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1.
Antimicrob Agents Chemother ; : AAC0150921, 2021 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-34633851

RESUMO

Mycobacterium abscessus is an opportunistic pathogen notorious for its resistance to most classes of antibiotics and low cure rates. M. abscessus carries an array of mostly unexplored defence mechanisms. A deeper understanding of antibiotic resistance and tolerance mechanisms is pivotal in development of targeted therapeutic regimens. We provide the first description of all major transcriptional mechanisms of tolerance to all antibiotics recommended in current guidelines, using RNA sequencing-guided experiments. M. abscessus ATCC 19977 bacteria were subjected to sub-inhibitory concentrations of clarithromycin, amikacin, tigecycline, cefoxitin and clofazimine for 4- and 24-hours, followed by RNA sequencing. To confirm key mechanisms of tolerance suggested by transcriptomic responses, we performed time-kill kinetic analysis using bacteria after pre-exposure to clarithromycin, amikacin or tigecycline for 24-hours and we constructed isogenic knockout and knockdown strains. To assess strain specificity, pan-genome analysis of 35 strains from all three subspecies was performed. Mycobacterium abscessus shows both drug-specific and common transcriptomic responses to antibiotic exposure. Ribosome-targeting antibiotics clarithromycin, amikacin and tigecycline elicit a common response characterized by upregulation of ribosome structural genes, the WhiB7 regulon and transferases, accompanied by downregulation of respiration through NuoA-N. Exposure to any of these drugs decreases susceptibility to ribosome-targeting drugs from multiple classes. The cytochrome bd-type quinol oxidase contributes to clofazimine tolerance in M. abscessus and the sigma factor sigH but not anti-sigma factor MAB_3542c is involved in tigecycline resistance. The observed transcriptomic responses are not strain-specific, as all genes involved in tolerance, except erm(41), are found in all included strains.

2.
Nat Microbiol ; 6(10): 1279-1288, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34545208

RESUMO

Mycobacterium abscessus, a multidrug-resistant nontuberculous mycobacterium, has emerged as a major pathogen affecting people with cystic fibrosis (CF). Although originally thought to be acquired independently from the environment, most individuals are infected with one of several dominant circulating clones (DCCs), indicating the presence of global transmission networks of M. abscessus. How and when these clones emerged and spread globally is unclear. Here, we use evolutionary analyses of isolates from individuals both with and without CF to reconstruct the population history, spatiotemporal spread and recent transmission networks of the DCCs. We demonstrate synchronous expansion of six unrelated DCCs in the 1960s, a period associated with major changes in CF care and survival. Each of these clones has spread globally as a result of rare intercontinental transmission events. We show that the DCCs, but not environmentally acquired isolates, exhibit a specific smoking-associated mutational signature and that current transmission networks include individuals both with and without CF. We therefore propose that the DCCs initially emerged in non-CF populations but were then amplified and spread through the CF community. While individuals with CF are probably the most permissive host, non-CF individuals continue to play a key role in transmission networks and may facilitate long-distance transmission.

3.
Life Sci Alliance ; 4(10)2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34353886

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic caused by the new coronavirus (SARS-CoV-2) is currently responsible for more than 3 million deaths in 219 countries across the world and with more than 140 million cases. The absence of FDA-approved drugs against SARS-CoV-2 has highlighted an urgent need to design new drugs. We developed an integrated model of the human cell and SARS-CoV-2 to provide insight into the virus' pathogenic mechanism and support current therapeutic strategies. We show the biochemical reactions required for the growth and general maintenance of the human cell, first, in its healthy state. We then demonstrate how the entry of SARS-CoV-2 into the human cell causes biochemical and structural changes, leading to a change of cell functions or cell death. A new computational method that predicts 20 unique reactions as drug targets from our models and provides a platform for future studies on viral entry inhibition, immune regulation, and drug optimisation strategies. The model is available in BioModels (https://www.ebi.ac.uk/biomodels/MODEL2007210001) and the software tool, findCPcli, that implements the computational method is available at https://github.com/findCP/findCPcli.


Assuntos
COVID-19/tratamento farmacológico , COVID-19/metabolismo , Desenvolvimento de Medicamentos/métodos , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/metabolismo , COVID-19/epidemiologia , Biologia Computacional/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Modelos Biológicos , Pandemias
4.
Science ; 372(6541)2021 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-33926925

RESUMO

Although almost all mycobacterial species are saprophytic environmental organisms, a few, such as Mycobacterium tuberculosis, have evolved to cause transmissible human infection. By analyzing the recent emergence and spread of the environmental organism M. abscessus through the global cystic fibrosis population, we have defined key, generalizable steps involved in the pathogenic evolution of mycobacteria. We show that epigenetic modifiers, acquired through horizontal gene transfer, cause saltational increases in the pathogenic potential of specific environmental clones. Allopatric parallel evolution during chronic lung infection then promotes rapid increases in virulence through mutations in a discrete gene network; these mutations enhance growth within macrophages but impair fomite survival. As a consequence, we observe constrained pathogenic evolution while person-to-person transmission remains indirect, but postulate accelerated pathogenic adaptation once direct transmission is possible, as observed for M. tuberculosis Our findings indicate how key interventions, such as early treatment and cross-infection control, might restrict the spread of existing mycobacterial pathogens and prevent new, emergent ones.


