Mycobacterium tuberculosis subverts innate immunity to evade specific effectors.

The macrophage is the niche of the intracellular pathogen Mycobacterium tuberculosis. Induction of macrophage apoptosis by CD4(+) or CD8(+) T cells is accompanied by reduced bacterial counts, potentially defining a host defense mechanism. We have already established that M. tuberculosis-infected primary human macrophages have a reduced susceptibility to Fas ligand (FasL)-induced apoptosis. To study the mechanisms by which M. tuberculosis prevents apoptotic signaling, we have generated a cell culture system based on PMA- and IFN-gamma-differentiated THP-1 cells recapitulating the properties of primary macrophages. In these cells, nucleotide-binding oligomerization domain 2 or TLR2 agonists and mycobacterial infection protected macrophages from apoptosis and resulted in NF-kappaB nuclear translocation associated with up-regulation of the antiapoptotic cellular FLIP. Transduction of a receptor-interacting protein-2 dominant-negative construct showed that nucleotide-binding oligomerization domain 2 is not involved in protection in the mycobacterial infection system. In contrast, both a dominant-negative construct of the MyD88 adaptor and an NF-kappaB inhibitor abrogated the protection against FasL-mediated apoptosis, showing the implication of TLR2-mediated activation of NF-kappaB in apoptosis protection in infected macrophages. The apoptosis resistance of infected macrophages might be considered as an immune escape mechanism, whereby M. tuberculosis subverts innate immunity signaling to protect its host cell against FasL(+)-specific cytotoxic lymphocytes.

Mykobakteerien lajinmääritykseen käytettävän kitin validointi

Tämä opinnäytetyö tehtiin Elintarviketurvallisuusvirasto Eviran Helsingin yksikön eläintautibakteriologian ryhmälle. Työn tarkoituksena oli löytää ja ottaa käyttöön tämänhetkisen AccuProbe-menetelmän lisäksi monipuolisempi menetelmä mykobakteerien lajinmääritykseen. AccuProbe-testillä pystytään tunnistamaan suurin osa Eviraan tulleista näytteistä eristetyistä mykobakteereista, mutta muun muassa kalanäytteistä eristettyjen mykobakteerien tunnistamiseen ei ole ollut menetelmää. Työhön valittiin GenoType® Mycobacterium CM -kitti. Mykobakteerien lajinmääritykseen kehitetyn GenoType® Mycobacterium CM -kitin käyttöä opeteltiin aluksi Mycobacterium avium ATCC 25291 -kannalla. Tämän jälkeen kittiä kokeiltiin eläimistä eristetyillä sekä M. intracellulare ATCC 13950- ja M. tuberculosis ATCC 25177 -kannoilla. Kyseessä on hybridisaatioon perustuva DNA-liuska-menetelmä, joka sisältää kolme päävaihetta: DNA:n eristäminen, monistaminen PCR:llä ja hybridisaatio. Työn aikana optimoitiin substraatin vaikutusaikoja, seurattiin hybrisaatiovaiheen lämpötiloja ja kehitettiin kitin valmistajan suosittelemaa ravistelevaa lämpöhaudetta / inkubaattoria vastaavia laitteita. Työn tuloksena kitti on käyttövalmis, mutta menetelmän soveltuvuutta eläinkannoille täytyy testata lisää. Osassa tulosliuskoissa oli vääriä vaaleita viivoja ja osa oikeista vaaleista viivoista puuttui. Saadut tulokset antavat kuitenkin hyvän pohjan jatkaa kitin validointia.

Safety of human immunisation with a live-attenuated Mycobacterium tuberculosis vaccine: a randomised, double-blind, controlled phase I trial.

