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1.
Cell ; 175(4): 1045-1058.e16, 2018 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-30388443

RESUMEN

Protein N-glycosylation is a widespread post-translational modification. The first committed step in this process is catalysed by dolichyl-phosphate N-acetylglucosamine-phosphotransferase DPAGT1 (GPT/E.C. 2.7.8.15). Missense DPAGT1 variants cause congenital myasthenic syndrome and disorders of glycosylation. In addition, naturally-occurring bactericidal nucleoside analogues such as tunicamycin are toxic to eukaryotes due to DPAGT1 inhibition, preventing their clinical use. Our structures of DPAGT1 with the substrate UDP-GlcNAc and tunicamycin reveal substrate binding modes, suggest a mechanism of catalysis, provide an understanding of how mutations modulate activity (thus causing disease) and allow design of non-toxic "lipid-altered" tunicamycins. The structure-tuned activity of these analogues against several bacterial targets allowed the design of potent antibiotics for Mycobacterium tuberculosis, enabling treatment in vitro, in cellulo and in vivo, providing a promising new class of antimicrobial drug.


Asunto(s)
Antibióticos Antituberculosos/farmacología , Trastornos Congénitos de Glicosilación/metabolismo , Inhibidores Enzimáticos/farmacología , N-Acetilglucosaminiltransferasas/química , Animales , Antibióticos Antituberculosos/química , Sitios de Unión , Trastornos Congénitos de Glicosilación/genética , Inhibidores Enzimáticos/química , Femenino , Células HEK293 , Células Hep G2 , Humanos , Metabolismo de los Lípidos , Ratones , Simulación del Acoplamiento Molecular , Mutación , N-Acetilglucosaminiltransferasas/antagonistas & inhibidores , N-Acetilglucosaminiltransferasas/genética , N-Acetilglucosaminiltransferasas/metabolismo , Unión Proteica , Células Sf9 , Spodoptera , Tunicamicina/química , Tunicamicina/farmacología , Uridina Difosfato Ácido Glucurónico/química , Uridina Difosfato Ácido Glucurónico/metabolismo
3.
J Am Chem Soc ; 143(21): 8056-8068, 2021 06 02.
Artículo en Inglés | MEDLINE | ID: mdl-34028251

RESUMEN

Among the ribosomally synthesized and post-translationally modified peptide (RiPP) natural products, "graspetides" (formerly known as microviridins) contain macrocyclic esters and amides that are formed by ATP-grasp ligase tailoring enzymes using the side chains of Asp/Glu as acceptors and Thr/Ser/Lys as donors. Graspetides exhibit diverse patterns of macrocylization and connectivities exemplified by microviridins, that have a caged tricyclic core, and thuringin and plesiocin that feature a "hairpin topology" with cross-strand ω-ester bonds. Here, we characterize chryseoviridin, a new type of multicore RiPP encoded by Chryseobacterium gregarium DS19109 (Phylum Bacteroidetes) and solve a 2.44 Å resolution crystal structure of a quaternary complex consisting of the ATP-grasp ligase CdnC bound to ADP, a conserved leader peptide and a peptide substrate. HRMS/MS analyses show that chryseoviridin contains four consecutive five- or six-residue macrocycles ending with a microviridin-like core. The crystal structure captures respective subunits of the CdnC homodimer in the apo or substrate-bound state revealing a large conformational change in the B-domain upon substrate binding. A docked model of ATP places the γ-phosphate group within 2.8 Å of the Asp acceptor residue. The orientation of the bound substrate is consistent with a model in which macrocyclization occurs in the N- to C-terminal direction for core peptides containing multiple Thr/Ser-to-Asp macrocycles. Using systematically varied sequences, we validate this model and identify two- or three-amino acid templating elements that flank the macrolactone and are required for enzyme activity in vitro. This work reveals the structural basis for ω-ester bond formation in RiPP biosynthesis.


Asunto(s)
Adenosina Trifosfato/metabolismo , Productos Biológicos/metabolismo , Ligasas/metabolismo , Péptidos/metabolismo , Adenosina Trifosfato/química , Amidas/química , Amidas/metabolismo , Productos Biológicos/química , Ésteres/química , Ésteres/metabolismo , Ligasas/química , Compuestos Macrocíclicos/química , Compuestos Macrocíclicos/metabolismo , Conformación Molecular , Péptidos/química , Procesamiento Proteico-Postraduccional
4.
ACS Infect Dis ; 8(3): 612-625, 2022 03 11.
Artículo en Inglés | MEDLINE | ID: mdl-35143160

RESUMEN

Natural products provide a rich source of potential antimicrobials for treating infectious diseases for which drug resistance has emerged. Foremost among these diseases is tuberculosis. Assessment of the antimycobacterial activity of nargenicin, a natural product that targets the replicative DNA polymerase of Staphylococcus aureus, revealed that it is a bactericidal genotoxin that induces a DNA damage response in Mycobacterium tuberculosis (Mtb) and inhibits growth by blocking the replicative DNA polymerase, DnaE1. Cryo-electron microscopy revealed that binding of nargenicin to Mtb DnaE1 requires the DNA substrate such that nargenicin is wedged between the terminal base pair and the polymerase and occupies the position of both the incoming nucleotide and templating base. Comparative analysis across three bacterial species suggests that the activity of nargenicin is partly attributable to the DNA binding affinity of the replicative polymerase. This work has laid the foundation for target-led drug discovery efforts focused on Mtb DnaE1.


