M. fortuitum-induced CNS-pathology: Deciphering the role of canonical Wnt signaling, blood brain barrier components and cytokines.

Molecular underpinning of mycobacteria-induced CNS-pathology is not well understood. In the present study, zebrafish were infected with Mycobacterium fortuitum and the prognosis of CNS-pathogenesis studied. We observed M. fortuitum triggers extensive brain-pathology. Evans blue extravasation demonstrated compromised blood-brain barrier (BBB) integrity. Further, decreased expression in tight-junction (TJ) and adherens junction complex (AJC) genes were noted in infected brain. Wnt-signaling has emerged as a major player in host-mycobacterial immunity but its involvement/role in brain-infection is not well studied. Sustained expression of wnt2, wnt3a, fzd5, lrp5/6 and ß-catenin, with concordant decline in degradation complex components axin, gsk3ß and ß-catenin regulator capn2a were observed. The surge in ifng1 and tnfa expression preceding il10 and il4 suggested cytokine-interplay critical in M. fortuitum-induced brain-pathology. Therefore, we suggest adult zebrafish as a viable model for studying CNS-pathology and using the same, conclude that M. fortuitum infection is associated with repressed TJ-AJC gene expression and compromised BBB permeability. Our results implicate Wnt/ß-catenin pathway in M. fortuitum-induced CNS-pathology wherein Th1-type signals facilitate bacterial clearance and Th2-type signals prevent the disease sequel.

mtROS Induced via TLR-2-SOCE Signaling Plays Proapoptotic and Bactericidal Role in Mycobacterium fortuitum-Infected Head Kidney Macrophages of Clarias gariepinus.

The mechanisms underlying Mycobacterium fortuitum-induced mycobacteriosis remain unexplored. Using head kidney macrophages (HKM) from catfish (Clarias gariepinus), we report that Ca2+ surge across mitochondrial-Ca2+ uniporter (MICU), and consequent mitochondrial ROS (mtROS) production, is imperative for mycobactericidal activity. Inhibition of mtROS alleviated HKM apoptosis and enhanced bacterial survival. Based on RNA interference (RNAi) and inhibitor studies, we demonstrate that the Toll-like receptor (TLR)-2-endoplasmic reticulum (ER) stress-store-operated calcium entry (SOCE) axis is instrumental for activating the mt-Ca2+/mtROS cascade in M. fortuitum-infected HKM. Additionally, pharmacological inhibition of mtROS attenuated the expression of CHOP, STIM1, and Orai1, which suggests a positive feedback loop between ER-stress-induced SOCE and mtROS production. Elevated tumor necrosis factor alpha (TNF-α) levels and caspase-8 activity were observed in HKM consequent to M. fortuitum infection, and our results implicate that mtROS is crucial in activating the TNF-mediated caspase-8 activation. Our results for the first time demonstrate mitochondria as an innate immune signaling center regulating mycobacteriosis in fish. We conclude that M. fortuitum-induced persistent SOCE signaling leads to mtROS production, which in turn activates the TNF-α/caspase-8 axis culminating in HKM apoptosis and bacterial clearance.

Mycobacterium fortuitum induces A20 expression that impairs macrophage inflammatory responses.

Mycobacterium fortuitum is a rapidly growing mycobacterium that has been regarded as an etiological agent of a variety of human infections. However, little is known about the host inflammatory responses and the molecular mechanisms by which MF-induced inflammation is regulated in macrophages. In this study, we report that MF infection leads to the induction of an anti-inflammatory molecule, A20 (also known as TNFAIP3), which is essential for the regulation of MF-induced inflammatory responses in murine bone marrow-derived macrophages (BMDMs). MF triggered the expression of tumor necrosis factor-α and interleukin-6 in BMDMs through signaling of the Toll-like receptor 2 (TLR2)-myeloid differentiation primary response gene 88. Additionally, MF rapidly induced the expression of A20, which inhibited proinflammatory cytokine expression and nuclear factor (NF)-κB reporter gene activities in BMDMs. Notably, MF-induced activation of NF-κB signaling was required for A20 expression and proinflammatory responses in BMDMs. Furthermore, the rough morphotype of the MF clinical strain induced a higher level of proinflammatory signaling activation, but less A20 induction in BMDMs, compared to the smooth morphotype. Taken together, these results suggest that MF-induced activation of host proinflammatory responses is negatively regulated through TLR2-dependent A20 expression.

