Structural Basis of RNA Recognition by Mycobacterium tuberculosis MazF Homologues

The MazEF toxin-antitoxin (TA) system consists of the antitoxin MazE and the toxin MazF. MazF is a sequence-specific endoribonuclease that upon activation causes cellular growth arrest and increass the level of persisters. Moreover, MazF-induced cells are in a quasi-dormant state that cells remain metabolically active while stop dividing. The quasi-dormancy is similar to the nonreplicating state of M. tuberculosis during latent tuberculosis, thus suggesting the role of mazEF in M. tuberculosis dormancy and persistence. M. tuberculosis has nine mazEF TA modules, each with different RNA cleavage specificities and implicated in selective gene expression during stress conditions. To date only the Bacillus subtilis MazF-RNA complex structure has been determined. As M. tuberculosis MazF homologues recognize distinct RNA sequences, their molecular mechanisms of substrate specificity remain unclear. By taking advantage of X-ray crystallography, we have determined structures of two M. tuberculosis MazF-RNA complexes, MazF-mt1 (Rv2801c) and MazF-mt3 (Rv1991c) in complex with an uncleavable RNA substrate. These structures have provided the molecular basis of sequence-specific RNA recognition and cleavage by MazF toxins.

Both MazF-mt1-RNA and MazF-mt3-RNA complexes showed similar structural organization with one molecule of RNA bound to a MazF-mt1 or MazF-mt3 dimer and occupying the same pocket within the MazF dimer interface. Similar to B. subtilis MazF-RNA complex, MazF-mt1 and MazF-mt3 displayed a conserved active site architecture, where two highly conserved residues, Arg and Thr, form hydrogen bonds with the scissile phosphate group in the cleavage site of the bound RNA. The MazF-mt1-RNA complex also showed specific interactions with its three-base RNA recognition element. Compared with the B. subtilis MazF-RNA complex, our structures showed that residues involved in sequence-specific recognition of target RNA vary between the MazF homologues, therefore explaining the molecular basis for their different RNA recognition sequences. In addition, local conformational changes of the loops in the RNA binding site of MazF-mt1 appear to play a role in MazF targeting different RNA lengths and sequences. In contrast, the MazF-mt3-RNA complex is in a non-optimal RNA binding state with a symmetry-related MazF-mt3 molecule found to make interactions with the bound RNA in the crystal. The crystal-packing interactions were further examined by isothermal titration calorimetry (ITC) studies on selected MazF-mt3 mutants. Our attempts to utilize a MazF-mt3 mutant bearing mutations involved in crystal contacts all crystallized with few nucleotides, which are still found to interact with a symmetry mate. However, these different crystal forms revealed the conformational flexibility of loops in the RNA binding interface of MazF-mt3, suggesting their role in RNA binding and recognition, which will require further studies on additional MazF-mt3-RNA complex interactions.

In conclusion, the structures of the MazF-mt1-RNA and MazF-mt3-RNA complexes provide the first structural information on any M. tuberculosis MazF homologues. Supplemented with structure-guided mutational studies on MazF toxicity in vivo, this study has addressed the structural basis of different RNA cleavage specificities among MazF homologues. Our work will guide future studies on the function of other M. tuberculosis MazF and MazE-MazF homologues, and will help delineate their physiological roles in M. tuberculosis stress responses and pathogenesis.

Evaluación del desempeño de pruebas diagnósticas de tuberculosis en pacientes VIH positivo en dos hospitales públicos de Bogotá