Asunto(s)
Mycobacterium tuberculosis , Tuberculosis , Antibacterianos/farmacología , Microscopía por Crioelectrón , ADN Polimerasa Dirigida por ADN , Humanos , Mycobacterium tuberculosis/genética , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiología
5.
Mucosal Immunol ; 14(5): 1055-1066, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-34158594

RESUMEN

Targeting MAIT cells holds promise for the treatment of different diseases and infections. We previously showed that treatment of Mycobacterium tuberculosis infected mice with 5-OP-RU, a major antigen for MAIT cells, expands MAIT cells and enhances bacterial control. Here we treated M. tuberculosis infected rhesus macaques with 5-OP-RU intratracheally but found no clinical or microbiological benefit. In fact, after 5-OP-RU treatment MAIT cells did not expand, but rather upregulated PD-1 and lost the ability to produce multiple cytokines, a phenotype resembling T cell exhaustion. Furthermore, we show that vaccination of uninfected macaques with 5-OP-RU+CpG instillation into the lungs also drives MAIT cell dysfunction, and PD-1 blockade during vaccination partly prevents the loss of MAIT cell function without facilitating their expansion. Thus, in rhesus macaques MAIT cells are prone to the loss of effector functions rather than expansion after TCR stimulation in vivo, representing a significant barrier to therapeutically targeting these cells.


Asunto(s)
Pulmón/efectos de los fármacos , Pulmón/inmunología , Pulmón/metabolismo , Células T Invariantes Asociadas a Mucosa/efectos de los fármacos , Células T Invariantes Asociadas a Mucosa/inmunología , Células T Invariantes Asociadas a Mucosa/metabolismo , Ribitol/análogos & derivados , Uracilo/análogos & derivados , Animales , Biomarcadores , Citocinas/biosíntesis , Manejo de la Enfermedad , Susceptibilidad a Enfermedades , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Inmunofenotipificación , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Macaca mulatta , Enfermedades de los Monos/diagnóstico , Enfermedades de los Monos/tratamiento farmacológico , Enfermedades de los Monos/etiología , Enfermedades de los Monos/metabolismo , Mycobacterium tuberculosis/inmunología , Tomografía de Emisión de Positrones , Ribitol/administración & dosificación , Tomografía Computarizada por Rayos X , Tuberculosis/veterinaria , Uracilo/administración & dosificación
6.
Front Microbiol ; 11: 591866, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33362741

RESUMEN

Mycobacterium tuberculosis resides in the lungs in various lesion types with unique microenvironmental conditions. This diversity is in line with heterogeneous disease progression and divergent drug efficiency. Fluorescent reporter strains can be used to decipher the micromilieu and to guide future treatment regimens. Current reporters using replicating plasmids, however, are not suitable for long-term mouse infections or studies in non-human primates. Using a combination of recombinant DNA and protein optimization techniques, we have developed reporter strains based on integrative plasmids, which exhibit stimulus-response characteristics and fluorescence intensities comparable to those based on replicating plasmids. We successfully applied the concepts by constructing a multi-color reporter strain able to detect simultaneous changes in environmental pH, Mg2+ concentrations, and protein expression levels.

7.
Sci Rep ; 7(1): 8853, 2017 08 18.
Artículo en Inglés | MEDLINE | ID: mdl-28821804

RESUMEN

During active TB in humans a spectrum of pulmonary granulomas with central necrosis and hypoxia exists. BALB/c mice, predominantly used in TB drug development, do not reproduce this complex pathology thereby inaccurately predicting clinical outcome. We found that Nos2 -/- mice incapable of NO-production in immune cells as microbial defence uniformly develop hypoxic necrotizing lung lesions, widely observed in human TB. To study the impact of hypoxic necrosis on the efficacy of antimycobacterials and drug candidates, we subjected Nos2 -/- mice with TB to monotherapy before or after establishment of human-like pathology. Isoniazid induced a drug-tolerant persister population only when necrotic lesions were present. Rifapentine was more potent than rifampin prior to development of human-like pathology and equally potent thereafter, in agreement with recent clinical trials. Pretomanid, delamanid and the pre-clinical candidate BTZ043 were bactericidal independent of pulmonary pathology. Linezolid was bacteriostatic in TB-infected Nos2 -/- mice but significantly improved lung pathology. Hypoxic necrotizing lesions rendered moxifloxacin less active. In conclusion, Nos2 -/- mice are a predictive TB drug development tool owing to their consistent development of human-like pathology.


