Increased Virulence of an Epidemic Strain of Mycobacterium massiliense in Mice

Chronic pulmonary disease and skin/soft tissue infections due to non-tuberculous mycobacteria (NTM) of the Mycobacterium chelonae-abscessus-massiliense group is an emerging health problem worldwide. Moreover, the cure rate for the infections this group causes is low despite aggressive treatment. Post-surgical outbreaks that reached epidemic proportions in Brazil recently were caused by M. massiliense isolates resistant to high-level disinfection with glutaraldehyde (GTA). Understanding the differences in the virulence and host immune responses induced by NTM differing in their sensitivity to disinfectants, and therefore their relative threat of causing outbreaks in hospitals, is an important issue.We compared the replication and survival inside macrophages of a GTA-susceptible reference Mycobacterium massiliense clinical isolate CIP 108297 and an epidemic strain from Brazil, CRM-0019, and characterized the immune responses of IFNγ knockout mice exposed to a high dose aerosol with these two isolates. CRM-0019 replicated more efficiently than CIP 108297 inside mouse bone marrow macrophages. Moreover, the animals infected with CRM-0019 showed a progressive lung infection characterized by a delayed influx of CD4+ and CD8+ T cells, culminating in extensive lung consolidation and demonstrated increased numbers of pulmonary CD4+ Foxp3+ regulatory T cells compared to those infected with the reference strain. Immunosuppressive activity of regulatory T cells may contribute to the progression and worsening of NTM disease by preventing the induction of specific protective immune responses.These results provide the first direct evidence of the increased virulence in macrophages and mice and pathogenicity in vivo of the Brazilian epidemic isolate and the first observation that NTM infections can be associated with variable levels of regulatory T cells which may impact on their virulence and ability to persist in the host

Cell Surface Remodeling of Mycobacterium abscessus under Cystic Fibrosis Airway Growth Conditions.

Understanding the physiological processes underlying the ability of Mycobacterium abscessus to become a chronic pathogen of the cystic fibrosis (CF) lung is important to the development of prophylactic and therapeutic strategies to better control and treat pulmonary infections caused by these bacteria. Gene expression profiling of a diversity of M. abscessus complex isolates points to amino acids being significant sources of carbon and energy for M. abscessus in both CF sputum and synthetic CF medium and to the bacterium undergoing an important metabolic reprogramming in order to adapt to this particular nutritional environment. Cell envelope analyses conducted on the same representative isolates further revealed unexpected structural alterations in major cell surface glycolipids known as the glycopeptidolipids (GPLs). Besides showing an increase in triglycosylated forms of these lipids, CF sputum- and synthetic CF medium-grown isolates presented as yet unknown forms of GPLs representing as much as 10% to 20% of the total GPL content of the cells, in which the classical amino alcohol located at the carboxy terminal of the peptide, alaninol, is replaced with the branched-chain amino alcohol leucinol. Importantly, both these lipid changes were exacerbated by the presence of mucin in the culture medium. Collectively, our results reveal potential new drug targets against M. abscessus in the CF airway and point to mucin as an important host signal modulating the cell surface composition of this pathogen

Functional drug screening reveals anticonvulsants as enhancers of mTOR-independent autophagic killing of Mycobacterium tuberculosis through inositol depletion.

Mycobacterium tuberculosis (MTB) remains a major challenge to global health made worse by the spread of multidrug resistance. We therefore examined whether stimulating intracellular killing of mycobacteria through pharmacological enhancement of macroautophagy might provide a novel therapeutic strategy. Despite the resistance of MTB to killing by basal autophagy, cell-based screening of FDA-approved drugs revealed two anticonvulsants, carbamazepine and valproic acid, that were able to stimulate autophagic killing of intracellular M. tuberculosis within primary human macrophages at concentrations achievable in humans. Using a zebrafish model, we show that carbamazepine can stimulate autophagy in vivo and enhance clearance of M. marinum, while in mice infected with a highly virulent multidrug-resistant MTB strain, carbamazepine treatment reduced bacterial burden, improved lung pathology and stimulated adaptive immunity. We show that carbamazepine induces antimicrobial autophagy through a novel, evolutionarily conserved, mTOR-independent pathway controlled by cellular depletion of myo-inositol. While strain-specific differences in susceptibility to in vivo carbamazepine treatment may exist, autophagy enhancement by repurposed drugs provides an easily implementable potential therapy for the treatment of multidrug-resistant mycobacterial infection

Stepwise pathogenic evolution of Mycobacterium abscessus.

