phagosome-enriched fractions from Mycobacterium bovis Bacillus Calmette-Guérin (BCG) mono-infected and HIV-M. bovis BCG co-infected THP-1 cells by LC-MALDI-MS/MS revealed differential distribution of 260 proteins. Validation of the proteomics data showed that HIV co-infection helped the survival of non-pathogenic mycobacteria by obstructing phagosome maturation, promoting lipid biogenesis and increasing intracellular ATP equivalents. In turn, mycobacterial co-infection up-regulated purinergic receptors in macrophages that are known to support HIV entry, explaining increased viral titers during co-infection. The mutualism was reconfirmed using clinically relevant opportunistic mycobacteria, Mycobacterium avium, Mycobacterium kansasii and Mycobacterium phlei that exhibited increased survival during co-infection, together with increase in HIV titers. Additionally, the catalogued proteins in the study provide new leads that will significantly add to the understanding of the biology of opportunistic mycobacteria and HIV coalition.
Assuntos
Coinfecção/microbiologia , Coinfecção/virologia , Infecções por HIV/microbiologia , Infecções por Mycobacterium/virologia , Trifosfato de Adenosina/metabolismo , Linhagem Celular , Coinfecção/metabolismo , Citocinas/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Macrófagos/microbiologia , Macrófagos/virologia , Mycobacterium/patogenicidade , Mycobacterium bovis/patogenicidade , Fagossomos/microbiologia , Fagossomos/virologia , Proteômica/métodos , Simbiose , Carga ViralRESUMO
BACKGROUND: Multiple techniques exist for detecting Mycobacteria, each having its own advantages and drawbacks. Among them, automated culture-based systems like the BACTEC-MGIT™ are popular because they are inexpensive, reliable and highly accurate. However, they have a relatively long "time-to-detection" (TTD). Hence, a method that retains the reliability and low-cost of the MGIT system, while reducing TTD would be highly desirable. METHODS: Living bacterial cells possess a membrane potential, on account of which they store charge when subjected to an AC-field. This charge storage (bulk capacitance) can be estimated using impedance measurements at multiple frequencies. An increase in the number of living cells during culture is reflected in an increase in bulk capacitance, and this forms the basis of our detection. M. bovis BCG and M. smegmatis suspensions with differing initial loads are cultured in MGIT media supplemented with OADC and Middlebrook 7H9 media respectively, electrical "scans" taken at regular intervals and the bulk capacitance estimated from the scans. Bulk capacitance estimates at later time-points are statistically compared to the suspension's baseline value. A statistically significant increase is assumed to indicate the presence of proliferating mycobacteria. RESULTS: Our TTDs were 60 and 36 h for M. bovis BCG and 20 and 9 h for M. smegmatis with initial loads of 1000 CFU/ml and 100,000 CFU/ml respectively. The corresponding TTDs for the commercial BACTEC MGIT 960 system were 131 and 84.6 h for M. bovis BCG and 41.7 and 12 h for M smegmatis, respectively. CONCLUSION: Our culture-based detection method using multi-frequency impedance measurements is capable of detecting mycobacteria faster than current commercial systems.
Assuntos
Técnicas Bacteriológicas/métodos , Espectroscopia Dielétrica , Mycobacterium/crescimento & desenvolvimento , Mycobacterium/isolamento & purificação , Meios de Cultura , Humanos , Mycobacterium/classificação , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
A Mycobacterium ulcerans human challenge model has the potential to fundamentally advance our understanding of early human immune responses to infection, while rapidly evaluating vaccines and other therapeutic interventions. Here, using a murine tail infection model, we tested a very well-characterized working cell bank of the proposed challenge isolate M. ulcerans JKD8049 in naïve and Mycobacterium bovis bacille Calmette-Guérin (BCG)-vaccinated BALB/c mice. All 10 naïve mice were successfully infected with 20 colony-forming units (CFU) of M. ulcerans [95% confidence interval (CI) 17-22 CFU] with a mean time to visible lesion of 86 days (95% CI 79-92 days). In the 10 vaccinated mice, there was a significant delay in the mean time to lesion compared to the naïve controls of 24 days (P = 0.0003), but all mice eventually developed ulcerative lesions. This study informs a future human infection model by demonstrating the successful application of the challenge agent in this in vivo model and highlights both the promise and the problems with trying to induce protective immunity against M. ulcerans. IMPORTANCE: In preparation for its proposed use in a controlled human infection model (CHIM), this study reports the successful infection of BALB/c mice using a carefully characterized, low-dose inoculum of Mycobacterium ulcerans JKD8049 (our proposed CHIM strain). We also demonstrate that Mycobacterium bovis bacille Calmette-Guérin delays the onset of disease but cannot alter the course of illness once a lesion becomes apparent. We also validate the findings of previous low-dose challenges that used less accurate methods to determine the inoculum, but our presented methodology is practical, accurate, and anticipated to be reproducible.
