RESUMEN
Tuberculous meningitis (TBM) is the most severe form of tuberculous with substantial mortality. In May 2015, 54 researchers from 10 countries met in Da Lat, Vietnam, to discuss advances in TBM. Among the attendees were researchers involved in pivotal studies on the use of Xpert MTB/Rif for TBM diagnosis. Attendees discussed the 2014 World Health Organization strong recommendation favoring the use of Xpert "in preference to conventional microscopy and culture as the initial diagnostic test for cerebrospinal fluid (CSF) if the sample volume is low or if additional specimens cannot be obtained to make a quick diagnosis." Attendees were concerned that the limitations of Xpert testing for TBM are not emphasized. Clear guidance is needed for the investigational pathway for TBM, including recommendations on the diagnostic package of investigations, which does not stop with Xpert testing. Second, emphasis on the large CSF volumes (ideally 8-10 mL) needed for Xpert testing is required. Guidelines should also emphasize that TBM is a medical emergency and early treatment reduces mortality.
Asunto(s)
Técnicas de Amplificación de Ácido Nucleico/métodos , Tuberculosis Meníngea/diagnóstico , ADN Bacteriano , Humanos , Mycobacterium tuberculosis/genéticaRESUMEN
The DevR/DosR regulator is believed to play a key role in dormancy adaptation mechanisms of Mycobacterium tuberculosis in response to a multitude of gaseous stresses, including hypoxia, which prevails within granulomas. DevR activates transcription by binding to target promoters containing a minimum of two binding sites. The proximal site overlaps with the SigA -35 element, suggesting that DevR-SigA interaction is required for activating transcription. We evaluated the roles of 14 charged residues of DevR in transcriptional activation under hypoxic stress. Seven of the 14 alanine substitution mutants were defective in regulon activation, of which K191A, R197A, and K179A+K168A (designated K179A*) mutants were significantly or completely compromised in DNA binding. Four mutants, namely, E154A, R155A, E178A, and K208A, were activation defective in spite of binding to DNA and were classified as positive-control (pc) mutants. The SigA interaction defect of the E154A and E178A proteins was established by in vitro and in vivo assays and implies that these substitutions lead to an activation defect because they disrupt an interaction(s) with SigA. The relevance of DevR interaction to the transcriptional machinery was further established by the hypoxia survival phenotype displayed by SigA interaction-defective mutants. Our findings demonstrate the role of DevR-SigA interaction in the activation mechanism and in bacterial survival under hypoxia and establish the housekeeping sigma factor SigA as a molecular target of DevR. The interaction of DevR and RNA polymerase suggests a new and novel interceptable molecular interface for future antidormancy strategies for Mycobacterium tuberculosis.
Asunto(s)
Proteínas Bacterianas/metabolismo , Inmunoglobulina A Secretora/metabolismo , Viabilidad Microbiana , Mycobacterium tuberculosis/fisiología , Proteínas Quinasas/metabolismo , Transcripción Genética , Anaerobiosis , Proteínas Bacterianas/genética , Análisis Mutacional de ADN , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN , Inmunoglobulina A Secretora/genética , Mycobacterium tuberculosis/metabolismo , Regiones Promotoras Genéticas , Mapeo de Interacción de Proteínas , Proteínas Quinasas/genéticaRESUMEN
BACKGROUND: Two-component systems have emerged as compelling targets for antibacterial drug design for a number of reasons including the distinct histidine phosphorylation property of their constituent sensor kinases. The DevR-DevS/DosT two component system of Mycobacterium tuberculosis (M. tb) is essential for survival under hypoxia, a stress associated with dormancy development in vivo. In the present study a combinatorial peptide phage display library was screened for DevS histidine kinase interacting peptides with the aim of isolating inhibitors of DevR-DevS signaling. RESULTS: DevS binding peptides were identified from a phage display library after three rounds of panning using DevS as bait. The peptides showed sequence similarity with conserved residues in the N-terminal domain of DevR and suggested that they may represent interacting surfaces between DevS and DevR. Two DevR mimetic peptides were found to specifically inhibit DevR-dependent transcriptional activity and restrict the hypoxic survival of M. tb. The mechanism of peptide action is majorly attributed to an inhibition of DevS autokinase activity. CONCLUSIONS: These findings demonstrate that DevR mimetic peptides impede DevS activation and that intercepting DevS activation at an early step in the signaling cascade impairs M. tb survival in a hypoxia persistence model.
