STING-Dependent 2'-5' Oligoadenylate Synthetase-Like Production Is Required for Intracellular Mycobacterium leprae Survival.

Cytosolic detection of nucleic acids elicits a type I interferon (IFN) response and plays a critical role in host defense against intracellular pathogens. Herein, a global gene expression profile of Mycobacterium leprae-infected primary human Schwann cells identified the genes differentially expressed in the type I IFN pathway. Among them, the gene encoding 2'-5' oligoadenylate synthetase-like (OASL) underwent the greatest upregulation and was also shown to be upregulated in M. leprae-infected human macrophage cell lineages, primary monocytes, and skin lesion specimens from patients with a disseminated form of leprosy. OASL knock down was associated with decreased viability of M. leprae that was concomitant with upregulation of either antimicrobial peptide expression or autophagy levels. Downregulation of MCP-1/CCL2 release was also observed during OASL knock down. M. leprae-mediated OASL expression was dependent on cytosolic DNA sensing mediated by stimulator of IFN genes signaling. The addition of M. leprae DNA enhanced nonpathogenic Mycobacterium bovis bacillus Calmette-Guerin intracellular survival, downregulated antimicrobial peptide expression, and increased MCP-1/CCL2 secretion. Thus, our data uncover a promycobacterial role for OASL during M. leprae infection that directs the host immune response toward a niche that permits survival of the pathogen.

STING-Dependent 2'-5' oligoadenylate synthetase-like production is required for intracellular Mycobacterium leprae survival

Cytosolic detection of nucleic acids elicits a type I interferon (IFN) response and plays a critical role in host defense against intracellular pathogens. Herein, a global gene expression profile of Mycobacterium leprae-infected primary human Schwann cells identified the genes differentially expressed in the type I IFN pathway. Among them, the gene encoding 2'-5' oligoadenylate synthetase-like (OASL) underwent the greatest upregulation and was also shown to be upregulated in M. leprae-infected human macrophage cell lineages, primary monocytes, and skin lesion specimens from patients with a disseminated form of leprosy. OASL knock down was associated with decreased viability of M. leprae that was concomitant with upregulation of either antimicrobial peptide expression or autophagy levels. Downregulation of MCP-1/CCL2 release was also observed during OASL knock down. M. leprae-mediated OASL expression was dependent on cytosolic DNA sensing mediated by stimulator of IFN genes signaling. The addition of M. leprae DNA enhanced nonpathogenic Mycobacterium bovis bacillus Calmette-Guerin intracellular survival, downregulated antimicrobial peptide expression, and increased MCP-1/CCL2 secretion. Thus, our data uncover a promycobacterial role for OASL during M. leprae infection that directs the host immune response toward a niche that permits survival of the pathogen.

ARC: automated resource classifier for agglomerative functional classification of prokaryotic proteins using annotation texts.

Functional classification of proteins is central to comparative genomics. The need for algorithms tuned to enable integrative interpretation of analytical data is felt globally. The availability of a general,automated software with built-in flexibility will significantly aid this activity. We have prepared ARC (Automated Resource Classifier), which is an open source software meeting the user requirements of flexibility. The default classification scheme based on keyword match is agglomerative and directs entries into any of the 7 basic non-overlapping functional classes: Cell wall, Cell membrane and Transporters (C), Cell division (D), Information (I), Translocation (L), Metabolism (M), Stress(R), Signal and communication (S) and 2 ancillary classes: Others (O) and Hypothetical (H). The keyword library of ARC was built serially by first drawing keywords from Bacillus subtilis and Escherichia coli K12. In subsequent steps,this library was further enriched by collecting terms from archaeal representative Archaeoglobus fulgidus, Gene Ontology, and Gene Symbols. ARC is 94.04% successful on 6,75,663 annotated proteins from 348 prokaryotes. Three examples are provided to illuminate the current perspectives on mycobacterial physiology and costs of proteins in 333 prokaryotes. ARC is available at http://arc.igib.res.in.