Assuntos
Doenças Transmissíveis Emergentes/microbiologia , Evolução Molecular , Aptidão Genética , Pulmão/microbiologia , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium abscessus/genética , Mycobacterium abscessus/patogenicidade , Pneumonia Bacteriana/microbiologia , Doenças Transmissíveis Emergentes/transmissão , Conjuntos de Dados como Assunto , Epigênese Genética , Transferência Genética Horizontal , Genoma Bacteriano , Humanos , Mutação , Infecções por Mycobacterium não Tuberculosas/transmissão , Pneumonia Bacteriana/transmissão , Virulência/genética
6.
Respir Med ; 173: 106164, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32992265

RESUMO

BACKGROUND: Nontuberculous mycobacterial pulmonary disease (NTM-PD) is an emerging opportunistic infection, but basic epidemiological data are lacking in most regions. We have investigated epidemiology and diagnostic and treatment practices in five EU countries (United Kingdom, Spain, Italy, France, Germany; EU5) and Japan. STUDY DESIGN: and methods: Annual prevalence in each country was established using a 2-round Delphi method in combination with a regional prevalence-estimation model that incorporated data obtained from a blinded physician screening survey (3154 physicians) and a real-world NTM-PD treating-physician/patient-chart observational study (619 physicians - 1429 patient charts). RESULTS: The annual prevalence of NTM-PD was estimated at 6.2/100,000 in the EU5 and 24.9/100,000 in Japan. Overall prevalence between the EU5 was comparable, while differences in regional prevalence were found to be pronounced in France and The United Kingdom. Regional differences were also found in Japan, with the majority of cases in Chubu and Kanto regions. CONCLUSION: This new methodology for obtaining often missing regional-level epidemiological data reveals dramatic variations in NTM-PD annual prevalence and helps pinpoint areas that may merit special preventative and treatment focus.


Assuntos
Métodos Epidemiológicos , Infecções por Mycobacterium não Tuberculosas , Micobactérias não Tuberculosas , Pneumonia Bacteriana/epidemiologia , Pneumonia Bacteriana/microbiologia , Europa (Continente)/epidemiologia , Feminino , Humanos , Japão/epidemiologia , Masculino , Infecções Oportunistas/complicações , Pneumonia Bacteriana/complicações , Pneumonia Bacteriana/prevenção & controle , Prevalência
7.
Front Immunol ; 11: 1733, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32849617

RESUMO

Inflammation-related progressive lung destruction is the leading causes of premature death in cystic fibrosis (CF), a genetic disorder caused by a defective cystic fibrosis transmembrane conductance regulator (CFTR). However, therapeutic targeting of inflammation has been hampered by a lack of understanding of the links between a dysfunctional CFTR and the deleterious innate immune response in CF. Herein, we used a CFTR-depleted zebrafish larva, as an innovative in vivo vertebrate model, to understand how CFTR dysfunction leads to abnormal inflammatory status in CF. We show that impaired CFTR-mediated inflammation correlates with an exuberant neutrophilic response after injury: CF zebrafish exhibit enhanced and sustained accumulation of neutrophils at wounds. Excessive epithelial oxidative responses drive enhanced neutrophil recruitment towards wounds. Persistence of neutrophils at inflamed sites is associated with impaired reverse migration of neutrophils and reduction in neutrophil apoptosis. As a consequence, the increased number of neutrophils at wound sites causes tissue damage and abnormal tissue repair. Importantly, the molecule Tanshinone IIA successfully accelerates inflammation resolution and improves tissue repair in CF animal. Our findings bring important new understanding of the mechanisms underlying the inflammatory pathology in CF, which could be addressed therapeutically to prevent inflammatory lung damage in CF patients with potential improvements in disease outcomes.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/imunologia , Imunidade Inata/imunologia , Inflamação/imunologia , Infiltração de Neutrófilos/imunologia , Cicatrização/imunologia , Proteínas de Peixe-Zebra/imunologia , Animais , Animais Geneticamente Modificados , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/genética
8.
Genome Biol ; 21(1): 180, 2020 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-32698896

RESUMO

Population-level comparisons of prokaryotic genomes must take into account the substantial differences in gene content resulting from horizontal gene transfer, gene duplication and gene loss. However, the automated annotation of prokaryotic genomes is imperfect, and errors due to fragmented assemblies, contamination, diverse gene families and mis-assemblies accumulate over the population, leading to profound consequences when analysing the set of all genes found in a species. Here, we introduce Panaroo, a graph-based pangenome clustering tool that is able to account for many of the sources of error introduced during the annotation of prokaryotic genome assemblies. Panaroo is available at https://github.com/gtonkinhill/panaroo .

