CtpB is a plasma membrane copper (I) transporting P-type ATPase of Mycobacterium tuberculosis

BACKGROUND: The intracellular concentration of heavy-metal cations, such as copper, nickel, and zinc is pivotal for the mycobacterial response to the hostile environment inside macrophages. To date, copper transport mediated by P-type ATPases across the mycobacterial plasma membrane has not been sufficiently explored. RESULTS: In this work, the ATPase activity of the putative Mycobacterium tuberculosis P1B-type ATPase CtpB was associated with copper (I) transport from mycobacterial cells. Although CtpB heterologously expressed in M. smegmatis induced tolerance to toxic concentrations of Cu2+ and a metal preference for Cu+, the disruption of ctpB in M. tuberculosis cells did not promote impaired cell growth or heavy-metal accumulation in whole mutant cells in cultures under high doses of copper. In addition, the Cu+ ATPase activity of CtpB embedded in the plasma mem-brane showed features of high affinity/slow turnover ATPases, with enzymatic parametersKM 0.19 ± 0.04 µM and Vmax 2.29 ± 0.10 nmol/mg min. In contrast, the ctpB gene transcription was activated in cells under culture conditions that mimicked the hostile intraphagosomal environment, such as hypoxia, nitrosative and oxidative stress, but not under high doses of copper. CONCLUSIONS: The overall results suggest that M. tuberculosis CtpB is associated with Cu+ transport from mycobacterial cells possibly playing a role different from copper detoxification.

Identification of the Mycobacterium tuberculosis Beijing lineage in Ecuador / Detección de Mycobacterium tuberculosis , linaje Beijing, en Ecuador

ABSTRACT Introducción. Los aislamientos de Mycobacterium tuberculosis pertenecientes al linaje Beijing se consideran especialmente virulentos y transmisibles, y con mayor tendencia a la adquisición de resistencia. El linaje Beijing se ha reportado en todo el mundo; sin embargo, en Latinoamérica los estudios al respecto son más escasos. En el único estudio multinacional llevado a cabo en la región, se detectó una distribución heterogénea del linaje, y no se le encontró en Chile, Colombia y Ecuador, aunque en estudios nacionales posteriores se identificaron aislamientos en Chile y Colombia. Objetivo. Rastrear la presencia del linaje Beijing de M. tuberculosis en Ecuador, único país en la región en el que aún no se reporta. Materiales y métodos. Se analizó una muestra de conveniencia (2006-2012) en dos hospitales que atendían poblaciones diferentes. La genotipificación de los aislamientos de M. tuberculosis se hizo mediante la plataforma 24-MIRU-VNTR. La asignación de linajes se hizo mediante la comparación de los patrones genotípicos con los incluidos en la plataforma MIRU-VNTRplus, y aquellos pertenecientes al linaje Beijing fueron confirmados mediante reacción en cadena de la polimerasa específica de alelo. Resultados. Se detectó el primer aislamiento Beijing en Ecuador, en una circunstancia epidemiológica inesperada: un paciente de la región andina, proveniente de una comunidad con escasa movilidad y alejada de las fronteras con los países limítrofes, Perú y Colombia, en los que ya se han identificado aislamientos de M. tuberculosis pertenecientes al linaje Beijing. Conclusiones. En este trabajo se reporta por primera vez la presencia del linaje Beijing de M. tuberculosis en Ecuador en un contexto epidemiológico inusual que merece especial atención.

RESUMEN Introduction: Mycobacterium tuberculosis Beijing lineage isolates are considered to be especially virulent, transmissible and prone to acquire resistances. Beijing strains have been reported worldwide, but studies in Latin America are still scarce. The only multinational study performed in the region indicated a heterogeneous distribution for this lineage, which was absent in Chile, Colombia and Ecuador, although further studies found the lineage in Chile and Colombia. Objective: To search for the presence of the Beijing lineage in Ecuador, the only country in the region where it remains unreported. Materials and methods: We obtained a convenience sample (2006-2012) from two hospitals covering different populations. The isolates were genotyped using 24-MIRU-VNTR. Lineages were assigned by comparing their patterns to those in the MIRU-VNTRplus platform. Isolates belonging to the Beijing lineage were confirmed by allele-specific PCR. Results: We identified the first Beijing isolate in Ecuador in an unexpected epidemiological scenario: A patient was infected in the Andean region, in a population with low mobility and far from the borders of the neighboring countries where Beijing strains had been previously reported. Conclusion: This is the first report of the presence of the Beijing lineage in Ecuador in an unusual epidemiological context that deserves special attention.