Localized cutaneous infections in immunocompetent individuals due to rapidly growing mycobacteria.

Rapidly growing mycobacteria (RGM) cause skin infections that are refractory to standard antibiotic regimens. Although typically associated with disseminated cutaneous or other systemic infections in immunocompromised patients, RGM sometimes cause localized cutaneous infections in immunocompetent hosts. These infections are almost always associated with precedent skin trauma and inoculation, and therefore have been implicated in outbreaks involving contaminated tattoo ink and inadequately sterilized acupuncture needles. Histologic features often include suppurative granulomatous inflammation, and microorganisms are rarely visualized with stains for acid-fast bacilli. The differential diagnosis includes granulomatous fungal and non-RGM bacterial infections as well as noninfectious suppurative or sarcoidlike conditions. Because no pathognomonic histologic features exist for cutaneous RGM infections, clinical suspicion and appropriate workup are essential to reach an accurate and timely diagnosis. Most localized cutaneous RGM infections in immunocompetent individuals respond well to either clarithromycin or amikacin, in combination with surgical debridement.

Control of Mycobacterium fortuitum and Mycobacterium intracellulare infections with respect to distinct granuloma formations in livers of BALB/c mice.

Mycobacterium fortuitum is a rapidly growing nontuberculous Mycobacterium that can cause a range of diseases in humans. Complications from M. fortuitum infection have been associated with numerous surgical procedures. A protective immune response against pathogenic mycobacterial infections is dependent on the granuloma formation. Within the granuloma, the macrophage effector response can inhibit bacterial replication and mediate the intracellular killing of bacteria. The granulomatous responses of BALB/c mice to rapidly and slowly growing mycobacteria were assessed in vivo and the bacterial loads in spleens and livers from M. fortuitum and Mycobacterium intracellulare-infected mice, as well as the number and size of granulomas in liver sections, were quantified. Bacterial loads were found to be approximately two times lower in M. fortuitum-infected mice than in M. intracellulare-infected mice and M. fortuitum-infected mice presented fewer granulomas compared to M. intracellulare-infected mice. These granulomas were characterized by the presence of Mac-1+ and CD4+ cells. Additionally, IFN-γmRNA expression was higher in the livers of M. fortuitum-infected mice than in those of M. intracellulare-infected mice. These data clearly show that mice are more capable of controlling an infection with M. fortuitum than M. intracellulare. This capacity is likely related to distinct granuloma formations in mice infected with M. fortuitum but not with M. intracellulare.

Control of Mycobacterium fortuitum and Mycobacterium intracellulare infections with respect to distinct granuloma formations in livers of BALB/c mice

Mycobacterium fortuitum is a rapidly growing nontuberculous Mycobacterium that can cause a range of diseases in humans. Complications from M. fortuitum infection have been associated with numerous surgical procedures. A protective immune response against pathogenic mycobacterial infections is dependent on the granuloma formation. Within the granuloma, the macrophage effector response can inhibit bacterial replication and mediate the intracellular killing of bacteria. The granulomatous responses of BALB/c mice to rapidly and slowly growing mycobacteria were assessed in vivo and the bacterial loads in spleens and livers from M. fortuitum and Mycobacterium intracellulare-infected mice, as well as the number and size of granulomas in liver sections, were quantified. Bacterial loads were found to be approximately two times lower in M. fortuitum-infected mice than in M. intracellulare-infected mice and M. fortuitum-infected mice presented fewer granulomas compared to M. intracellulare-infected mice. These granulomas were characterized by the presence of Mac-1+ and CD4+ cells. Additionally, IFN-γmRNA expression was higher in the livers of M. fortuitum-infected mice than in those of M. intracellulare-infected mice. These data clearly show that mice are more capable of controlling an infection with M. fortuitum than M. intracellulare. This capacity is likely related to distinct granuloma formations in mice infected with M. fortuitum but not with M. intracellulare.

Mycobacterial di-O-acyl trehalose inhibits Th-1 cytokine gene expression in murine cells by down-modulation of MAPK signaling.