Asunto(s)
Hipoxia/metabolismo , Necrosis/genética , Necrosis/metabolismo , Óxido Nítrico Sintasa de Tipo II/deficiencia , Tuberculosis Pulmonar/etiología , Tuberculosis Pulmonar/metabolismo , Animales , Antituberculosos/farmacología , Modelos Animales de Enfermedad , Fibrosis , Células Espumosas/inmunología , Células Espumosas/metabolismo , Células Espumosas/patología , Humanos , Hipoxia/patología , Isoniazida/farmacología , Ratones , Ratones Noqueados , Necrosis/patología , Rifampin/análogos & derivados , Rifampin/farmacología , Resultado del Tratamiento , Tuberculosis Pulmonar/tratamiento farmacológico , Tuberculosis Pulmonar/patología
8.
Gates Open Res ; 1: 9, 2017 Nov 06.
Artículo en Inglés | MEDLINE | ID: mdl-29528048

RESUMEN

Background: By the early 1980s, tuberculosis treatment was shortened from 24 to 6 months, maintaining relapse rates of 1-2%. Subsequent trials attempting shorter durations have failed, with 4-month arms consistently having relapse rates of 15-20%. One trial shortened treatment only among those without baseline cavity on chest x-ray and whose month 2 sputum culture converted to negative. The 4-month arm relapse rate decreased to 7% but was still significantly worse than the 6-month arm (1.6%, P<0.01).  We hypothesize that PET/CT characteristics at baseline, PET/CT changes at one month, and markers of residual bacterial load will identify patients with tuberculosis who can be cured with 4 months (16 weeks) of standard treatment. Methods: This is a prospective, multicenter, randomized, phase 2b, noninferiority clinical trial of pulmonary tuberculosis participants. Those eligible start standard of care treatment. PET/CT scans are done at weeks 0, 4, and 16 or 24. Participants who do not meet early treatment completion criteria (baseline radiologic severity, radiologic response at one month, and GeneXpert-detectable bacilli at four months) are placed in Arm A (24 weeks of standard therapy). Those who meet the early treatment completion criteria are randomized at week 16 to continue treatment to week 24 (Arm B) or complete treatment at week 16 (Arm C). The primary endpoint compares the treatment success rate at 18 months between Arms B and C. Discussion: Multiple biomarkers have been assessed to predict TB treatment outcomes. This study uses PET/CT scans and GeneXpert (Xpert) cycle threshold to risk stratify participants. PET/CT scans are not applicable to global public health but could be used in clinical trials to stratify participants and possibly become a surrogate endpoint. If the Predict TB trial is successful, other immunological biomarkers or transcriptional signatures that correlate with treatment outcome may be identified. TRIAL REGISTRATION: NCT02821832.

9.
Nat Med ; 22(10): 1090-1093, 2016 10.
Artículo en Inglés | MEDLINE | ID: mdl-27595321

RESUMEN

Tuberculosis is classically divided into states of latent infection and active disease. Using combined positron emission and computed tomography in 35 asymptomatic, antiretroviral-therapy-naive, HIV-1-infected adults with latent tuberculosis, we identified ten individuals with pulmonary abnormalities suggestive of subclinical, active disease who were substantially more likely to progress to clinical disease. Our findings challenge the conventional two-state paradigm and may aid future identification of biomarkers that are predictive of progression.


Asunto(s)
Infecciones por VIH/complicaciones , Tuberculosis Latente/diagnóstico por imagen , Tuberculosis Pulmonar/diagnóstico por imagen , Adulto , Coinfección/diagnóstico por imagen , Progresión de la Enfermedad , Femenino , Fluorodesoxiglucosa F18 , Humanos , Ensayos de Liberación de Interferón gamma , Tuberculosis Latente/complicaciones , Masculino , Mycobacterium tuberculosis/aislamiento & purificación , Tomografía Computarizada por Tomografía de Emisión de Positrones , Radiografía Torácica , Radiofármacos , Sudáfrica , Esputo/microbiología , Tuberculosis/complicaciones , Tuberculosis/diagnóstico por imagen , Tuberculosis Pulmonar/complicaciones
10.
J Med Case Rep ; 4: 156, 2010 May 26.
Artículo en Inglés | MEDLINE | ID: mdl-20504347

RESUMEN

INTRODUCTION: Pulmonary ossification has been rarely observed in pulmonary fibrosis and in some chronic respiratory diseases such as chronic obstructive pulmonary disease. We report here a metaplastic ossification in the bronchial cartilage of a patient with multi-drug resistant tuberculosis. CASE PRESENTATION: We report the case of a 41-year-old Asian man from Korea with chronic multi-drug resistant tuberculosis with a rare focus of bone formation from the cartilage of a bronchus subtending an active cavity. The patient had a large multi-lobed, thick-walled cavitary tuberculosis lesion in his left upper lobe. Severe infiltration of his lymphocytes and epithelioid cells, along with some giant cells and neutrophils, was observed in the patient's bronchial wall. Desquamated bronchial epithelium and acid-fast bacilli were found inside his bronchus. A small focus of bony metaplasia was found in the cartilage of his bronchial wall. Histopathological examination confirmed calcification and showed hematopoietic cells forming in his marrow cavity. CONCLUSIONS: Chronic inflammation in the lungs of our patient, caused by underlying tuberculosis, probably played a role in the development of osseous metaplasia from the associated cartilage of the bronchial wall.

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