M. leprae inhibits apoptosis in THP-1 cells by downregulation of Bad and Bak and upregulation of Mcl-1 gene expression.

BACKGROUND: Virulent Mycobacterium leprae interfere with host defense mechanisms such as cytokine activation and apoptosis. The mitochondrial pathway of apoptosis is regulated by the Bcl-2 family of proteins. Expression of Fas ligand and apoptotic proteins is found in leprosy lesions and M. leprae has been shown to activate pro-apoptotic Bcl-2 genes, Bak and Bax. However, the mechanism by which M. leprae modulates apoptosis is as yet unclear. We investigated expression of apoptotic genes in THP-1 monocytes in response to infection by M. leprae and non-pathogenic M. bovis BCG. RESULTS: M. leprae did not induce apoptosis in THP-1 cells, while BCG induced a significant loss of cell viability by 18 h post-infection at both (multiplicity of infection) MOI-10 and 20, with an increase by 48 h. BCG-induced cell death was accompanied by characteristic apoptotic DNA laddering in cells. Non-viable BCG had a limited effect on host cell death suggesting that BCG-induced apoptosis was a function of mycobacterial viability. M. leprae also activated lower levels of TNF-alpha secretion and TNF-alpha mRNA expression than BCG. Mycobacterium-induced activation of apoptotic gene expression was determined over a time course of infection. M. leprae reduced Bad and Bak mRNA expression by 18 h post-stimulation, with a further decrease at 48 h. Outcome of cell viability is determined by the ratio between pro- and anti-apoptotic proteins present in the cell. M. leprae infection resulted in downregulation of gene expression ratios, Bad/Bcl-2 mRNA by 39% and Bak/Bcl-2 mRNA by 23%. In contrast, live BCG increased Bad/Bcl-2 mRNA (29 %) but had a negligible effect on Bak/Bcl-2 mRNA. Heat killed BCG induced only a negligible (1-4 %) change in mRNA expression of either Bak/Bcl-2 or Bad/Bcl-2. Additionally, M. leprae upregulated the expression of anti-apoptotic gene Mcl-1 while, BCG downregulated Mcl-1 mRNA. CONCLUSION: This study proposes an association between mycobacterium-induced apoptosis in THP-1 cells and the regulation of Bcl-2 family of proteins. M. leprae restricts apoptosis in THP-1 cells by downregulation of Bad and Bak and upregulation of Mcl-1 mRNA expression.

Gelatinase activity in mycobacterium bovis protein extract

Proteases are well-recognized as virulence factors in different pathologies, resulting in tissue damage potential. Despite efforts over the past few years to identify mycobacterial protein antigens, there is little information regarding the role of mycobacterial proteinase activities. In this study, by zymography techniques, we have detected and partially studied some biochemical properties of Mycobacterium bovis proteases, such as pH dependency of activity and susceptibility to classical proteinase inhibitors. We observed optimal proteolytic activity at pH 8. Some proteinases were inhibited by classic inhibitors of serine proteases, such as PMSF, AEBSF, and 3-4 DCI. In some AEBSF pre-treated preparations we observed residual gelatinase activity in Rf 0.32. This gelatinase was stimulated by Zn2+ and inhibited by OPA (1 mM). This last effect was reversed by exposure to equimolar quantitative OPA/Zn+2 (1 mM/1 mM). These results suggest the existence of serine proteinase and metalloproteinase types in protein extracts of Mycobacterium bovis.

Intracellular signals triggered during association of Mycobacterium leprae and Mycobacterium bovis BCG with human monocytes.