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Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Materiales Biomiméticos/metabolismo , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Viabilidad Microbiana/efectos de los fármacos , Mycobacterium tuberculosis/efectos de los fármacos , Protamina Quinasa/antagonistas & inhibidores , Proteínas Quinasas/metabolismo , Proteínas de Unión al ADN , Inhibidores Enzimáticos/metabolismo , Mycobacterium tuberculosis/enzimología , Oxígeno/metabolismo , Biblioteca de Péptidos , Péptidos/metabolismo , Estrés FisiológicoRESUMEN
In view of WHO's "End-TB" strategy, we developed a non-invasive, urine-based ELISA, targeting 2 Mycobacterium tuberculosis antigens namely MPT51 and MPT64 for extrapulmonary TB (EPTB) diagnosis. Suspected EPTB patients (n = 137) [Pleural TB, Abdominal TB and Tuberculous meningitis] were categorized in "Definite" EPTB (n = 10) [Xpert-MTB/RIF and/or culture-positive], "Probable" EPTB (n = 77) and "Non-EPTB" (n = 50) groups using defined composite reference standards. ROC-curves were generated using ELISA results of "Definite" EPTB and "Non-EPTB" groups for both antigens independently and cut-off values were selected to provide 86.3% (95%CI:73.3-94.2) specificity for MPT51 and 92% (95%CI:80.8-97.8) for MPT64. The sensitivity of MPT51-ELISA and MPT64-ELISA was 70% (95%CI:34.7-93.3) and 90% (95%CI:55.5-99.7) for "Definite" EPTB group and 32.5% (95%CI:22.2-44.1) and 30.8% (95%CI:20.8-42.2) for "Probable" EPTB group, respectively. Combining the results of both ELISAs showed a 100% (95%CI:69.1-100) sensitivity in "Definite" EPTB group and 41.6% (95%CI:30.4-53.4) in "Probable" EPTB group, with an 80% (95%CI:66.3-89.9) specificity. The results demonstrated the potential of urine-based ELISAs as screening tests for EPTB diagnosis.
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Mycobacterium tuberculosis , Tuberculosis Extrapulmonar , Tuberculosis Meníngea , Humanos , Sensibilidad y Especificidad , Curva ROCRESUMEN
Among the two GroEL paralogs in Mycobacterium tuberculosis, GroEL1 and GroEL2, GroEL1 has a characteristic histidine-rich C terminus. Since histidine richness is likely to be involved in metal binding, we attempted to decipher the role of GroEL1 in chelating metals and the consequence on M. tuberculosis physiology. Isothermal titration calorimetry showed that GroEL1 binds copper and other metals. Mycobacterial viability assay, redox balance, and DNA protection assay concluded that GroEL1 protects from copper stress in vitro. Solution X-ray scattering and constrained modeling of GroEL1 -/+ copper ions showed reorientation of the apical domain as seen in functional assembly. We conclude that the duplication of chaperonin genes in M. tuberculosis might have led to their evolutionary divergence and consequent functional divergence of chaperonins.
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Chaperonina 60/metabolismo , Cobre/metabolismo , Homeostasis , Mycobacterium tuberculosis/metabolismo , Homología de Secuencia de Aminoácido , Secuencia de Aminoácidos , Naftalenosulfonatos de Anilina/metabolismo , Sitios de Unión , Chaperonina 60/química , Daño del ADN , Técnicas de Inactivación de Genes , Silenciador del Gen , Histidina/metabolismo , Modelos Biológicos , Modelos Moleculares , Oxidación-Reducción , Conformación Proteica , Dispersión del Ángulo Pequeño , Homología Estructural de Proteína , Termodinámica , Difracción de Rayos XRESUMEN
Dormancy is a key characteristic of the intracellular life-cycle of Mtb. The importance of sensor kinase DosS in mycobacteria are attributed in part to our current findings that DosS is required for both persistence and full virulence of Mtb. Here we show that DosS is also required for optimal replication in macrophages and involved in the suppression of TNF-α and autophagy pathways. Silencing of these pathways during the infection process restored full virulence in MtbΔdosS mutant. Notably, a mutant of the response regulator DosR did not exhibit the attenuation in macrophages, suggesting that DosS can function independently of DosR. We identified four DosS targets in Mtb genome; Rv0440, Rv2859c, Rv0994, and Rv0260c. These genes encode functions related to hypoxia adaptation, which are not directly controlled by DosR, e.g., protein recycling and chaperoning, biosynthesis of molybdenum cofactor and nitrogen metabolism. Our results strongly suggest a DosR-independent role for DosS in Mtb.