Descripción de las mutaciones de Mycobacterium tuberculosis que confieren resistencia a rifampicina e isoniacida detectadas mediante GenoType(r) MTBDR plus V.2 en Colombia / Description of Mycobacterium tuberculosis mutations conferring resistance to rifampicin and isoniazid detected by GenoType(r) MTBDR plus V.2 in Colombia

Resumen Introducción: La metodología de GenoType(r) MTBDRplus V.2 es una técnica molecular aprobada por la Organización Mundial de la Salud y la Organización Panamericana de la Salud para la detección de las mutaciones en el gen rpoβ del complejo Mycobacterium tuberculosis, las cuales confieren resistencia a la rifampicina, y las de los genes katG e inhA que la confieren frente a la isoniacida. Debido a la variación genética en las cepas circulantes a nivel mundial, los programas nacionales de control de la tuberculosis deben comprobar el desempeño de los nuevos métodos de diagnóstico para su aplicación como prueba rápida. Objetivo: Describir las mutaciones detectadas mediante la técnica GenoType(r) MTBDRplus V.2 en muestras pulmonares y aislamientos de M. tuberculosis procesados en el Laboratorio Nacional de Referencia del Instituto Nacional de Salud durante el 2014. Materiales y métodos: Se hizo un estudio retrospectivo descriptivo que determinó la expresión de los genes inhA, KatG y rpoβ responsables de la resistencia a isoniacida y rifampicina, utilizando la técnica GenoType(r) MTBDRplus V.2 en 837 muestras y aislamientos de casos de tuberculosis. Resultados. Se obtuvieron 689 resultados de pruebas: 581(84,3 %) sensibles, 58 (8,4 %) resistentes y 50 (7,2 %) multirresistentes. Se detectaron diversas mutaciones en el gen rpoβ, de las cuales la más frecuente fue la Ser531Leu (36,6 %), seguida por la Asp516Val (21,6 %), en tanto que en el gen katG la más frecuente fue la Ser315Thr1 (91,9 %). Conclusiones: Se detectaron varias mutaciones en los casos resistentes reportados en el país, con frecuencias similares a las reportadas en otros países de la región de América del Sur.

Abstract Introduction: The GenoType(r)MTBDRplusV.2 assay is a molecular technique endorsed by the World Health Organization and the Pan American Health Organization that allows for the identification of the Mycobacterium tuberculosis complex and the detection of mutations in the rpoβ gene for rifampicin resistance, and katG and inhA genes for isoniazid resistance. Due to the genetic variability in the circulating strains around the world, the national tuberculosis control programs should assess the performance of these new diagnostic technologies and their use under program conditions as rapid tests. Objective: To describe the mutations identified by the GenoType(r)MTBDRplusV.2 assay in pulmonary samples and Mycobacterium tuberculosis isolates in the Laboratorio Nacional de Referencia of the Instituto Nacional de Salud in 2014. Materials and methods. We conducted a retrospective, descriptive study to detect the expression of inhA, KatG and rpoβ genes, responsible for resistence against isoniazid and rifampicin using the GenoType(r) MTBDRplus V.2 assay in 837 samples and isolates from tuberculosis cases. Results: Several mutations in the rpoβ gene were identified. Ser531Leu was the most frequent (36.6%) followed by Asp516Val (21.6%), while Ser315Thr1 was the most frequent mutation in the katG gene (91.9%). Conclusions: We were able to identify different mutations present in MDR-TB strains in the country, with frequencies similar to those reported in other countries in the South American region.

A low molecular weight ES-20 protein released in vivo and in vitro with diagnostic potential in lymph node tuberculosis.