BACKGROUND: Tuberculosis remains one of the world's deadliest transmissible diseases despite widespread use of the BCG vaccine. MTBVAC is a new live tuberculosis vaccine based on genetically attenuated Mycobacterium tuberculosis that expresses most antigens present in human isolates of M tuberculosis. We aimed to compare the safety of MTBVAC with BCG in healthy adult volunteers. METHODS: We did this single-centre, randomised, double-blind, controlled phase 1 study at the Centre Hospitalier Universitaire Vaudois (CHUV; Lausanne, Switzerland). Volunteers were eligible for inclusion if they were aged 18-45 years, clinically healthy, HIV-negative and tuberculosis-negative, and had no history of active tuberculosis, chemoprophylaxis for tuberculosis, or BCG vaccination. Volunteers fulfilling the inclusion criteria were randomly assigned to three cohorts in a dose-escalation manner. Randomisation was done centrally by the CHUV Pharmacy and treatments were masked from the study team and volunteers. As participants were recruited within each cohort, they were randomly assigned 3:1 to receive MTBVAC or BCG. Of the participants allocated MTBVAC, those in the first cohort received 5 × 10(3) colony forming units (CFU) MTBVAC, those in the second cohort received 5 × 10(4) CFU MTBVAC, and those in the third cohort received 5 × 10(5) CFU MTBVAC. In all cohorts, participants assigned to receive BCG were given 5 × 10(5) CFU BCG. Each participant received a single intradermal injection of their assigned vaccine in 0·1 mL sterile water in their non-dominant arm. The primary outcome was safety in all vaccinated participants. Secondary outcomes included whole blood cell-mediated immune response to live MTBVAC and BCG, and interferon γ release assays (IGRA) of peripheral blood mononuclear cells. This trial is registered with ClinicalTrials.gov, number NCT02013245. FINDINGS: Between Jan 23, 2013, and Nov 6, 2013, we enrolled 36 volunteers into three cohorts, each of which consisted of nine participants who received MTBVAC and three who received BCG. 34 volunteers completed the trial. The safety of vaccination with MTBVAC at all doses was similar to that of BCG, and vaccination did not induce any serious adverse events. All individuals were IGRA negative at the end of follow-up (day 210). After whole blood stimulation with live MTBVAC or BCG, MTBVAC was at least as immunogenic as BCG. At the same dose as BCG (5×10(5) CFU), although no statistical significance could be achieved, there were more responders in the MTBVAC group than in the BCG group, with a greater frequency of polyfunctional CD4+ central memory T cells. INTERPRETATION: To our knowledge, MTBVAC is the first live-attenuated M tuberculosis vaccine to reach clinical assessment, showing similar safety to BCG. MTBVAC seemed to be at least as immunogenic as BCG, but the study was not powered to investigate this outcome. Further plans to use more immunogenicity endpoints in a larger number of volunteers (adults and adolescents) are underway, with the aim to thoroughly characterise and potentially distinguish immunogenicity between MTBVAC and BCG in tuberculosis-endemic countries. Combined with an excellent safety profile, these data support advanced clinical development in high-burden tuberculosis endemic countries. FUNDING: Biofabri and Bill & Melinda Gates Foundation through the TuBerculosis Vaccine Initiative (TBVI).

Genomic and transcriptomic analysis of the streptomycin-dependent Mycobacterium tuberculosis strain 18b.

The ability of Mycobacterium tuberculosis to establish a latent infection (LTBI) in humans confounds the treatment of tuberculosis. Consequently, there is a need to discover new therapeutic agents that can kill M. tuberculosis both during active disease and LTBI. The streptomycin-dependent strain of M. tuberculosis, 18b, provides a useful tool for this purpose since upon removal of streptomycin (STR) it enters a non-replicating state that mimics latency both in vitro and in animal models. The 4.41 Mb genome sequence of M. tuberculosis 18b was determined and this revealed the strain to belong to clade 3 of the ancient ancestral lineage of the Beijing family. STR-dependence was attributable to insertion of a single cytosine in the 530 loop of the 16S rRNA and to a single amino acid insertion in the N-terminal domain of initiation factor 3. RNA-seq was used to understand the genetic programme activated upon STR-withdrawal and hence to gain insight into LTBI. This revealed reconfiguration of gene expression and metabolic pathways showing strong similarities between non-replicating 18b and M. tuberculosis residing within macrophages, and with the core stationary phase and microaerophilic responses. The findings of this investigation confirm the validity of 18b as a model for LTBI, and provide insight into both the evolution of tubercle bacilli and the functioning of the ribosome.

Mycobacterium tuberculosis Infection in Young Children: Analyzing the Performance of the Diagnostic Tests