To gain a better understanding of mycobacteria-host cell interaction, the present study compared the signal transduction events triggered during the interaction of Mycobacterium leprae (the causative agent of leprosy) and of Mycobacterium bovis BCG (an attenuated strain used as a vaccine against leprosy and tuberculosis) with human monocytes. The assays consisted of pre-treating or not THP-1 cells (a human monocytic cell line) with different kinase inhibitors, followed by incubation with fluorescein-labelled bacteria and analysis of bacterial association via fluorescence microscopy. The specific tyrosine kinase (TK) inhibitor tyrphostin AG126 provided the highest rates of association inhibition (>90% for BCG and >65% for M. leprae). The early activation of TKs during mycobacteria-host cell interaction was confirmed by immunoblot analysis, demonstrating that in several host cell proteins mycobacteria stimulated tyrosine phosphorylation. The use of the drugs wortmannin and bisindolylmaleimide I which, respectively, inhibit phosphatidylinositide 3-kinase (PI 3-kinase) and protein kinase C (PKC), produced lower but consistent results within a 35--60% association inhibition range for both bacteria. Dose response curves with these inhibitors were obtained. Similar results were obtained when primary human monocytes were used as host cells, strongly suggesting that TK, PKC and PI 3-kinase signals are activated during the interaction of human monocytes with both pathogenic and attenuated species of mycobacteria.

Analise da conversao da reacao de mitsuda nas formas multibacilares da hanseniase / An analysis of mitsuda reactions conversion in multi-bacillar forms of hansen's disease

Com o objetivo de verificar o percentual de conversao imunologica atraves da reacao de Mitsuda e realizar a analise de fatores envolvidos nesse processo, selecionou-se ex-pacientes portadores de hanseniase atendidos na Fundacao Alfredo da Matta (Manaus-AM), com as seguintes caracteristicas: forma clinica multibacilar, reacao de Mitsuda negativo no diagnostico, alta por cura no periodo de 1989 a 1993, sem tratamento especifico anterior e que fizeram uso de esquemas poliquimioterapicos (PQT) com regularidade. Os participantes foram dispostos em dois grupos conforme esquema PQT: 22 haviam feito uso do esquema da OMS (PQT/OMS), isto e, ate negativacao baciloscopica, e 24 utilizaram o esquema de duracao fixa (PQT/DF), com ingestacao de 24 doses. Todos foram reavaliados, em 1997, atraves de exame clinico-dermatoneurologico, baciloscopia da pele, grau de incapacidade e aplicacao de novo reacao de Mitsuda com posterior leitura. Constatou-se que entre os individuos classificados como portadores de hanseniase Virchowiana nao ocorreu nenhuma variacao de resposta imunologica celular, medida pela reacao de Mitsuda. Entretanto, nas demais formas e principalmente nos pacientes que fizeram uso de PQT/DF, a conversao ocorreu com maior frequencia a medida que o paciente, na classificacao de Ridley e Jopling, estava mais proximo do polo imunocompetente. Em relacao ao Indice Baciloscopico verificou-se que a negativacao baciloscopica precoce pode ter enterferido na conversao da Reacao de Mitsuda. A correlacao dos dados retrospectivos e os obtidos na reavalicao contemporanea permitiu concluir que estas conversoes, embora frequentes, nao estao relacionadas com o esquema poliquimioterapico utilizado, a presenca ou nao de reacao reversa ou com o grau de incapacidade induzido pela hanseniase.

Mycobacterium lepraemurium, a well-adapted parasite of macrophages: I. Oxygen metabolites.

We measured the release of reactive oxygen intermediaries [ROI (hydrogen peroxide and superoxide anion)] by murine peritoneal macrophages challenged in vitro with Mycobacterium lepraemurium (MLM), complement-opsonized yeast, M. bovis BCG, M. phlei, or phorbol myristate acetate (PMA). We found that except for MLM, all of the other materials provoked the release of significant amounts of hydrogen peroxide and superoxide. MLM entered the macrophages without triggering their oxidative metabolism. Pre-infection of macrophages with MLM did not alter these cells' capacity to release the normal amounts of ROI in response to other microorganisms or PMA. Killing of MLM did not revert the macrophages' failure to release ROI upon ingestion of the microorganism, nor were macrophages able to produce these toxic metabolites when pre-incubated in the presence of murine gamma interferon (IFN-gamma). MLM has several attributes that allow it to survive within macrophages: a) it is a nontoxigenic microorganism (it does not harm its host), b) it resists the harsh conditions of the intraphagolysosomal milieu (a property perhaps dependent on its thick lipidic envelope), and c) it penetrates the macrophages without triggering their oxidative response (thus avoiding the generation of the toxic intermediaries of oxygen). For these attributes (and others discussed in this paper), we recognize MLM as a highly evolved, well-adapted parasite of macrophages. In addition, the results of the present study prompted the analysis of the biochemical pathways used by MLM and M. bovis BCG to penetrate into their cellular hosts, a subject now under investigation in our laboratory.