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Autofagosomas/metabolismo , Autofagosomas/microbiología , Proteínas Bacterianas/metabolismo , Mycobacterium tuberculosis/fisiología , Protamina Quinasa/metabolismo , Proteínas Quinasas/metabolismo , Tuberculosis/metabolismo , Tuberculosis/microbiología , Autofagosomas/inmunología , Autofagia , Proteínas Bacterianas/genética , Proteínas de Unión al ADN , Perfilación de la Expresión Génica , Silenciador del Gen , Interacciones Huésped-Patógeno/inmunología , Humanos , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/microbiología , Mutación , Mycobacterium tuberculosis/enzimología , Fagocitos/inmunología , Fagocitos/metabolismo , Fagocitos/microbiología , Fosforilación , Protamina Quinasa/genética , Proteínas Quinasas/genética , Tuberculosis/inmunología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , VirulenciaRESUMEN
BACKGROUND AND OBJECTIVES: The aim of the study is to evaluate the therapeutic efficacy and safety of Yttrium- 90 radiolabelled chimeric anti CD20 antibody-Rituximab in the treatment of patients with relapsed/ refractory B cell Non-Hodgkins Lymphoma (NHL). METHODS: Twenty patients with relapsed/refractory CD20+ NHL in progressive state were included in the study. These patients had undergone a median of 2 (range 2-5) prior standard chemotherapy ± immunotherapy regimens. All the patients received rituximab 250 mg/m2 on days 1 and 8, and either 14 MBq/kg (0.4 mCi/kg) or 11 MBq/kg (0.3 mCi/kg) of Y-90 Rituximab on day 8 (maximum dose, 32 mCi) depending upon their platelet count. The patients were observed for systemic toxicity and response for at least 12 weeks after therapy. RESULTS: No acute adverse effects were observed after the administration of 90Y-Rituximab. Overall response rate (ORR) was 45% of which complete response (CR) was observed in 2 patients, stable disease in 1 patient and partial response in 6 patients. The therapy was well tolerated with grade IV thrombocytopenia, neutropenia and anemia observed in 3, 4 and 2 patients respectively. CONCLUSION: 90Y-Rituximab therapy is safe and well tolerated in high risk extensively pretreated NHL patients. Toxicity is primarily hematologic, transient and reversible.
Asunto(s)
Antineoplásicos/uso terapéutico , Linfoma de Células B/radioterapia , Radioinmunoterapia , Rituximab/uso terapéutico , Radioisótopos de Itrio/uso terapéutico , Adulto , Anciano , Antígenos CD20/inmunología , Antineoplásicos/efectos adversos , Supervivencia sin Enfermedad , Estudios de Factibilidad , Femenino , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia , Rituximab/efectos adversos , Resultado del Tratamiento , Adulto Joven , Radioisótopos de Itrio/efectos adversosRESUMEN
DevR is a key regulator of the dormancy response in Mycobacterium tuberculosis (M. tb). Using DevR as bait to screen a phage display library, a peptide, DevRS1, was obtained. DevRS1 inhibited DevR-regulated transcription and survival of nonreplicating tubercle bacilli in a hypoxia model of dormancy. DevRS1 peptide-mediated inhibition demonstrates the efficacy of intercepting DevR function to block hypoxic adaptation of M. tb.
Asunto(s)
Antibacterianos/metabolismo , Proteínas Bacterianas/antagonistas & inhibidores , Mycobacterium tuberculosis/fisiología , Péptidos/metabolismo , Estrés Fisiológico , Adaptación Fisiológica , Anaerobiosis , Antibacterianos/aislamiento & purificación , Proteínas de Unión al ADN , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Humanos , Viabilidad Microbiana/efectos de los fármacos , Biblioteca de Péptidos , Péptidos/aislamiento & purificación , Proteínas QuinasasRESUMEN
The importance of inter- and intracellular signal transduction in all forms of life cannot be underestimated. A large number of genes dedicated to cellular signalling are found in almost all sequenced genomes, and Mycobacteria are no exception. What appears to be interesting in Mycobacteria is that well characterized signalling mechanisms used by bacteria, such as the histidine-aspartate phosphorelay seen in two-component systems, are found alongside signalling components that closely mimic those seen in higher eukaryotes. This review will describe the important contribution made by researchers in India towards the identification and characterization of proteins involved in two-component signalling, protein phosphorylation and cyclic nucleotide metabolism.
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Mycobacterium tuberculosis/metabolismo , Transducción de Señal , Investigación Biomédica , Humanos , India , Mycobacterium tuberculosis/genética , Fosforilación/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/genéticaRESUMEN
Tuberculosis (TB) is the leading cause of death worldwide attributable to a single infectious disease agent. India has more new TB cases annually than any other country. In 2008, India accounted for a fifth of the estimated 9.4 million TB cases globally. There is an overwhelming need for improving TB diagnostics in India through the use of cost effective, patient-friendly methods appropriate to different tiers of the country health system. Substantial progress has been made in India in the field of TB diagnosis and serious efforts have been made to herald the development of diagnostic tests for pulmonary TB, extra pulmonary TB and MDR-TB. Diverse approaches have been attempted towards improving smear microscopy, rapid culture and for differentiation between the Mycobacterium tuberculosis complex and non-tuberculous mycobacteria. Several laboratories have developed in-house PCR assays for diagnosing TB with high accuracy. Approaches for distinguishing M. tuberculosis and/or Mycobacterium bovis infection and disseminated Mycobacterium avium complex infection in HIV-AIDS patients have also been described. Serological tests to detect antigens or antibodies to M. tuberculosis specific components by using cocktails of Excretory/Secretory protein antigens, Ag85 complex antigens, Hsp 65 antigen, RD1 antigens and Rapid Reverse Line Blot Hybridization assays to detect MDR-TB (mutations to rifampicin, isoniazid and streptomycin) have also been developed. Other methods like measurement of adenosine deaminase activity and use of luciferase reporter phages have also been explored for TB diagnosis. These advances in the Indian context are detailed in the present chapter. The validation and application of these methods in laboratory and public health settings is likely to result in improved TB diagnosis and contribute to effective disease management in India.