This study evaluated the performance of the Tuberculin Skin Test (TST) and Quantiferon-TB Gold in-Tube (QFT) and the possible association of factors which may modify their results in young children (0-6 years) with recent contact with an index tuberculosis case. Materials and Methods: A cross-sectional study including 135 children was conducted in Manaus, Amazonas-Brazil. The TST and QFT were performed and the tests results were analyzed in relation to the personal characteristics of the children studied and their relationship with the index case. Results: The rates of positivity were 34.8% (TST) and 26.7% (QFT), with 14.1% of indeterminations by the QFT. Concordance between tests was fair (Kappa = 0.35 P<0.001). Both the TST and QFT were associated with the intensity of exposure (Linear OR = 1.286, P = 0.005; Linear OR = 1.161, P = 0.035 respectively) with only the TST being associated with the time of exposure (Linear OR = 1.149, P = 0.009). The presence of intestinal helminths in the TST+ group was associated with negative QFT results (OR = 0.064, P = 0.049). In the TST- group lower levels of ferritin were associated with QFT+ results (Linear OR = 0.956, P = 0.036). Conclusions: Concordance between the TST and QFT was lower than expected. The factors associated with the discordant results were intestinal helminths, ferritin levels and exposure time to the index tuberculosis case. In TST+ group, helminths were associated with negative QFT results suggesting impaired cell-mediated immunity. The TST-&QFT+ group had a shorter exposure time and lower ferritin levels, suggesting that QFT is faster and ferritin may be a potential biomarker of early stages of tuberculosis infection.

Role of PheE15 gate in ligand entry and nitric oxide detoxification function of Mycobacterium tuberculosis truncated hemoglobin N

The truncated hemoglobin N, HbN, of Mycobacterium tuberculosis is endowed with a potent nitric oxide dioxygenase (NOD) activity that allows it to relieve nitrosative stress and enhance in vivo survival of its host. Despite its small size, the protein matrix of HbN hosts a two-branched tunnel, consisting of orthogonal short and long channels, that connects the heme active site to the protein surface. A novel dual-path mechanism has been suggested to drive migration of O(2) and NO to the distal heme cavity. While oxygen migrates mainly by the short path, a ligand-induced conformational change regulates opening of the long tunnel branch for NO, via a phenylalanine (PheE15) residue that acts as a gate. Site-directed mutagenesis and molecular simulations have been used to examine the gating role played by PheE15 in modulating the NOD function of HbN. Mutants carrying replacement of PheE15 with alanine, isoleucine, tyrosine and tryptophan have similar O(2)/CO association kinetics, but display significant reduction in their NOD function. Molecular simulations substantiated that mutation at the PheE15 gate confers significant changes in the long tunnel, and therefore may affect the migration of ligands. These results support the pivotal role of PheE15 gate in modulating the diffusion of NO via the long tunnel branch in the oxygenated protein, and hence the NOD function of HbN.

Evaluation of anti-Mycobacterium tuberculosis activity of Campomanesia adamantium (Myrtaceae)

The anti-Mycobacterium tuberculosis activity of Campomanesia adamantium fruits extracts were evaluated. Six compounds, identified as flavanones and chalcones were quantified by HPLC-DAD-UV. Promising antitubercular activity was observed with ethyl acetate extract (MIC 62.5 µg/mL) and their fractions (MIC values ranging from 39 to above 250 µg/mL). The better MIC result of 39 µg/mL was associated with two fractions that contain bigger amounts of 5,7-dihydroxy-6, 8-di-C-methylflavanone and 2',4'-dihydroxy-3',5'-dimethyl-6'-methoxychalcone. These compounds exhibited MICs >250 and 62.5 µg/mL, respectively, while their mixtures showed values ranging from 62.5 to 7.8 µg/mL, demonstrating a synergism between them.

Evaluation of Mycobacterium avium subsp. paratuberculosis faecal culture protocols and media

Paratuberculosis is an important enteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis (Map). The disease is officially considered exotic in Brazil, but recent serological surveys and the isolation of the agent suggest it may occur in our herds. The aim of this study was to evaluate three different formulations of Herrold's egg yolk agar with mycobactin J (HEYM) and four faecal culture protocols considering their ability for Map growth as well as cost and ease of application. Three formulations of HEYM were inoculated with two suspensions of Map. Spiked faeces and naturally contaminated faecal samples were treated by the four faecal culture protocols. Centrifugation protocol and HEYM recommended by OIE showed the best results on the recovery of Map.

Resposta imune específica de bovinos experimentalmente sensibilizados com inóculos inativados de Mycobacterium bovis e Mycobacterium avium

O diagnóstico presuntivo da tuberculose bovina é baseado na análise da resposta imune celular a antígenos micobacterianos. Procedeu-se à simulação experimental de sensibilização por Mycobacterium bovis e Mycobacterium avium inativados em bovinos a fim de acompanhar a resposta imune a partir do teste cervical comparativo e da evolução da produção específica de interferon-gama, além de identificar a interferência de reações inespecíficas por M. avium nos resultados dos testes. Verificou-se que os animais desencadearam resposta de hipersensibilidade tardia contra os bacilos inativados, e que ambos os testes diagnósticos da tuberculose bovina foram eficientes na identificação dos animais sensibilizados com M. bovis e na discriminação das reações geradas pela inoculação dos bovinos com M. avium.