Assuntos
Vacinas Bacterianas , Úlcera de Buruli , Modelos Animais de Doenças , Camundongos Endogâmicos BALB C , Mycobacterium ulcerans , Animais , Camundongos , Mycobacterium ulcerans/imunologia , Projetos Piloto , Feminino , Humanos , Úlcera de Buruli/imunologia , Úlcera de Buruli/prevenção & controle , Úlcera de Buruli/microbiologia , Vacinas Bacterianas/imunologia , Vacinas Bacterianas/administração & dosagem , Mycobacterium bovis/imunologia , Vacinação , Vacina BCG/imunologia , Vacina BCG/administração & dosagemRESUMO
Microorganisms present in the gut modulate host defence responses against infections in order to maintain immune homeostasis. This host-microbe crosstalk is regulated by gut metabolites. Butyrate is one such small chain fatty acid produced by gut microbes upon fermentation that has the potential to influence immune responses. Here we investigated the role of butyrate in macrophages during mycobacterial infection. Results demonstrate that butyrate significantly suppresses the growth kinetics of mycobacteria in culture medium as well as inhibits mycobacterial survival inside macrophages. Interestingly, butyrate alters the pentose phosphate pathway by inducing higher expression of Glucose-6-Phosphate Dehydrogenase (G6PDH) resulting in a higher oxidative burst via decreased Sod-2 and increased Nox-2 (NADPH oxidase-2) expression. Butyrate-induced G6PDH also mediated a decrease in mitochondrial membrane potential. This in turn lead to an induction of apoptosis as measured by lower expression of the anti-apoptotic protein Bcl-2 and a higher release of Cytochrome C as a result of induction of apoptosis. These results indicate that butyrate alters the metabolic status of macrophages and induces protective immune responses against mycobacterial infection.
Assuntos
Butiratos , Infecções por Mycobacterium , Humanos , Butiratos/farmacologia , Glucosefosfato Desidrogenase/metabolismo , Explosão Respiratória , Macrófagos/microbiologia , Infecções por Mycobacterium/metabolismo , ApoptoseRESUMO
Mycobacterium tuberculosis attenuates many defence responses from alveolar macrophages to create a niche at sites of infection in the human lung. Levels of Heat Shock Proteins have been reported to increase many folds in the serum of active TB patients than in latently infected individuals. Here we investigated the regulation of key defence responses by HSPs during mycobacterial infection. We show that infection of macrophages with M. bovis BCG induces higher expression of HSP-27 and HSP-70. Inhibiting HSP-27 and HSP-70 prior to mycobacterial infection leads to a significant decrease in mycobacterial growth inside macrophages. Further, inhibiting HSPs resulted in a significant increase in intracellular oxidative burst levels. This was accompanied by an increase in the levels of T cell activation molecules CD40 and IL-12 receptor and a concomitant decrease in the levels of T cell inhibitory molecules PD-L1 and IL-10 receptor. Furthermore, inhibiting HSPs significantly increased the expression of key proteins in the autophagy pathway along with increased activation of pro-inflammatory promoting transcription factors NF-κB and p-CREB. Interestingly, we also show that both HSP-27 and HSP-70 are associated with anti-apoptotic proteins Bcl-2 and Beclin-1. These results point towards a suppressive role for host HSP-27 and HSP-70 during mycobacterial infection.