Protection against tuberculosis (TB) is based on cell-mediated immune responses. TB is often characterized by immunological dysfunction of peripheral blood mononuclear cells, especially at chronic stages. Lipids from the Mycobacterium tuberculosis cell wall have been shown to produce various suppressive effects on cell-mediated immunity. The cell-surface lipid di-O-acyl-trehalose (DAT) is able to inhibit T-cell proliferation and cytokine secretion in cells from naïve mice. In the present study, we addressed the mechanisms involved in the suppressive effect caused by DAT. We found that DAT decreased the proliferation of spleen cells induced with PMA-ionomycin, suggesting that the suppressive mechanisms target intracellular functions just after phospholipase C-gamma activation. Addressing this possibility, the effect of DAT was found to involve down-modulation of the di-acyl glycerol-dependent activation of the MAPK-ERK1/2 pathway, one of the crucial signaling pathways leading to adaptive cell immune response against TB. Moreover, the inhibitory effect of DAT on proliferation was reproduced in antigen-stimulated T cells from M. tuberculosis-infected mice, involving the lowering of Th1-type cytokine transcription levels. The present findings thus reveal a new kind of bioactivity for a long-known M. tuberculosis cell wall lipid, DAT.

Estudo in situ e in vivo da resposta imuno-inflamatória induzida por M.fortuitum ou M. intracellulare em camundongos balb/c / Study in situ and in vivo immune-inflammatory response induced by M.fortuitum or M. intracellulare in BALB/c

Micobactérias ambientais estão amplamente distribuídas no meio, podendo ser encontradas em água, solo, poeira, alimentos e animais. Com o aumento do número de casos de micobacterioses ocorrendo simultaneamente com infecções por HIV o estudo de micobactérias ambientais tem assumido importância. As manifestações clínicas das infecções causadas por esses patógenos vão desde a abscessos localizados até doenças pulmonares, ou mesmo doenças disseminadas em indivíduos imunocomprometidos. A manifestação da doença, assim como a manutenção da infecção micobacteriana, dependem da interação entre a micobactéria e o sistema imune do hospedeiro. A patogênese da infecção micobacteriana é associada à formação de granulomas, onde pode ocorrer uma resposta protetora do hospedeiro, concomitante com lesão tecidual. Em estudo anterior demonstramos que em macrófagos da linhagem J774E clone estimulados com IFN-y há uma diminuição da viabilidade intracelular de M intracellulare, num processo dependente de NO, enquanto que não houve diferença na viabilidade intracelular de M fortuitum em células estimuladas. O presente trabalho teve como objetivo avaliar a resposta imuno-inflamatória in vivo em camundongos BALB/c infectados por M intracellulare ou M fortuitum. A carga bacilar no baço e fígado desses camundongos foi determinada pelas unidades formadoras de colônias (UFC) nos tempos de 7, 14, 28 e 60 dias após a infecção por uma ou outra espécie de micobactéria. Foi realizada a caracterização da lesão tecidual presente no fígado dos animais infectados, bem corno a expressão de RNAm para citocinas envolvidas na resposta granulomatosa contra micobactérias (IFN-y, TNF-a e IL-10) e iNOS. Essa enzima é importante na eliminação de micobactérias intracelulares, pois induz a produção da molécula microbicida óxido nítrico (NO) pelos macrófagos infectados. Os dados obtidos nesse trabalho mostram que as duas espécies de micobactérias ambientais induzem respostas distintas em camundongos BALB/c. Em todos os tempos avaliados a carga bacilar no baço ou fígados dos animais infectados foi sempre maior no grupo infectado por M intracellulare. Também, nesse grupo, observou-se a presença de numerosos granulomas a partir do 14° dia de infecção, em contraste com o grupo infectado por M fortuitum. A expressão de RNAm para as citocinas IFN-ye IL-I0 foi sempre maior no fígado de animais infectados por Mfortuitum, comparado com o grupo de animais infectados M intracellulare.

Mycobacterial trehalose-containing glycolipid with immunomodulatory activity on human CD4+ and CD8+ T-cells.