La tuberculosis TB es una de las principales causas de muerte en el mundo en individuos con infección por VIH. En Colombia esta coinfección soporta una carga importante en la población general convirtiéndose en un problema de salud pública. En estos pacientes las pruebas diagnósticas tienen sensibilidad inferior y la enfermedad evoluciona con mayor frecuencia hacia formas diseminadas y rápidamente progresivas y su diagnóstico oportuno representa un reto en Salud. El objetivo de este proyecto es evaluar el desempeño de las pruebas diagnósticas convencionales y moleculares, para la detección de TB latente y activa pacientes con VIH, en dos hospitales públicos de Bogotá. Para TB latente se evaluó la concordancia entre las pruebas QuantiFERON-TB (QTF) y Tuberculina (PPD), sugiriendo superioridad del QTF sobre la PPD. Se evaluaron tres pruebas diagnósticas por su sensibilidad y especificidad, baciloscopia (BK), GenoType®MTBDR plus (Genotype) y PCR IS6110 teniendo como estándar de oro el cultivo. Los resultados de sensibilidad (S) y especificidad (E) de cada prueba con una prevalencia del 19,4 % de TB pulmonar y extrapulmonar en los pacientes que participaron del estudio fue: BK S: 64% E: 99,1%; Genotype S: 77,8% E: 94,5%; PCRIS6110 S: 73% E: 95,5%, de la misma forma se determinaron los valores predictivos positivos y negativos (VPP y VPN) BK: 88,9% y 94,8%, Genotype S: 77,8% E: 94,5%; PCRIS6110 S: 90% y 95,7%. Se concluyó bajo análisis de curva ROC que las pruebas muestran un rendimiento diagnóstico similar por separado en el diagnóstico de TB en pacientes con VIH, aumentando su rendimiento diagnostico cuando se combinan

Avaliação de marcadores biológicos com potencial para detecção da evolução para doença em tuberculose

A tuberculose (TB) é uma doença infecto-contagiosa obtida a partir da inalação de aerossóis contendo seu agente etiológico, o Mycobacterium tuberculosis. A TB acomete principalmente os pulmões e é a patologia bacteriana líder em causar mortes no mundo. No Brasil, por ano, são notificados 69 mil casos de tuberculose, dos quais 4,6 mil evoluem para o óbito. Durante a infecção pelo M. tuberculosis, 90% dos indivíduos permanece na forma latente assintomática, e aproximadamente 10% evolui para doença. Este trabalho estudou parâmetros de resposta imune e inflamatória, em indivíduos de ambos os sexos, com idades de 18 a 65 anos, com diferentes graus de exposição ao M. tuberculosis (indivíduos não-expostos ao M. tuberculosis, TST < 5 mm, n= 30; indivíduos com tuberculose latente, TST ≥ 5 mm, n=29; pacientes com tuberculose pulmonar n= 22). Nossos resultados mostraram que o TST isoladamente falhou em detectar todos os indivíduos expostos ao M. tuberculosis, e em 1/3 dos TST positivos não foi observada resposta in vitro a antígenos específicos de M. tuberculosis, avaliada com os biomarcadores IFN-γ e CXCL10. Houve uma alta correlação entre os biomarcadores IFN-γ e CXCL10 em culturas de sangue não fracionado estimuladas com antígenos específicos de M. tuberculosis. A utilização combinada destes 2 biomarcadores mostrou positividade para M. tuberculosis em 94,4% dos pacientes. Foram observadas diferenças marcantes de nível de expressão de RNA mensageiro específicos para CD64, GTPase associada a Ras, lactoferrina, PDL-1 e CXCL10, mas não para OASL em leucócitos sanguíneos, quando os pacientes com tuberculose pulmonar foram comparados com os dois outros grupos de voluntários. Da mesma forma, os níveis de expressão dos receptores CD64 e CD163 foram significativamente mais elevados em neutrófilos dos pacientes quando comparados com os grupos-controle. Tomadas em conjunto, nossas observações sugerem que o uso de mais de um biomarcador aumenta a sensibilidade e especificidade dos métodos para detecção de infecção latente por M. tuberculosis e tuberculose.

Custo-efetividade da prova tuberculínica versus QuantiFERON-TB Gold-In-Tube no diagnóstico e tratamento da infecção latente tuberculosa em profissionais de saúde da Atenção Básica no Brasil.