Assuntos
Proteínas de Choque Térmico HSP27 , Proteínas de Choque Térmico HSP70 , Macrófagos , Infecções por Mycobacterium , Mycobacterium tuberculosis , Humanos , Proteínas de Choque Térmico/metabolismo , Macrófagos/microbiologia , Infecções por Mycobacterium/metabolismo , Mycobacterium tuberculosis/patogenicidade , Linfócitos T , Proteínas de Choque Térmico HSP70/imunologia , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP27/imunologia , Proteínas de Choque Térmico HSP27/metabolismoRESUMO
MALP-2, a synthetic lipopeptide is a Toll-like receptor 2 and -6 ligand and agonist. MALP-2 stimulates immune cells at different sites. Local stimulation in the lungs has beneficial effects in experimental pneumococci infection. The presented study investigated local effects of MALP-2 in the mycobacterial infection of lungs. MALP-2 was applied prior, simultaneously or after the pulmonary infection with Mycobacterium bovis BCG. Colony forming units were determined in the bronchoalveolar lavage fluid and lung homogenate. Numbers of Mycobacterium bovis BCG colony forming units were found to be reduced in two compartments, bronchoalveolar lavage fluid and lung homogenate after treatment with MALP-2 simultaneously to the infection for up to eight weeks. Reduction of the bacterial load in both compartments was also found up to two weeks after local treatment before and after the infection. Thus, macrophage activating lipopeptide-2 enhances the host defence in the lung in acute and long term bacterial infections.
Assuntos
Pulmão , Pneumonia , Líquido da Lavagem Broncoalveolar , Humanos , Lipopeptídeos , MacrófagosRESUMO
We previously reported that M. tb on its own as well as together with HIV inhibits macrophage apoptosis by upregulating the expression of Bcl2 and Inhibitor of Apoptosis (IAP). In addition, recent reports from our lab showed that stimulation of either macrophages or BMDCs results in the significant upregulation of Bcl2. In this report, we delineate the role of Bcl2 in mediating defense responses from dendritic cells (BMDCs) during mycobacterial infection. Inhibiting Bcl2 led to a significant decrease in intracellular bacterial burden in BMDCs. To further characterize the role of Bcl2 in modulating defense responses, we inhibited Bcl2 in BMDCs as well as human PBMCs to monitor their activation and functional status in response to mycobacterial infection and stimulation with M. tb antigen Rv3416. Inhibiting Bcl2 generated protective responses including increased expression of co-stimulatory molecules, oxidative burst, pro-inflammatory cytokine expression and autophagy. Finally, co-culturing human PBMCs and BMDCs with antigen-primed T cells increased their proliferation, activation and effector function. These results point towards a critical role for Bcl2 in regulating BMDCs defense responses to mycobacterial infection.
Assuntos
Células Dendríticas/imunologia , Imunidade , Macrófagos/imunologia , Infecções por Mycobacterium/imunologia , Mycobacterium bovis/imunologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Apoptose , Autofagia , Citocinas/metabolismo , Células Dendríticas/metabolismo , Células Dendríticas/microbiologia , Células Dendríticas/patologia , Feminino , Humanos , Macrófagos/metabolismo , Macrófagos/microbiologia , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Mycobacterium/metabolismo , Infecções por Mycobacterium/microbiologia , Infecções por Mycobacterium/patologia , Proteínas Proto-Oncogênicas c-bcl-2/genéticaRESUMO
Biofilm formed in vitro by mycobacteria has been associated with increased antibiotic tolerance as compared with planktonic cells. Cellulose has been identified as a component of DTT-exposed biofilms formed by M. tuberculosis. The celA1 gene of M. tuberculosis encodes a cellulase, which could affect the formation of biofilm by slow-growing mycobacteria. In this work, the celA1 gene of M. tuberculosis was cloned into the integrative pMV361 plasmid and then transformed into M. bovis BCG Pasteur to produce BCG:celA1, to have celA1 expressed from the strong promoter hsp60. We compared planktonic and biofilm growth, possible presence of CelA1 in whole protein extracts, quantitated biofilm, presence of monosaccharides, and bacillary burden in lungs after aerosol infection in BALB/c mice. Differences in the appearance of the surface pellicle and of the biofilm attached to the substrate were observed. In biofilms, we observed a significant decrease of glucosamine in BCG:celA1 compared with BCG:pMV361. Finally, BCG:celA1 had lower viable bacteria than the BCG:pMV361 strain after 24 h and 3 weeks post-infection, but no difference was found at 9 weeks post-infection.