Linfadenite granulomatosa em suínos: linfonodos afetados e diagnóstico patológico da infecção causada por agentes do Complexo Mycobacterium avium

Realizou-se um estudo com o objetivo de avaliar a distribuição de lesões granulomatosas nos linfonodos das carcaças de suínos abatidos e de comparar os métodos de isolamento do Complexo Mycobacterium avium (MAC), coloração de Ziehl-Neelsen (ZN), exames histopatológico e de imunohisto-química (IHQ) para o diagnóstico da linfadenite granulomatosa causada por micobactérias do MAC. Foram utilizadas 431 amostras de linfonodos colhidos de 394 carcaças de suínos abatidos em 12 frigoríficos da Região Sul do Brasil, com o Serviço de Inspeção Federal (SIF). Os linfonodos que apresentavam lesões granulomatosas foram submetidos aos exames histológicos, ZN e IHQ com anticorpo monoclonal produzido com extrato celular de M. avium. A concordância entre os exames foi medida pelo teste Kappa, com nível de confiança de 95%. O exame macroscópico realizado pelo SIF identifica corretamente 90,3% das lesões granulomatosas, quando comparado com o exame histológico e a maioria das carcaças (92,5) apresentam lesões apenas nos linfonodos cadeia alimentar. O exame histológico confirmou a presença de lesões granulo-matosas em 90,3% dos linfonodos. As concordâncias entre os exames histopatológico e coloração de ZN (Kappa: 0,342) e de IHQ e o isolamento do MAC (Kappa: 0,102) foram baixas, porém alta entre os exames de IHQ e histológico com a presença de granulomas típicos nos linfonodos (Kappa: 0,973). O exame de IHQ associado ao exame histopatológico mostrou-se eficiente na identificação das lesões de linfadenite granulomatosa causadas pelo MAC.

Comparação de duas técnicas de isolamento do Mycobacterium avium subsp. paratuberculosis em amostras de fezes de ovinos com suspeita clínica de paratuberculose

A paratuberculose é uma enterite crônica granulomatosa causada por Mycobacterium avium subsp. paratuberculosis que afeta principalmente os ruminantes. A cultura de bactérias a partir de amostras de fezes e tecidos constitui um dos métodos mais eficazes de diagnóstico, sendo ainda o único método disponível para obtenção de isolamentos e estirpes de micobactérias. Contudo, este método apresenta baixa sensibilidade e requer meses de incubação antes do crescimento de colônias. Neste estudo, utilizou-se a cultura fecal como método de diagnóstico em ovinos de diferentes raças portuguesas, com sinais compatíveis com a doença. Fez-se ainda a comparação entre os meios de cultura Löwenstein Jensen® com micobactina® J e o de Middlebrook® 7H11 com OADC®, utilizados no isolamento da bactéria. As percentagens de isolamento em cada um os meios foram de 2,0% (6/300) para Löwenstein Jensen® com micobactina J e 1,0% (3/300) para Middlebrook® 7H11/OADC. As três amostras positivas no meio de Middlebrook® 7H11/OADC também foram positivas no meio de Löwenstein Jensen® com micobactina J e nenhuma foi somente positiva no meio de Middlebrook® 7H11/OADC. Os resultados deste estudo sugerem que o meio de Löwenstein-Jensen® com micobactina® J é mais efetivo para a obtenção de estirpes ovinas em Portugal.

A seroepidemiological survey of Mycobacterium avium subsp. paratuberculosis in sheep from North of Portugal

The aim of this study was evaluate the risk factors for Mycobacterium avium subsp. paratuberculosis (Map) seroprevalence in sheep in the North of Portugal. The effects on seroprevalence of several variables such as individual characteristics, management practices, farm characteristics, animal health, and available veterinary services were evaluated. This information was then used in a multivariable logistic regression model in order to identify risk factors for Map seropositivity. Univariable analysis was used to screen the variables used in the logistic regression model. Variables that showed p values of <0.15 were retained for the multivariable analysis. Fifteen variables were associated with paratuberculosis in univariable analysis. The multivariable logistic regression model identified a number of variables as risk factors for seropositivity like sheep pure local and/or a cross of a local breed (OR=2.02), herd size with 31-60 head (OR=2.14), culling during the Spring-Summer season (OR=1.69) and the use of an anti-parasitic treatment such as Ivermectin as the only anti-parasitic medication (OR=5.60). Potential risk factors identified in this study support current recommendations for the control of paratuberculosis.