Os profissionais da área da saúde formam um dos grupos mais vulneráveis à infecção pelo Mycobacterium tuberculosis (Mtb). Segundo estimativas da Organização Mundial de Saúde (OMS), 8,8 milhões de pessoas estavam infectadas pelo Mtb e ocorreram 1,4 milhão de óbitos por tuberculose (TB) em 2010. A identificação de pessoas com Infecção Latente Tuberculosa (ILTB) é considerada pela OMS como uma prioridade no controle da doença, especialmente em países em desenvolvimento em que a incidência da doença ativa tem apresentado redução. O objetivo do presente trabalho foi avaliar, no Brasil, o custo-efetividade dos testes Prova Tuberculínica (PT) e Quantiferon TB Gold-In-Tube (QTF-GIT) no diagnóstico e tratamento da ILTB em profissionais de saúde atuantes na atenção básica, sob a perspectiva do Sistema Único de Saúde (SUS), comparando cinco estratégias que incluem o QTF-GIT, distintos pontos de corte para a PT e uso sequencial dos dois testes; e analisar o impacto do tabagismo sobre o risco de ILTB entre os profissionais de saúde, destacando-se a categoria da Enfermagem. Foi realizada uma avaliação econômica completa do tipo custo-efetividade, conduzida considerando uma coorte hipotética de 10.000 profissionais de saúde atuantes na atenção básica, com horizonte temporal restrito a um ano. Um modelo analítico de decisão, caracterizado por uma árvore de probabilidades de eventos, foi desenvolvido utilizando o software TreeAge ProTM 2013 para simular os resultados clínicos e impactos econômicos em saúde da nova tecnologia diagnóstica (QTF-GIT) versus a PT tradicional. Esse modelo simulou cinco estratégias diagnósticas para detecção e tratamento da ILTB: (a) PT, usando ponto de corte de 5mm; (b) PT, usando ponto de corte de 10 mm; (c) teste QTF-GIT; (d) PT, com ponto de corte de 5mm, seguida de teste QTF-GIT quando PT positiva; (e) PT, com ponto de corte de 10mm, seguida de teste QTF-GIT quando PT positiva. Foi realizada análise de sensibilidade determinística univariada. Na determinação dos fatores associados à ILTB, foi elaborado um modelo de regressão logística múltipla com seleção hierarquizada, utilizando o software Stata. A estratégia mais custo-efetiva foi a PT no ponto de corte ≥10mm, considerando como medida de desfecho tanto o número de indivíduos corretamente classificados pelos testes assim como o número de casos de TB evitados. A utilização isolada do QTF-GIT revelou-se a estratégia de menor eficiência, com RCEI= R$ 343,24 por profissional corretamente classificado pelo teste. Encontrou-se risco à ILTB significantemente maior para sexo masculino [OR=1,89; IC 95%:1,11-3,20], idade ≥ 41 anos [OR=1,56; IC 95%: 1.09-2,22], contato próximo com familiar com TB [OR=1,55; IC 95%: 1.02-2,36], status do tabagismo fumante [OR=1,75; IC 95%: 1.03-2,98] e categoria profissional da Enfermagem [OR=1,44; IC 95%: 1.02-2,03]. Concluiu-se que a PT no ponto de corte de 10mm é a estratégia diagnóstica mais custo-efetiva para ILTB entre os profissionais de saúde na atenção básica e que a ILTB está associada ao hábito do tabagismo e à categoria profissional de Enfermagem.

Vulnérabilité, observance et adhésion thérapeutique : quels risques? : la prévention de la tuberculose chez les enfants immigrants à Montréal

Si le nombre de nouveaux cas de tuberculose au Québec a considérablement baissé au cours des dernières décennies, l’épidémiologie mondiale rappelle toutefois que cette maladie est responsable de plus de deux millions de morts par an. Au Canada, certains groupes seraient plus vulnérables, notamment les immigrants provenant de pays où la tuberculose est endémique. La Clinique de tuberculose du Centre hospitalier universitaire Sainte-Justine est un outil de lutte active contre cette maladie, entre autres grâce à son programme de dépistage scolaire auprès des enfants immigrants. Ce dépistage vise à identifier les porteurs de la tuberculose latente, c’est-à-dire la forme non contagieuse de la maladie. Un traitement préventif de neuf mois est offert aux enfants qui présentent un résultat positif afin de prévenir le développement de la tuberculose maladie (forme active). Dans 28 % des cas, ce traitement n’est pas adéquatement complété et dans 11 % des cas, il est refusé. La présente étude porte à la fois sur la question de l’observance thérapeutique et sur les conditions de vie post-migratoires. L’observation de consultations à la Clinique de tuberculose et les entrevues auprès des soignants et des familles ont engendré une réflexion sur la prévention de la tuberculose en contexte migratoire de même que sur le caractère multifactoriel de la non-observance thérapeutique. L’analyse des données fait ressortir l’impact du vécu migratoire et des conditions de vie (le logement, l’emploi, la maîtrise de la langue, etc.) sur la prise irrégulière du médicament, permettant une meilleure compréhension de ce comportement (chapitre 4). Il a également été possible de documenter une distinction entre les conduites (l’observance) et les attitudes (l’adhésion) nuançant la compréhension des diverses trajectoires thérapeutiques à l’aide de rationalités plurielles et diversifiées (chapitres 5 et 6). Il s’en dégage une réflexion sur le caractère normatif de la catégorisation de « groupe » et de « comportement » à risque laissant place aux différents univers référentiels et, plus globalement, aux conditions de vie des familles (chapitre 7).