Assuntos
Vacina BCG/farmacologia , Biofilmes/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Glucosamina/metabolismo , Mycobacterium tuberculosis/genética , Elastase Pancreática/genética , Tuberculose Pulmonar/microbiologia , Adjuvantes Imunológicos/farmacologia , Animais , Biofilmes/efeitos dos fármacos , DNA Bacteriano/genética , Modelos Animais de Doenças , Feminino , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Elastase Pancreática/biossíntese , Tuberculose Pulmonar/tratamento farmacológicoRESUMO
Lipid droplets (LDs) are organelles that have multiple roles in inflammatory and infectious diseases. LD act as essential platforms for immunometabolic regulation, including as sites for lipid storage and metabolism, inflammatory lipid mediator production, and signaling pathway compartmentalization. Accumulating evidence indicates that intracellular pathogens may exploit host LDs as source of nutrients and as part of their strategy to promote immune evasion. Notably, numerous studies have demonstrated the interaction between LDs and pathogen-containing phagosomes. However, the mechanism involved in this phenomenon remains elusive. Here, we observed LDs and PLIN2 surrounding M. bovis BCG-containing phagosomes, which included observations of a bacillus cell surrounded by lipid content inside a phagosome and LAM from mycobacteria co-localizing with LDs; these results were suggestive of exchange of contents between these compartments. By using beads coated with M.tb lipids, we demonstrated that LD-phagosome associations are regulated through the mycobacterial cell wall components LAM and PIM. In addition, we demonstrated that Rab7 and RILP, but not Rab5, localizes to LDs of infected macrophages and observed the presence of Rab7 at the site of interaction with an infected phagosome. Moreover, treatment of macrophages with the Rab7 inhibitor CID1067700 significantly inhibited the association between LDs and LAM-coated beads. Altogether, our data demonstrate that LD-phagosome interactions are controlled by mycobacterial cell wall components and Rab7, which enables the exchange of contents between LDs and phagosomes and may represent a fundamental aspect of bacterial pathogenesis and immune evasion.
Assuntos
Gotículas Lipídicas/metabolismo , Infecções por Mycobacterium/metabolismo , Mycobacterium tuberculosis/metabolismo , Fagossomos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis/citologia , proteínas de unión al GTP Rab7RESUMO
PURPOSE: The prevalence of Mycobacterium tuberculosis (M. tb) and the status of M. bovis BCG vaccination may affect host immune responses to M. tb antigens. Understanding of the predominant local M. tb strain and immune signatures induced by its strain-specific antigens may contribute to an improved diagnosis of tuberculosis (TB). The aim of this study was to determine immune responses to M. tb antigen which was identified from the hyper-virulent Beijing/K strain in South Korea. MATERIALS AND METHODS: Pulmonary TB patients (n=52) and healthy subjects (n=92) including individuals with latent TB infection (n=31) were recruited, and QuantiFERON-TB Gold In-Tube tests were performed. The Beijing/K-antigen specific immune signatures were examined by diluted whole blood assays and multiplex bead arrays in a setting where nationwide BCG vaccination is employed. RESULTS: Statistical analyses demonstrated that three [C-X-C motif chemokine (CXCL10), interleukin (IL)-6, interferon (IFN)-α] of 17 cytokines/chemokines distinguished active cases from healthy controls following stimulation with the Beijing/K-specific antigen. IFN-α also differentiated between active diseases and latent TB infection (p<0.01), and the detection rate of TB was dramatically increased in combination with IL-6 and CXCL10 at the highest levels of specificity (95-100%). CONCLUSION: Our data indicate that immune signatures to the M. tb Beijing/K-specific antigen can provide useful information for improved TB diagnostics. The antigen may be developed as a diagnostic marker or a vaccine candidate, particularly in regions where the M. tb Beijing/K strain is endemic.