Although almost all mycobacterial species are saprophytic environmental organisms, a few, such as Mycobacterium tuberculosis, have evolved to cause transmissible human infection. By analyzing the recent emergence and spread of the environmental organism M. abscessus through the global cystic fibrosis population, we have defined key, generalizable steps involved in the pathogenic evolution of mycobacteria. We show that epigenetic modifiers, acquired through horizontal gene transfer, cause saltational increases in the pathogenic potential of specific environmental clones. Allopatric parallel evolution during chronic lung infection then promotes rapid increases in virulence through mutations in a discrete gene network; these mutations enhance growth within macrophages but impair fomite survival. As a consequence, we observe constrained pathogenic evolution while person-to-person transmission remains indirect, but postulate accelerated pathogenic adaptation once direct transmission is possible, as observed for M. tuberculosis Our findings indicate how key interventions, such as early treatment and cross-infection control, might restrict the spread of existing mycobacterial pathogens and prevent new, emergent ones

Non-Tuberculous Mycobacteria Interference with BCG-Current Controversies and Future Directions

The global tuberculosis (TB) epidemic caused by the bacterial pathogen Mycobacterium tuberculosis (M.tb) continues unabated. The Mycobacterium bovis bacillus Calmette–Guérin (BCG) vaccination is widely utilized worldwide to protect against infection with M.tb. BCG vaccine protection against TB has had widely varying results for reasons that are not well understood. BCG vaccine interference by non-tuberculosis (NTM) mycobacterial species has been implicated as the potential cause of reduced BCG vaccine efficacy against M.tb. Ongoing efforts to develop new vaccines for TB requires a thorough understanding of the effect of NTM exposure on BCG vaccine efficacy, which may ultimately be a critical determinant of success. We reviewed the conflicting reports on whether NTM interferes with the BCG vaccine, potential explanations to help resolve the controversy, and strategies for developing better animal models. Further studies are needed to longitudinally track the effects of NTM exposure on BCG vaccine-induced host-protective anti-TB immunity

[Increased IL-4 production in response to virulent Mycobacterium tuberculosis in tuberculosis patients with advanced disease].

The study was designed to compare immune responses to Mycobacterium tuberculosis bacilli and antigens in healthy Portuguese subjects and pulmonary tuberculosis patients (TB), and to correlate immune status with clinical severity of tuberculosis disease. PBMC were cultured and stimulated with live and killed M. tuberculosis H37Rv and purified protein derivative (PPD) and lymphoproliferation and production of IFN-gamma and IL-5/IL-4 by these cultures were evaluated by the use of ELISA and multi-parameter flow cytometry. PBMC from 30 tuberculosis patients demonstrated significantly reduced amounts of proliferation and IFN-gamma when stimulated with live M. tuberculosis compared the control group. Of 15 tuberculosis patients tested for intracellular IL-4 following stimulation with M. tuberculosis, 7 showed greatly increased IL-4 production in CD8+ and gammadelta+ T cells. Tuberculosis patients demonstrated an increase of intracellular IL-4 after PBMC were stimulated with live M. tuberculosis in the CD4+ phenotype, but more notably in CD8+ and gammadelta TCR+ subsets. Increased production of IL-4 in tuberculosis patients was primarily in individuals with advanced involvement of lung parenchymal with high bacterial loads in sputum. These results suggest that an alteration in type 1 and type 2 cytokine balance can occur in patients with tuberculosis at an advanced clinical stage of disease

Gr1(int)CD11b+ myeloid-derived suppressor cells in Mycobacterium tuberculosis infection.