Shikimate kinase: A potential target for development of novel antitubercular agents

Tuberculosis (TB) remains the leading cause of mortality due to a bacterial pathogen, Mycobacterium tuberculosis. However, no new classes of drugs for TB have been developed in the past 30 years. Therefore there is an urgent need to develop faster acting and effective new antitubercular agents, preferably belonging to new structural classes, to better combat TB, including MDR-TB, to shorten the duration of current treatment to improve patient compliance, and to provide effective treatment of latent tuberculosis infection. The enzymes in the shikimate pathway are potential targets for development of a new generation of antitubercular drugs. The shikimate pathway has been shown by disruption of aroK gene to be essential for the Mycobacterium tuberculosis. The shikimate kinase (SK) catalyses the phosphorylation of the 3-hydroxyl group of shikimic acid (shikimate) using ATP as a co-substrate. SK belongs to family of nucleoside monophosphate (NMP) kinases. The enzyme is an alpha/beta protein consisting of a central sheet of five parallel beta-strands flanked by alpha-helices. The shikimate kinases are composed of three domains: Core domain, Lid domain and Shikimate-binding domain. The Lid and Shikimate-binding domains are responsible for large conformational changes during catalysis. More recently, the precise interactions between SK and substrate have been elucidated, showing the binding of shikimate with three charged residues conserved among the SK sequences. The elucidation of interactions between MtSK and their substrates is crucial for the development of a new generation of drugs against tuberculosis through rational drug design.

Current advances in antitubercular drug discovery: potent prototypes and new targets

Tuberculosis (TB) is an infectious disease caused by bacterium of the Mycobacterium genus, mainly by Mycobacterium tuberculosis (MTB). The World Health Organization aims to substantially reduce the number of cases in the coming years; however, the increased number of multidrug-resistant (MDR) and extremely drug-resistant (XDR) forms of the bacterium and the lack of treatment for latent tuberculosis are challenges to be overcome. In this review, we have identified the most potent compounds described in the literature during recent years with MIC values < 7 µM, low toxicity and a high selective index. In addition, emerging targets in MTB are presented to provide new perspectives for the discovery of new antitubercular drugs. This review aims to summarize the current advances in and promote insights into antitubercular drug discovery.

IGRA-positive patients and interferon-gamma/interleukin-2 signatures: can the Fluorospot assay provide further information?

A goal of testing for latent tuberculosis (TB) infection is to identify individuals who are at increased risk for the development of active TB. No laboratory tool is currently available to distinguish between individuals in the process of progressing from latent TB infection towards active disease and those who are not. Determination of the interferon-gamma and interleukin-2 T cell signature might provide an additional and rapid tool to evaluate treatment necessity and clinical management of a patient. Here, we present three cases of interferon-gamma release assay-positive patients with differing interferon-gamma and interleukin-2 signatures when analyzed by the Fluorospot assay.