Assuntos
Tuberculose Latente/diagnóstico , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose Pulmonar/diagnóstico , Adolescente , Adulto , Antígenos de Bactérias/sangue , Antígenos de Bactérias/genética , Antígenos de Superfície/sangue , Antígenos de Superfície/genética , Proteínas de Bactérias , Pequim , Estudos de Casos e Controles , Citocinas/sangue , Feminino , Humanos , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/metabolismo , República da Coreia , Sensibilidade e EspecificidadeRESUMO
Mycobacterium ensures its survival inside macrophages and long-term infection by subverting the innate and adaptive immune response through the modulation of cytokine gene expression profiles. Different Mycobacterium species promote the expression of TGFß and IL-10, which, at the early stages of infection, block the formation of the phagolysosome, thereby securing mycobacterial survival upon phagocytosis, and at later stages, antagonize IFNγ production and functions. Despite the key role of IL-10 in mycobacterium infection, the signal transduction pathways leading to IL-10 expression in infected macrophages are poorly understood. Here, we report that Mycobacterium bovis BCG promotes IL-10 expression and cytokine production by establishing a SYK/PKCα/ß positive feedback loop that leads to STAT3 activation.
Assuntos
Interleucina-10/biossíntese , Monócitos/imunologia , Mycobacterium bovis/imunologia , Proteína Quinase C beta/metabolismo , Proteína Quinase C-alfa/metabolismo , Fator de Transcrição STAT3/metabolismo , Quinase Syk/metabolismo , Expressão Gênica , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Células THP-1RESUMO
The aim of this study was to evaluate the survival of mice inoculated with M. bovis BCG Pasteur recombinant expressing the PLD protein and challenged with a C. pseudotuberculosis virulent strain. Four groups were immunized with a sterile 0.9% saline solution (G1), 106â¯CFU of M. bovis BCG Pasteur (G2), 106â¯CFU of M. bovis BCG/pld (G3) or 106â¯CFU of M. bovis BCG/pld with a booster with rPLD (G4) and challenged with 104 CFU of C. pseudotuberculosis MIC-6 strain. The highest survival rate of 88% was observed in G4, followed by 77% in G3 and 66% in G2. A significant statistical difference was observed in the levels of cytokines IFN-γ and IL-10 in vaccinated groups (G3 and G4) when compared with the control group (G1) (pâ¯<â¯0.05). The results seem promising as the recombinant vaccine elicited a cellular immune response and provided significant survival after a high virulent challenge.
Assuntos
Vacina BCG/genética , Proteínas de Bactérias/imunologia , Infecções por Corynebacterium/prevenção & controle , Fosfolipase D/imunologia , Vacinação/métodos , Animais , Vacina BCG/administração & dosagem , Vacina BCG/imunologia , Proteínas de Bactérias/genética , Clonagem Molecular , Infecções por Corynebacterium/imunologia , Infecções por Corynebacterium/microbiologia , Infecções por Corynebacterium/mortalidade , Corynebacterium pseudotuberculosis/imunologia , Corynebacterium pseudotuberculosis/patogenicidade , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Engenharia Genética , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Imunização Secundária , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium bovis/genética , Mycobacterium bovis/imunologia , Fosfolipase D/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Análise de SobrevidaRESUMO
In vitro and ex vivo efficacies of four series of benzo[b]thiophene-2-carboxylic acid derivatives were studied against Mycobacterium tuberculosis H37Ra (MTB). Benzo[b]thiophenes were also tested in vitro against multidrug resistant Mycobacterium tuberculosis H37Ra (MDR-MTB), and 7b was found to be highly active against A- and D-MDR-MTB/MTB (MIC ranges 2.73-22.86 µg/mL). The activity of all benzo[b]thiophenes against M. bovis BCG (BCG) was also assessed grown under aerobic and under conditions of oxygen depletion. Compounds 8c and 8g showed significant activity with MICs of 0.60 and 0.61 µg/mL against dormant BCG. The low cytotoxicity and high selectivity index data against human cancer cell lines, HeLa, Panc-1, and THP-1 indicate the potential importance of the development of benzo[b]thiophene-based 1,3-diketones and flavones as lead candidates to treat mycobacterial infections. Molecular docking studies into the active site of DprE1 (Decaprenylphosphoryl-ß-d-ribose-2'-epimerase) enzyme revealed a similar binding mode to native ligand in the crystal structure thereby helping to understand the ligand-protein interactions and establish a structural basis for inhibition of MTB. In summary, its good activity in in vitro and ex vivo model, as well as its activity against multidrug-resistant M. tuberculosis H37Ra in a potentially latent state, makes 7b an attractive drug candidate for the therapy of tuberculosis.