Protection against Mycobacterium tuberculosis is based on cell-mediated immunity, most importantly involving CD4+ and CD8+ T-cell subsets. One of the key features of the tubercle bacillus is its cell envelope, characterized by extremely abundant and specific lipids. The cell-surface glycolipid 2,3-di-O-acyl-trehalose (DAT) has been consistently found in M. tuberculosis strains. In this study, analysis of proliferation, activation markers and cytokine release was performed in human peripheral blood mononuclear cells (PBMC) activated in the presence and absence of DAT. We present evidence that mycobacterial DAT is able to reduce antigen-induced proliferation of human CD4+ and CD8+ T-cell subsets. We show that the effect is associated with a decrease of cells expressing the T-cell surface activation markers CD25 and CD69, and down-modulation of IL-2, IL-12, TNF-alpha and IL-10 cytokines. Data indicating that fine acyl chain structural variations in the trehalose-containing lipid may be involved in the degree of immune modulation are also presented.

Plantas do cerrado brasileiro com atividade contra Mycobacterium fortuitum / Brazilian cerrado plants active against Mycobacterium fortuitum

Mycobacterium fortuitum é uma micobactéria de crescimento rápido, ubíquo na natureza e relacionada a micobacteriose de importância médica.Ela tem sido isolada de bacteremias, abscessos, endocardites, feridas cirúrgicas e traumáticas.De difícil tratamento, o bacilo é reconhecido na literatura como resistente inclusive aos medicamentos utilizados na terapêutica da tuberculose.O objetivo deste trabalho foi pesquisar extratos vegetais do Cerrado brasileiro com atividade contra M. fortuitum, empregando a técnica do Microplate Alamar Blue Assay (MABA) como método analítico.Dos 26 extratos testados frente ao M.fortuitum, o extrato apolar de Quassia amara (extrato diclorometanico) foi o que apresentou melhor resultado com valor de CIM de 62,5mg/mL seguidos pelos extratos apolares de Syngonanthus macrolepsis, Davilla elliptica, Turnera ulmifolia com CIM de 125g/mL.Para as mesmas plantas analisadas, utilizando-se agentes extratores polares (etanol e metanol), foram verificados CIM superiores a 500g/mL.Os valores foram semelhantes aos de extratos de outras plantas analisadas sendo considerados não promissores.

Failure of the Mycobacterium bovis BCG vaccine: some species of environmental mycobacteria block multiplication of BCG and induction of protective immunity to tuberculosis.

The efficacy of Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine against pulmonary tuberculosis (TB) varies enormously in different populations. The prevailing hypothesis attributes this variation to interactions between the vaccine and mycobacteria common in the environment, but the precise mechanism has so far not been clarified. Our study demonstrates that prior exposure to live environmental mycobacteria can result in a broad immune response that is recalled rapidly after BCG vaccination and controls the multiplication of the vaccine. In these sensitized mice, BCG elicits only a transient immune response with a low frequency of mycobacterium-specific cells and no protective immunity against TB. In contrast, the efficacy of TB subunit vaccines was unaffected by prior exposure to environmental mycobacteria. Six different isolates from soil and sputum samples from Karonga district in Northern Malawi (a region in which BCG vaccination has no effect against pulmonary TB) were investigated in the mouse model, and two strains of the Mycobacterium avium complex were found to block BCG activity completely.

Mycobacterial di-O-acyl-trehalose inhibits mitogen- and antigen-induced proliferation of murine T cells in vitro.

2,3-Di-O-acyl-trehalose (DAT) is a glycolipid located on the outer layer of the Mycobacterium tuberculosis cell envelope. Due to its noncovalent linkage to the mycobacterial peptidoglycan, DAT could easily interact with host cells located in the focus of infection. The aim of the present work was to study the effects of DAT on the proliferation of murine spleen cells. DAT was purified from reference strains of M. tuberculosis, or M. fortuitum as a surrogate source of the compound, by various chromatography and solvent extraction procedures and then chemically identified. Incubation of mouse spleen cells with DAT inhibited in a dose-dependent manner concanavalin A-stimulated proliferation of the cells. Experiments, including the propidium iodide exclusion test, showed that these effects were not due to death of the cells. Tracking of cell division by labeling with 5,6-carboxyfluorescein diacetate succinimidyl ester revealed that DAT reduces the rounds of cell division. Immunofluorescence with an anti-CD3 monoclonal antibody indicated that T lymphocytes were the population affected in our model. Our experiments also suggest that the extent of the suppressive activity is strongly dependent on the structural composition of the acyl moieties in DATs. Finally, the inhibitory effect was also observed on antigen-induced proliferation of mouse spleen cells specific for Toxoplasma gondii. All of these data suggest that DAT could have a role in the T-cell hyporesponsiveness observed in chronic tuberculosis.