Indeterminate QunatiFERON-TB Gold In-Tube assay results in association with procedural specimen collection in children

BACKGROUND. The development of interferon-gamma release assays (IGRA) has introduced powerful tools in diagnosing latent tuberculosis infection (LTBI) and may play a critical role in the future of tuberculosis diagnosis. However, there have been reports of high indeterminate results in young patient populations (0-18 years). This study investigated results of the QunatiFERON-TB Gold In-Tube (QFT-GIT) IGRA in a population of children (0-18 years) at Texas Children's Hospital in association with specimen collection procedures using surrogate variables. ^ METHODS. A retrospective case-control study design was used for this investigation. Cases were defined as having QFT-GIT indeterminate results. Controls were defined as having either positive or negative results (determinates). Patients' admission status, staff performing specimen collection, and specific nurse performing specimen collection were used as surrogates to measure specimen collection procedures. ^ To minimize potential confounding, abstraction of patients' electronic medical records was performed. Abstracted data included patients' medications and evaluation at the time of QFT-GIT specimen collection in addition to their medical history. QFT-GIT related data was also abstracted. Cases and controls were characterized using chi-squared tests or Fisher's exact tests across categorical variables. Continuous variables were analyzed using one-way ANOVA and t-tests for continuous variables. A multivariate model was constructed by backward stepwise removal of statistically significant variables from univariate analysis. ^ RESULTS. Patient data was abstracted from 182 individuals aged 0-18 years from July 2010 to August 2011 at Texas Children's Hospital. 56 cases (indeterminates) and 126 controls (determinates) were enrolled. Cancer was found to be an effect modifier with subsequent stratification resulting in a cancer patient population too small to analyze (n=13). Subsequent analyses excluded these patients. ^ The exclusion of cancer patients resulted in a population of 169 patients with 49 indeterminates (28.99%) and 120 determinates (71.01%), with mean ages of 9.73 (95% CI: 8.03, 11.43) years and 11.66 (95% CI: 10.75, 12.56) years (p = 0.033), respectively. Median age of patients who were indeterminates and determinates were 12.37 and 12.87 years, respectively. Lack of data for our specific nurse surrogate (QFTNurse) resulted in its exclusion from analysis. The final model included only our remaining surrogate variables (QFTStaff and QFTInpatientOutpatient). The staff collecting surrogate (QFTStaff) was found to be modestly associated with indeterminates when nurses collected the specimen (OR = 1.54, 95% CI: 0.51, 4.64, p = 0.439) in the final model. Inpatients were found to have a strong and statistically significant association with indeterminates (OR = 11.65, 95% CI: 3.89, 34.9, p < 0.001) in the final model. ^ CONCLUSION. Inpatient status was used as a surrogate for indication of nurse drawn blood specimens. Nurses have had little to no training regarding shaking of tubes versus phlebotomists regarding QFT-GIT testing procedures. This was also measured by two other surrogates; specifically a medical note stating whether a nurse or phlebotomist collected the specimen (QFTStaff) and the name and title of the specific nurse if collection was performed by a nurse (QFTNurse). Results indicated that inpatient status was a strong and statistically significant factor for indeterminates, however, nurse collected specimens and indeterminate results had no statistically significant association in non-cancer patients. The lack of data denoting the specific nurse performing specimen collection excluded the QFTNurse surrogate in our analysis. ^ Findings suggests training of staff personnel in specimen procedures may have little effect on the number of indeterminates while inpatient status and thus possibly illness severity may be the most important factor for indeterminate results in this population. The lack of congruence between our surrogate measures may imply that our inpatient surrogate gauged illness severity rather than collection procedures as intended. ^ Despite the lack of clear findings, our analysis indicated that more than half of indeterminates were found in specimens drawn by nurses and as such staff training may be explored. Future studies may explore methods in measuring modifiable variables during pre-analytical QFT-GIT procedures that can be discerned and controlled. Identification of such measures may provide insight into ways to lowering indeterminate QFT-GIT rates in children.^

Eritema induratum de Bazin : a propósito de um caso clínico

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Serodiagnosis of Tuberculosis in Asian Elephants (Elephas maximus) in Southern India: A Latent Class Analysis