Green fluorescent protein as a marker for gene expression and cell biology of mycobacterial interactions with macrophages.

The green fluorescent protein (GFP) of the jellyfish Aequorea victoria offers certain advantages over other bioluminescence systems because no exogenously added substrate or co-factors are necessary, and fluorescence can be elicited by irradiation with blue light without exposing the cells producing GFP to invasive treatments. A mycobacterial shuttle-plasmid vector carrying gfp cDNA was constructed and used to generate transcriptional fusions with promoters of interest and to examine their expression in Mycobacterium smegmatis and Mycobacterium bovis BCG grown in macrophages or on laboratory media. The promoters studied were: (i) ahpC from Mycoosis and Mycobacterium leprae, a gene encoding alkyl hydroperoxide reductase which, along with the divergently transcribed regulator oxyR, are homologues of corresponding stress-response systems in enteric bacteria and play a role in isoniazid sensitivity; (ii) mtrA, an M. tuberculosis response regulator belonging to the superfamily of bacterial two-component signal-transduction systems; (iii) hsp60, a previously characterized heat-shock gene from M. bovis; and (iv) tbprc3, a newly isolated promoter from M. tuberculosis. Expression of these promoters in mycobacteria was analysed using epifluorescence microscopy, laser scanning confocal microscopy, fluorescence spectroscopy, and flow cytometry. These approaches permitted assessment of fluorescence prior to and after macrophage infection, and analyses of promoter expression in individual mycobacteria and its distribution within populations of bacterial cells. Bacteria expressing GFP from a strong promoter could be separated by fluorescence-activated cell sorting from cells harbouring the vector used to construct the fusion. In addition, the stable expression of mtrA-gfp fusion in M. bovis BCG facilitated localization and isolation of phagocytic vesicles containing mycobacteria. The experiments presented here suggest that GFP will be a useful tool for analysis of mycobacterial gene expression and a convenient cell biology marker to study mycobacterial interactions with macrophages.

Suppression of monocyte oxidative response by phenolic glycolipid I of Mycobacterium leprae.

Mycobacterium leprae synthesizes a unique phenolic glycolipid (PGL-I) in abundant quantities. We studied the effect of PGL-I on the generation of superoxide anion (O2-) by stimulated human monocytes. Peripheral blood monocytes pretreated with PGL-I released less O2- when stimulated with M. leprae than did control monocytes. Monocytes pretreated with dimycocerosyl phthiocerol, mycoside A of Mycobacterium kansasii, or mycoside B of Mycobacterium microti, on the other hand, released O2- in quantities comparable to control monocytes in response to M. leprae stimulation. Monocyte O2- release in response to other stimuli of the oxidative metabolic burst, such as PMA, zymosan, Mycobacterium bovis Bacille Calmette-Guérin, or M. kansasii, was unaffected by lipid pretreatment. These findings demonstrate that PGL-I has a direct effect on monocyte O2- generation in response to M. leprae and suggest that PGL-I is a modulator of phagocytic cell function.

Human phagocytic cell responses to Mycobacterium leprae and Mycobacterium bovis Bacillus Calmette-Guérin. An in vitro comparison of leprosy vaccine components.