Tuberculosis is one of the world's leading killers, stealing 1.4 million lives and causing 8.7 million new and relapsed infections in 2011. The only vaccine against tuberculosis is BCG which demonstrates variable efficacy in adults worldwide. Human infection with Mycobacterium tuberculosis results in the influx of inflammatory cells to the lung in an attempt to wall off bacilli by forming a granuloma. Gr1(int)CD11b(+) cells are called myeloid-derived suppressor cells (MDSC) and play a major role in regulation of inflammation in many pathological conditions. Although MDSC have been described primarily in cancer their function in tuberculosis remains unknown. During M. tuberculosis infection it is crucial to understand the function of cells involved in the regulation of inflammation during granuloma formation. Understanding their relative impact on the bacilli and other cellular phenotypes is necessary for future vaccine and drug design.We compared the bacterial burden, lung pathology and Gr1(int)CD11b(+) myeloid-derived suppressor cell immune responses in M. tuberculosis infected NOS2-/-, RAG-/-, C3HeB/FeJ and C57/BL6 mice. Gr-1(+) cells could be found on the edges of necrotic lung lesions in NOS2-/-, RAG-/-, and C3HeB/FeJ, but were absent in wild-type mice. Both populations of Gr1(+)CD11b(+) cells expressed high levels of arginase-1, and IL-17, additional markers of myeloid derived suppressor cells. We then sorted the Gr1(hi) and Gr1(int) populations from M. tuberculosis infected NOS-/- mice and placed the sorted both Gr1(int) populations at different ratios with naïve or M. tuberculosis infected splenocytes and evaluated their ability to induce activation and proliferation of CD4+T cells. Our results showed that both Gr1(hi) and Gr1(int) cells were able to induce activation and proliferation of CD4+ T cells. However this response was reduced as the ratio of CD4(+) T to Gr1(+) cells increased. Our results illustrate a yet unrecognized interplay between Gr1(+) cells and CD4(+) T cells in tuberculosis

Respostas Th1 e Th2 desencadeadas por Mycobacterium tuberculosis virulento em doentes com tuberculose pulmonar

RESUMO: Analisaram-se as respostas Th1 e Th2 desencadeadas por Mycobacterium tuberculosis virulento em doentes com tuberculose pulmonar (TP) e em dadores saudáveis vacinados pela BCG. Efectuaramse comparações entre a capacidade que as células T apresentavam para proliferar e para produzir IFN-γ e IL-5 em resposta aos derivados de proteíns purificada (PPD), M. tuberculosis H37Rv (Mtb), e M. tuberculosis H37Rv inactivado pelo calor (hk Mtb).Este estudo demoostrou que os individuos saudá-vels vacínados com BCG evidenciaram um máximo de proliferação e produção de IFN-γ em resposta ao painel de antigénios, e que Mtb vívo destencadeou uma resposta significativamente mais forte que a obtida pelo Mtb inactivado pelo calor. Embora os doentes com tuberculose pulmonary mostrassem respostas medias mais baxies de proliferação e produção de IFN-γ cm relação aos eontrolos saúdaveis, a resposta proliferativa ao PPD não foi significativamente reduzida, enquanto que a resposta ao Mtb e hk Mtb foram, significativas estatisticamitante. Em conclusão na tuberculose pulmonar a produção de IFN-γ pode estar reduzida sem um concomitante aumento de IL-5, confirmandose assim não existir uma mudança de resposta Th1 para Th2 na tuberculose pulmonar.REV PORT PNEUMOL 1998; IV (4):393-402 ABSTRACT: The contribution of Th1 and Th2 responses elicited by virulent Mycobacterium tuberculosis was investigated, in healthy BCG vaccinated individuals and pulmonary tuberculosis patients. Comparisons were made between the T cell capacity to proliferate, produce IFN-γ and IL-5 in response to the soluable antigen purified protein derivative (PPD), live M. tuberculosis H37Rv (Mtb) and heat killed M tuberculosis H37Rv (hk Mtb). These studies demonstrated that control individuals showed the strongest mean proliferative and IFN-γ responses towards the antigen antigen panel and Mth elicited a significantly stronger response than hk Mtb. Although, pulmonary tuberculosis patients showed lower mean proliferation and IFN-γ towards all the antigen when compared to that control group, proliferative responses to PPD were found not to be significantly reduced, while the reduction in the response to Mtb and hk Mtb preparations were statistically significant. In conclusion, in pulmonary tuberculose infection mean prodution of IFN-γ may be reduced but no concomitant increase in IL-5 production occurred. These data confirm that there is no switch from a Th1 response to a Th2 response in tuberculosis infection.REV PORT PNEUMOL 1998; IV (4): 393-402 Key-words: Cellular Immunity, Cytokines, Pulmonary tuberculoses, Th1 and Th2 responses, Palavaras-chave: Imunidade Celular, Citocinas, Tuberculose Pulmonar, respostas Th1 e Th