Background: Mycobacterium tuberculosis, a causative agent of chronic tuberculosis disease, is widespread among some animal species too. There is paucity of information on the distribution, prevalence and true disease status of tuberculosis in Asian elephants (Elephas maximus). The aim of this study was to estimate the sensitivity and specificity of serological tests to diagnose M. tuberculosis infection in captive elephants in southern India while simultaneously estimating sero-prevalence. Methodology/Principal Findings: Health assessment of 600 elephants was carried out and their sera screened with a commercially available rapid serum test. Trunk wash culture of select rapid serum test positive animals yielded no animal positive for M. tuberculosis isolation. Under Indian field conditions where the true disease status is unknown, we used a latent class model to estimate the diagnostic characteristics of an existing (rapid serum test) and new (four in-house ELISA) tests. One hundred and seventy nine sera were randomly selected for screening in the five tests. Diagnostic sensitivities of the four ELISAs were 91.3-97.6% (95% Credible Interval (CI): 74.8-99.9) and diagnostic specificity were 89.6-98.5% (95% CI: 79.4-99.9) based on the model we assumed. We estimate that 53.6% (95% CI: 44.6-62.8) of the samples tested were free from infection with M. tuberculosis and 15.9% (97.5% CI: 9.8 - to 24.0) tested positive on all five tests. Conclusions/Significance: Our results provide evidence for high prevalence of asymptomatic M. tuberculosis infection in Asian elephants in a captive Indian setting. Further validation of these tests would be important in formulating area-specific effective surveillance and control measures.

Effector functions of heparin-binding hemagglutinin-specific CD8+ T lymphocytes in latent human tuberculosis.

BACKGROUND: Most individuals infected with Mycobacterium tuberculosis do not develop tuberculosis (TB) and can be regarded as being protected by an appropriate immune response to the infection. The characterization of the immune responses of individuals with latent TB may thus be helpful in the definition of correlates of protection and the development of new vaccine strategies. The highly protective antigen heparin-binding hemagglutinin (HBHA) induces strong interferon (IFN)- gamma responses during latent, but not active, TB. Because of the recently recognized importance of CD8(+) T lymphocytes in anti-TB immunity, we characterized the CD8(+) T lymphocyte responses to HBHA in subjects with latent TB. RESULTS: HBHA-specific CD8(+) T lymphocytes expressed memory cell markers and synthesized HBHA-specific IFN- gamma .They also restricted mycobacterial growth and expressed cytotoxicity by a granule-dependent mechanism. This activity was associated with the intracellular expression of HBHA-induced perforin. Surprisingly, the perforin-producing CD8(+) T lymphocytes were distinct from the IFN- gamma -producing CD8(+) T lymphocytes. CONCLUSION: During latent TB, the HBHA-specific CD8(+) T lymphocyte population expresses all 3 effector functions associated with CD8(+) T lymphocyte-mediated protective immune mechanisms, which supports the notion that HBHA may be protective in humans and suggests that markers of HBHA-specific CD8(+) T lymphocyte responses may be useful in the monitoring of protection.

Heparin-Binding Haemagglutinin, a New Tool for the Detection of Latent Mycobacterium tuberculosis Infection in Hemodialysis Patients

Background:Patients with end-stage renal disease (ESRD) and latently infected with Mycobacterium tuberculosis (LTBI) are at higher risk to develop tuberculosis (TB) than healthy subjects. Interferon-gamma release assays (IGRAs) were reported to be more sensitive than tuberculin skin tests for the detection of infected individuals in dialysis patients.Methods:On 143 dialysis patients prospectively enrolled, we compared the results from the QuantiFERON®-TB Gold assay (QFT), to those of an IGRA in response to in vitro stimulation of circulating mononuclear cells with the mycobacterial latency antigen Heparin-Binding Haemagglutinin purified from Mycobacterium bovis BCG (native HBHA, nHBHA).Results:Seven patients had a past history of active TB and 1 had an undetermined result with both IGRAs. Among the other 135 patients, 94 had concordant results with the QFT and nHBHA-IGRA, 40.0% being negative and therefore not latently infected, and 29.6% being positive and thus LTBI. Discrepant results between these tests were found for 36 patients positive only with the nHBHA-IGRA and 5 only with the QFT.Conclusions:The nHBHA-IGRA is more sensitive than the QFT for the detection of LTBI dialysis patients, and follow-up of the patients will allow us to define the clinical significance of discrepant results between the nHBHA-IGRA and the QFT. © 2013 Dessein et al.

HBHA-induced interferon-gamma release assay for the diagnosis of both latent and active tuberculosis.

info:eu-repo/semantics/nonPublished