Skin test reactivity to mycobacterial antigens parallels the phylogenetic structure of their genus.

SETTING: City of Manaus, Amazonas, Brazil. OBJECTIVE: To explore the relationship between positivity to tuberculin and other environmental mycobacteria sensitins, according to a range of criteria and presence of BCG scar. DESIGN: Dual skin testing with tuberculin and four mycobacterial sensitins, and BCG scar recording of 1070 schoolchildren aged 7-14. Four criteria for positivity were used: simple and dominant, with 5 and 10 mm cut-off points. RESULTS: The standardised prevalence of reactions > or = 5 mm for BCG scar negative children was 58.3% for Mycobacterium avium, 54.2% for M. scrofulaceum, 26.8% for M. fortuitum, 17.9% for M. tuberculosis and 7.6% for M. kansasii. Correlations between tuberculin and each sensitin, for BCG scar negative children, were 0.47 for M. avium, 0.53 for M. scrofulaceum, 0.60 for M. kansasii and 0.22 for M. fortuitum (all with P < 0.01). BCG effect was particularly significant for tuberculin (odds ratio = 3.44 for reactions > or = 5 mm, P < 0.001) and influenced the balance between dominant/non-dominant reactions for all sensitins. CONCLUSION: The correlation between tuberculin and each sensitin confirmed the separation of the rapidly (M. fortuitum) and slowly growing mycobacteria (M. tuberculosis, M. avium, M. scrofulaceum and M. kansasii). The influence of BCG on tuberculin reactions was more marked than on other mycobacterial sensitins.

Mycobacterium fortuitum-chelonae complex infection in a child with complete interleukin-12 receptor beta 1 deficiency.

A 10-year-old boy had chronic diarrhea, abdominal pain, severe weight loss and hepatomegaly; multiple enlarged para-aortic and mesenteric lymph nodes. Mycobacterium fortuitum-chelonae complex was identified in the culture of the lymph nodes. Interleukin-12 receptor beta 1 expression could not be observed in phytohemagglutinin-driven T cell blasts. A homozygous missense interleukin-12 receptor beta 1 mutation was found (R173P).

Development of tuberculin reactivity and sensitization to M. scrofulaceum and M. fortuitum in children BCG-vaccinated at birth.

Since the incidence of tuberculosis is steadily declining in Finland and infections by environmental mycobacteria may be increasing, the aim of the present study was to evaluate the development of tuberculin reactivity and sensitization to environmental mycobacteria. Healthy Finnish schoolchildren aged 10.4-12.4 yrs (n=201) were tested with tuberculin purified protein derivative RT23, Mycobacterium scrofulaceum RS95 and M. fortuitum RS20 sensitins. The same children had been previously tested with the same antigens and methods at the age of 4-6 yrs in 1989. Rapid waning of tuberculin reactivity and decrease in sensitization to environmental mycobacteria were observed between 4-6 yrs. Both tuberculin and sensitin skin reaction sizes decreased significantly over the 6-yrs period. The mean tuberculin skin reaction size was 3.2 mm in diameter, which was significantly (p<0.001) smaller than the mean induration size (4.8 mm) at the age of 4-6 yrs. Similarly, the mean skin reaction sizes to M. scrofulaceum and M. fortuitum sensitins were 3.4 and 1.7 mm, respectively, which were significantly (p<0.001) smaller than 6 yrs earlier (mean 4.5 and 3.1 mm). The number of zero reactions to all antigens increased significantly during the follow-up period. Contacts with pets or farm animals were associated with larger reactions. In contrast, children suffering from allergic symptoms had smaller reactions. Contacts with mycobacteria, either with Mycobacterium tuberculosis or environmental mycobacteria, seem to be too rare to maintain tuberculin responsiveness and a high sensitivity to other mycobacteria. Different bacille Calmette-Guérin vaccine products and dosages used, the declining incidence of tuberculosis and geographical factors, which can influence environmental mycobacterial exposure, may explain the disparity between the present and previous Finnish studies.