PURPOSE: To determine role of antigens released in vivo and in vitro in immunodiagnosis of tuberculosis (TB). METHODS: In vivo released circulating tuberculosis antigen (CTA) was obtained from TB sera by ammonium sulphate precipitation and in vitro released excretory-secretory (ES) antigens from Mycobacterium tuberculosis culture filtrate. CTA and ES antigens were fractionated by SDS-PAGE and electro-eluted gel fractions were analysed for antigen by ELISA. RESULTS: Low molecular weight proteins CTA-9 and ES-9 showed high titre of antigen activity. To explore the diagnostic potential of low molecular weight ES antigen, M. tuberculosis ES antigen was further fractionated by gel filtration chromatography followed by purification on anion exchange column using fast protein liquid chromatography and a highly seroreactive ESG-5D (ES-20) antigen was obtained. Competitive inhibition showed that CTA-9 and ES-9 antigens inhibit the binding of ES-20 antigen to its antibody. Seroanalysis showed sensitivity of 83 and 80% for ES-20 antigen and antibody detection, respectively, in pulmonary TB and 90% in lymph node TB. CONCLUSIONS: Seroreactivity studies using M. tuberculosis ES-20 antigen showed usefulness in detection of TB; in particular, lymph node TB.

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.

Analysis of mycobacterial strains by two-dimensional gel electrophoresis.

This study pertains to analysis of the protein profile of different mycobacterial strains by two-dimensional gel electrophoresis (2DE). The strains were selected as they exhibit different phenotypic behaviour. TCA-acetone precipitated proteins were resolved by 2DE using immobilized pH gradient (IPG) strips. This study demonstrates that 2DE may be used as a tool for characterization of mycobacterial strains. Visual examination of the electrophoretograms was sufficient for characterization. Detailed characterization of specific proteins might lead to development of novel targets, diagnostic probes or sub-unit vaccine(s) against tuberculosis.

Análisis cromatográfico de cepas de Mycobacterium tuberculosis aisladas de un brote en pacientes VIH en Cuba

Se realiza el estudio mediante técnicas cromatográficas, de un grupo de cepas de Mycobacterium tuberculosis aisladas de un brote en pacientes infectados con el virus VIH. Se utilizaron como referencia un grupo de cepas de M. tuberculosis de pacientes sintomáticos respiratorios (SR+14) y cepas patrones de la colección del laboratorio, con el objetivo de comparar cualitativamente los patrones cromatográficos descritos por las cepas aisladas de pacientes. Se obtuvieron y compararon los perfiles cromatográficos de las cepas aisladas de pacientes (ST+) y de VIH+ por la técnica de cromatografía en capa delgada. Se identificó cada uno de los ácidos grasos presentes por la técnica de cromatografía de gases acoplada a espectrometría de masas. Todas las cepas estudiadas fueron clasificadas como Mycobacterium tuberculosis. Por resultados obtenidos se demuestra la utilidad de las técnicas cromatográficas como técnicas alternativas para el diagnóstico micobacteriano.

A group of strains of Mycobacterium tuberculosis isolated from an outbreak in HIV-infected patients was studied by chromatographic techniques.A group of strains of M. Tuberculosis from symptomatic respiratory patients (SR + 14) and patterns strains from the laboratory collection were used as a reference aimed at making a qualitative comparison of the chromatographic patterns described by the strains isolated from patients. The chromatographic profiles of the strains isolated from patients (SR +) and fro HIV + were obtained and comparede by thin layer chromatography (TLC). Each of the present fatty acids was identified by using the gas chromatography technique (GC) coupled to mass spectrum analysis. All the studied strains were classified as Mycobacterium tuberculosis. According to the results attained, the usefulness of the chromatographic techniques as alternative techniques for the mycobacterial diagnosis is demonstrated.

Tuberculosis: new strategies for the development of diagnostic tests and vaccines

New diagnostic tests and vaccines for tuberculosis are being developed by means of a strategy based on the study of antigens exclusive to Mycobacterium tuberculosis. These antigens were initially identified by Western blots using sera from active pulmonary tuberculosis patients against sonic extracts from M. tuberculosis and M. bovis BCG. Several proteins present in the M. tuberculosis but absent in the Mycobacterium bovis BCG sonic extracts were selected and are currently under investigation. One of these, denoted MTP40, has been extensively studied. The nucleotide sequence of the mtp40 gene has been obtained; hybridization studies have shown that this DNA fragment is exclusive to M. tuberculosis. Using this genomic fragment, a polymerase chain reaction (PCR)-based diagnostic test which allows the specific identification of a minimum of 10 fg of M. tuberculosis DNA was developed. The diagnostic assay is now being tested on uncultured clinical samples in order to determine its usefulness in routine diagnosis. Peptides synthesized from the derived sequence for the MTP40 protein and also from other M. tuberculosis proteins are now being studied as possible candidates for a new generation of synthetic vaccines against tuberculosis