Components of current vaccines for Hansen's disease include Mycobacterium bovis Bacillus Calmette-Guérin (BCG) and killed Mycobacterium leprae. BCG infections in humans are rare and most often occur in immune-compromised individuals. M. leprae on the other hand, although not causing clinical disease in most exposed individuals, is capable of infecting and replicating within mononuclear phagocytes. Lymphocytes from patients with the lepromatous form of Hansen's disease exhibit defective lymphokine production when challenged in vitro with M. leprae. This may result in inefficient mononuclear phagocyte activation for oxidative killing. To study the ability of normal phagocytes to ingest and respond oxidatively to BCG and M. leprae, we measured phagocytic cell O2- release and fluorescent oxidative product formation and visually confirmed the ingestion of the organisms. BCG stimulated a vigorous O2- generation in neutrophils and monocytes and flow cytometric oxidative product generation by neutrophils occurred in the majority of cells. M. leprae, stimulated a weak but significant O2- release requiring a high concentration of organisms and long exposure. By flow cytometric analysis, most neutrophils were able to respond to both organisms with the generation of fluorescent oxidative products. Neutrophil oxidative responses to M. leprae were substantially less than responses seen from neutrophils exposed to BCG. By microscopic examination of neutrophils phagocytizing FITC-labeled bacteria, it was shown that both M. leprae and BCG were slowly ingested but that more BCG appeared to be associated with the cell membrane of more of the cells. When phagocytic cells were incubated with BCG and M. leprae for 30 min and subsequently examined by electron microscopy, few organisms were seen in either neutrophils or monocytes. This suggests that BCG are easily recognized and slowly ingested by normal phagocytic cells, the majority of which respond with a strong oxidative burst. M. leprae appeared to only weakly stimulate phagocyte oxidative responses and were also slowly phagocytized.

Lack of Mycobacterium leprae-specific uptake in Schwann cells.

Among mycobacteria, Mycobacterium leprae have a unique property to infect peripheral nerves, which is the cause of a variety of debilities seen in leprosy. The possibility of selective uptake of M. leprae by Schwann cells was studied using a rat Schwannoma cell line 33B and rat sciatic nerve-derived Schwann cells. M. leprae were phagocytosed by 33B cells but so also were seven other mycobacteria ("Mycobacterium w," BCG, M. tuberculosis H37Rv, M. nonchromogenicum, M. vaccae, ICRC bacillus, and M. smegmatis) which do not involve peripheral nerves. All three mycobacteria tested (M. leprae, M. tuberculosis and "Mycobacterium w") were phagocytosed by sciatic nerve-derived Schwann cells. Both Schwannoma and Schwann cells phagocytosed even inert latex particles. These results fail to demonstrate any M. leprae-specific uptake system in Schwann cells.

Viragem da lepromino-reação em função de diferentes estímulos. Influência da idade, nessa viragem, no grupo etário de 6 a 43 meses

112 crianças, 56 de cada sexo, com idades variando de 6 a 34 meses, tuberculino-negativas, foram alotadas em quatro grupos experimentais, a saber: Grupo A - recebeu uma dose de BCG por via intradérmica; Grupo B - recebeu três inoculações do ant¡geno de Mitsuda com intervalos de 1 mês; Grupo C - recebeu três doses de BCG por via oral, com intervalo de 1 semana; Grupo D - testemunha. O alotamento foi feito sorteando-se pelos quatro grupos as 4 crianças mais velhas de cada sexo, em seguida as 4 mais velhas das restantes e assim por diante. Um mês após a última inoculação do ant¡geno de Mitsuda, foi feita a reação de Mitsuda em tôdas as crianças, sendo os resultados lidos por três leprólogos experimentados, idependentemente, e sem conhecimento do grupo experimental a que pertencia cada criança. Tôda a análise estat¡stica foi feita por métodos não paramétricos. Seus resultados mostraram, desde logo, que não pode ser aceita, ao n¡vel de 5% pré-fixado, a hipótese de igualdade de intensidade da reação nos quatro grupos. Verificando-se que pode ser aceita a hipótese de igualdade, quanto á intensidade da reação, entre os sexos, e considerando que, do ponto de vista prático, interessa primordialmente a distinção entre reações expressas por ++ ou +++, de um lado, e...