9.
ACS Infect Dis ; 6(8): 2143-2154, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32551551

RESUMO

Understanding the physiological processes underlying the ability of Mycobacterium abscessus to become a chronic pathogen of the cystic fibrosis (CF) lung is important to the development of prophylactic and therapeutic strategies to better control and treat pulmonary infections caused by these bacteria. Gene expression profiling of a diversity of M. abscessus complex isolates points to amino acids being significant sources of carbon and energy for M. abscessus in both CF sputum and synthetic CF medium and to the bacterium undergoing an important metabolic reprogramming in order to adapt to this particular nutritional environment. Cell envelope analyses conducted on the same representative isolates further revealed unexpected structural alterations in major cell surface glycolipids known as the glycopeptidolipids (GPLs). Besides showing an increase in triglycosylated forms of these lipids, CF sputum- and synthetic CF medium-grown isolates presented as yet unknown forms of GPLs representing as much as 10% to 20% of the total GPL content of the cells, in which the classical amino alcohol located at the carboxy terminal of the peptide, alaninol, is replaced with the branched-chain amino alcohol leucinol. Importantly, both these lipid changes were exacerbated by the presence of mucin in the culture medium. Collectively, our results reveal potential new drug targets against M. abscessus in the CF airway and point to mucin as an important host signal modulating the cell surface composition of this pathogen.


Assuntos
Fibrose Cística , Infecções por Mycobacterium não Tuberculosas , Mycobacterium abscessus , Glicolipídeos , Humanos , Mycobacterium abscessus/genética , Escarro
10.
Brain Behav Immun ; 87: 473-488, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32006615

RESUMO

The innate immune system is implicated in Parkinson's disease (PD), but peripheral in-vivo clinical evidence of the components and driving mechanisms involved and their relationship with clinical heterogeneity and progression to dementia remain poorly explored. We examined changes in peripheral innate immune-related markers in PD cases (n = 41) stratified according to risk of developing early dementia. 'Higher Risk'(HR) (n = 23) and 'Lower Risk' (LR) (n = 18) groups were defined according to neuropsychological predictors and MAPT H1/H2 genotype, and compared to age, gender and genotype-matched controls. Monocyte subsets and expression of key surface markers were measured using flow cytometry. Serum markers including alpha-synuclein, inflammasome-related caspase-1 and bacterial translocation-related endotoxin were measured using quantitative immuno-based assays. Specific markers were further investigated using monocyte assays and validated in plasma samples from a larger incident PD cohort (n = 95). We found that classical monocyte frequency was elevated in PD cases compared to controls, driven predominantly by the HR group, in whom Toll-Like Receptor (TLR)4+ monocytes and monocyte Triggering Receptor Expressed on Myeloid cells-2 (TREM2) expression were also increased. Monocyte Human Leukocyte Antigen (HLA)-DR expression correlated with clinical variables, with lower levels associated with worse cognitive/motor performance. Notably, monocyte changes were accompanied by elevated serum bacterial endotoxin, again predominantly in the HR group. Serum alpha-synuclein and inflammasome-related caspase-1 were decreased in PD cases compared to controls regardless of group, with decreased monocyte alpha-synuclein secretion in HR cases. Further, alpha-synuclein and caspase-1 correlated positively in serum and monocyte lysates, and in plasma from the larger cohort, though no associations were seen with baseline or 36-month longitudinal clinical data. Principal Components Analysis of all monocyte and significant serum markers indicated 3 major components. Component 1 (alpha-synuclein, caspase-1, TLR2+ monocytes) differentiated PD cases and controls in both groups, while Component 2 (endotoxin, monocyte TREM2, alpha-synuclein) did so predominantly in the HR group. Component 3 (classical monocytes, alpha-synuclein) also differentiated cases and controls overall in both groups. These findings demonstrate that systemic innate immune changes are present in PD and are greatest in those at higher risk of rapid progression to dementia. Markers associated with PD per-se (alpha-synuclein, caspase-1), differ from those related to cognitive progression and clinical heterogeneity (endotoxin, TREM2, TLR4, classical monocytes, HLA-DR), with mechanistic and therapeutic implications. Alpha-synuclein and caspase-1 are associated, suggesting inflammasome involvement common to all PD, while bacterial translocation associated changes may contribute towards progression to Parkinson's dementia. Additionally, HLA-DR-associated variations in antigen presentation/clearance may modulate existing clinical disease.