Serotaxonomic analysis of glycolipids from Mycobacterium chelonae-M. fortuitum complex and bovine farcy strains.

The antigenicity and cross-reactivity of glycolipids from strains of bovine farcy and the Mycobacterium chelonae-M. fortuitum complex were analyzed using the ELISA technique. Purified alkali-stable glycopeptidolipids (GPLs) with a characteristic dimethylrhamnosyl sugar unit extracted from M. abscessus, M. chelonae, M. peregrinum and M. senegalense, gave very strong reactions with sera against members of the same four species. Particularly strong cross-reactions were evident between M. peregrinum and M. senegalense. These GPLs reacted more weakly with antisera against the other mycobacteria tested, though clear reactions were noticed with M. farcinogenes and M. fortuitum and also with M. bovis BCG, M. phlei, and M. tuberculosis strains. Alkali-labile diacyl trehalose (DAT) and triacyl trehalose (TAT) from M. fortuitum reacted with homologous sera, and with that against M. tuberculosis. Traces of uncharacterized acyl trehaloses isolated from two strains of M. farcinogenes gave comparatively weak reactions. Mycobacteria labeled M. farcinogenes and M. senegalense produced glucosylated trehalose-based glycolipids (GTs) and the studies showed that the major type was antigenic. These glycolipids cross-reacted strongly with M. senegalense NCTC 4524 but not with the type strain of M. senegalense. On the basis of the chemical patterns and the antigenicity of the GPLs it is evident that M. peregrinum and M. senegalense are particularly closely related and these species show a very close affinity to M. abscessus-M. chelonae.

Patterns of phosphoantigen stimulation of human Vgamma9/Vdelta2 T cell clones include Th0 cytokines.

This paper examines functional properties of human Vgamma9/Vdelta2 T cell lines and clones generated by in vitro culture with synthetic and natural (mycobacterial) phosphoantigenic molecules. It confirms the broad reactivity of Vgamma9/Vdelta2 T cell lines and clones toward phosphoantigens. Optimal recognition of phosphoantigens by Vgamma9/Vdelta2 T cells required accessory cells to occur, but did not require specialized antigen presenting cells. However, species origin of the APC was irrelevant as proliferation of Vgamma9/Vdelta2 T cells occurred in the presence of syngeneic, allogeneic or xenogeneic APC and was not restricted to APC of particular tissue origin. Moreover antigen uptake and processing was not required for recognition by Vgamma9/ Vdelta2 cells, as evidenced by the ability of fixed APCs to present phosphoantigens. Similarly, the expression of classical MHC class I and class II molecules was not required for phosphoantigen recognition by gammadelta T cells. However, gammadelta T cell clones responded to stimulation by several cytokines including IL-12, IFNgamma and TNFalpha. Finally, Vgamma9/Vdelta2 T cell clones preferentially produced both IFN-gamma and IL-4 in response to PHA or TUBAg stimulation, revealing that a Th0 pattern of cytokine production is frequent among these cells.

CD94/NKG2 inhibitory receptor complex modulates both anti-viral and anti-tumoral responses of polyclonal phosphoantigen-reactive V gamma 9V delta 2 T lymphocytes.

Viral, bacterial, protozoal, and cancer-associated Ags elicit strong responses in human gammadelta T lymphocytes. The majority of these cells in the peripheral blood express the Vgamma9Vdelta2-encoded TCR and recognize nonpeptidic phosphoantigens without an apparent MHC restriction. We have shown that Vgamma9Vdelta2 T cells express the inhibitory CD94/NKG2 receptor for HLA class I molecules. The anti-CD94 mAb inhibits 1) the Vgamma9Vdelta2 T cell proliferation in response mycobacterial phosphoantigens and 2) the HIV-induced Vgamma9Vdelta2 T cell expansion. Vgamma9Vdelta2 T cells stimulated with nonpeptidic mycobacterial antigens produce IFN-gamma and TNF-alpha. Signaling through the CD94/NKG2 receptor interferes with the synthesis of these cytokines. The CD94/HLA class I interaction is also involved in the cytotoxic activity of Vgamma9Vdelta2 T cells. The Vgamma9Vdelta2 T cell regulation through the CD94 receptor may be important for the potentially dual function in innate immunity, i.e., 1) NK-like and 2) TCR ligand-induced cytolytic activities.