A reação lepromínica em cobaios após prévia inoculação do BCG e de Mycobacterium tuberculosis morto por irradiação

Os autores estudaram nessa experiência a capacidade dos cobaios se tornarem lepromino-positivos; após a inoculação da vacina Parke Davis (Mycobacterium tuberculosis morto por irradiação) ou do BCG. I)- Cobaios inoculados subcutâneamente com a vacina Parke Davis (1 dose de 0,5 ml., semanalmente, durante 3 semanas) e testados com a lepromina integral mostraram os seguintes resultados: 80% de negatividade 3 20% de reações duvidosas. II)- Cobaios inoculados com BCG., por via oral, testados com lepromina integral, forneceram percentuais de positividade variáveis com as doses: a) com 60 mg. de BCG., por kg. de peso, os resultados foram: 47,0% reações positivas; 41,2% reações duvidosas; 11,8% negativas. b) com 120 mg. de BCG. por kg. de peso, os resultados foram: 25,0% reações positivas; 33,3% reações duvidosas; 41,7% reações negativas. c) o grupo contrôle fornecau os seguintes resultados: 12,5% reações duvidosas; 87,5% reações negativas. Os resultados desta experiência mostram: I)- A vacina Parke Davis preparada com Mycobacterium tuberculosis mortos por irradiação não induz positividade leprom¡nica, no cobaio. II)- O BCG administrado oralmente ‚ capaz de provocar a positividade lepromínica, no cobaio. III)- A julgar pelos resultados de experiência anteriores dos autores mostrando que a dose de 30 mg. de BCG por kg. de peso provocou, no cobaio, a mais elevada positividade lepromínica (87,5%) e doses maiores e menores do que 30 mg. (experiência anterior e atual) forneceram percentuais mais baixos de positividade, os autores pensam que aquela dose deve ser a ideal. IV- É interessante referir que a dose de 30 mg. por kg. de peso ‚ muito próxima da usada no Brasil (28 mg. por kg. de peso) para a premunição de recem-nascidos.

Fator N de resistência à lepra e relações com a reatividade lepromínica e tuberculínica: valor duvidoso do BCG na imunização antileprosa / Factor \"N\" of resistance to leprosy and relations with the lepromin and tuberculin reactivity: doubtful value of BCG in immunization antileprosy

O A. resume os fatos que o levaram a sugerir, em 1937, a hipótese da existência de um "Fator N" (natural), específico de resistência à lepra: A lepromino-reação (LR) positiva indica resistência à lepra. A grande maioria das crianças é lepromino-negativa, mas adquire espontâneamente a capacidade de reagir à lepromina e à lepra (alta freqüência de LR positivas no adulto). A capacidade de reação não ‚ atribuível a fator alguns conhecido; a pequena fração da população que permanece LR negativa mesmo na idade adulta ("margem anérgica") e suscetível de lepromatização, não se distingue da maioria LR positiva a não ser pelo próprio resultado da LR. As diferenças de reatividade podem aparecer já  na infância, entre os comunicantes de doentes contagiantes. Nessas condições pode-se supor que haja fator constitucional conferindo ao individuo a capacidade de ragir ao bacilo de Hansen: "Fator N" mas também pode tratar-se de fator adquirido, a investigar. A "margem anérgica", isto, é a minoria desprovida do "Fator N", é a que apresenta interêsse do ponto de vista epidemiológico e profilático, pois ‚ dessa minoria que surgem os casos lepromatosos, por ação de fatôres vários, agindo secundáriamente, e que o A. denomina "acessórios": moléstias, carências, debilitações e outros. Revendo as relações imunológicas entre a lepra e a tuberculose julga o A. que: 1) A infecção tuberculosa ou tuberculose ativa, por si só, sem o "Fator N" básico específico da imunidade antileprosa, não determina LR positivas. Éste seria o motivo pelo qual os lepromatosos, todos êles práticamente LR negativos, são, contudo, tuberculino-positivos em 50-80% dos casos com tuberculose ativa ou Tbk positiva e que permanecem LR negativos: essa percentagem, variável de autor para autor, é, contudo, em média, aproximadamente igual à "margem anérgica"...