Respostas das citocinas T 2 desencadeadas por Mycobacterium tuberculosis virulento nos doentes com tuberculose pulmonar em estado avançado

RESUMO: Avaliaram-se in vitro as respostas das citocinas Tipo 1 e Tipo 2 de dadores portugueses saudáveis vacinados à nascença com BCG, com Mantoux positivo (induraçãoâ¥5 mm) e de doentes com tuberculose pulmonar (TP). Foi efectuado o estudo por ELISA da produção de IFN-γ e IL-5 por Células Mononucleares do Sangue Periférico (CMSP) dos dadores após estimulação com M. tuberculosis e antigénio solúvel PPD. Foi confirmada a presença intracelular de IFN-γ e de IL-4 em subpopulações de células T por análise multiparamétrica, em citometria de fluxo. As CMSP dos doentes com tuberculose estimuladas com PPD e M. tuberculosis demonstraram uma diminuição na produção de IFN-γ sem aumento da produção de IL-5 em resposta ao painel de antigénios. Nos doentes com tuberculose observou-se uma frequência diminuída de IFN-γ intracelular nas células T CD4+ e CD8+, em comparação com os grupos controlos. Após estimulação das CMSP com M. tuberculosis, os doentes demonstraram um aumento médio de IL-4 intracelular no fenótipo T CD4+, mais evidente na subpopulação T CD8+. O aumento da secreção de IL-4 nos doentes com tuberculose verificou-se sobretudo nos indivíduos em estado avançado da doença. Os resultados obtidos por Citometria de Fluxo contradizem de alguma forma os obtidos por ELISA. A secreção de IL-4 intracelular representa uma medida mais sensível da produção de citocinas tipo 2 do que a quantificação de IL-5 por técnicas de ELISA. Estes novos resultados sugerem que pode ocorrer uma mudança de T 1 para T 2 em doentes com tuberculose em estado avançado.REV PORT PNEUMOL 2001; VII (2): ABSTRACT: In vitro Type 1 and Type 2 cytokine responses were assessed in healthy Portuguese donors who were BGC vaccinated at birth and Mantoux positive (induration 5â¥mm) and pulmonary tuberculosis patients (TB). We evaluated the production of IFN-γ and IL-5, after PBMC from donors were stimulated with live M. tuberculosis H37Rv and the soluble antigen PPD, by standard ELISA techniques. These studies were extended to confirm intracellular presence of IFN-γ and IL-4 in specific T cell subsets by multi-parameter flow cytometry. PBMC from tuberculosis patients demonstrated significantly reduced amounts of IFN-γ when stimulated with PPD and M. tuberculosis, with no increase in IL-5 production towards all the antigens, when compared to the control group. Intracellular staining for IFN-γ in tuberculosis patients showed reduced frequencies of CD4+ and CD8+ T cell intracellular IFN-γ in comparison to healthy subjects. Tuberculosis patients demonstrated a mean increase of intracellular IL-4 after PBMC were stimulated with M. tuberculosis in the CD4+ phenotype, but more notably in the CD8+ subset. The increased secretion of IL-4 in tuberculosis patients was primarily in individuals with an advanced clinical form of the disease. Interestingly the findings using flow cytometry techniques somewhat contradicts the results obtained by ELISA. Intracellular IL-4 secretion is therefore a more sensitive measure of Type 2 cytokine production than quantitation of IL-5 by ELISA. These results suggest that a type 1 switch to a type 2 can occur, in patients with tuberculosis in an advanced stage.REV PORT PNEUMOL 2001; VII (2): Palavras-chave: Imunidade Celular, Tuberculose Pulmonar, Respostas de citocinas Tipo 1 e Tipo 2, Key-words: Cellular Immunity, Pulmonary tuberculosis, Type 1 and Type 2 Cytokine response