Assuntos
Doença de Parkinson , Biomarcadores , Humanos , Imunidade Inata , Glicoproteínas de Membrana , Monócitos , Receptores Imunológicos , alfa-Sinucleína
11.
Database (Oxford) ; 2019(1)2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31681953

RESUMO

Mycobacterium abscessus, a rapid growing, multidrug resistant, nontuberculous mycobacteria, can cause a wide range of opportunistic infections, particularly in immunocompromised individuals. M. abscessus has emerged as a growing threat to patients with cystic fibrosis, where it causes accelerated inflammatory lung damage, is difficult and sometimes impossible to treat and can prevent safe transplantation. There is therefore an urgent unmet need to develop new therapeutic strategies. The elucidation of the M. abscessus genome in 2009 opened a wide range of research possibilities in the field of drug discovery that can be more effectively exploited upon the characterization of the structural proteome. Where there are no experimental structures, we have used the available amino acid sequences to create 3D models of the majority of the remaining proteins that constitute the M. abscessus proteome (3394 proteins and over 13 000 models) using a range of up-to-date computational tools, many developed by our own group. The models are freely available for download in an on-line database, together with quality data and functional annotation. Furthermore, we have developed an intuitive and user-friendly web interface (http://www.mabellinidb.science) that enables easy browsing, querying and retrieval of the proteins of interest. We believe that this resource will be of use in evaluating the prospective targets for design of antimicrobial agents and will serve as a cornerstone to support the development of new molecules to treat M. abscessus infections.


Assuntos
Proteínas de Bactérias , Bases de Dados Genéticas , Genoma Bacteriano , Modelos Moleculares , Mycobacterium abscessus , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Estudo de Associação Genômica Ampla , Infecções por Mycobacterium não Tuberculosas/genética , Infecções por Mycobacterium não Tuberculosas/metabolismo , Mycobacterium abscessus/química , Mycobacterium abscessus/genética , Mycobacterium abscessus/metabolismo
12.
J Control Release ; 314: 116-124, 2019 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-31647980

RESUMO

Mycobacterium tuberculosis (Mtb) remains a major challenge to global health, made worse by the spread of multi-drug resistance. Currently, the efficacy and safety of treatment is limited by difficulties in achieving and sustaining adequate tissue antibiotic concentrations while limiting systemic drug exposure to tolerable levels. Here we show that nanoparticles generated from a polymer-antibiotic conjugate ('nanobiotics') deliver sustained release of active drug upon hydrolysis in acidic environments, found within Mtb-infected macrophages and granulomas, and can, by encapsulation of a second antibiotic, provide a mechanism of synchronous drug delivery. Nanobiotics are avidly taken up by infected macrophages, enhance killing of intracellular Mtb, and are efficiently delivered to granulomas and extracellular mycobacterial cords in vivo in an infected zebrafish model. We demonstrate that isoniazid (INH)-derived nanobiotics, alone or with additional encapsulation of clofazimine (CFZ), enhance killing of mycobacteria in vitro and in infected zebrafish, supporting the use of nanobiotics for Mtb therapy and indicating that nanoparticles generated from polymer-small molecule conjugates might provide a more general solution to delivering co-ordinated combination chemotherapy.


Assuntos
Antituberculosos/administração & dosagem , Isoniazida/administração & dosagem , Mycobacterium tuberculosis/efeitos dos fármacos , Nanopartículas , Animais , Antituberculosos/farmacologia , Clofazimina/administração & dosagem , Clofazimina/farmacologia , Preparações de Ação Retardada , Modelos Animais de Doenças , Combinação de Medicamentos , Sistemas de Liberação de Medicamentos , Humanos , Isoniazida/farmacologia , Macrófagos/microbiologia , Polímeros/química , Tuberculose/tratamento farmacológico , Tuberculose/microbiologia , Peixe-Zebra
13.
mBio ; 10(5)2019 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-31551336

RESUMO

Both intracellular immune sensing and extracellular innate immune sensing have been implicated in initiating macrophage proinflammatory cytokine responses to Streptococcus pneumoniae The S. pneumoniae capsule, a major virulence determinant, prevents phagocytosis, and we hypothesized that this would reduce activation of host innate inflammatory responses by preventing activation of intracellular proinflammatory signaling pathways. We investigated this hypothesis in human monocyte-derived macrophages stimulated with encapsulated or isogenic unencapsulated mutant S. pneumoniae Unexpectedly, despite strongly inhibiting bacterial internalization, the capsule resulted in enhanced inflammatory cytokine production by macrophages infected with S. pneumoniae Experiments using purified capsule material and a Streptococcus mitis mutant expressing an S. pneumoniae serotype 4 capsule indicated these differences required whole bacteria and were not due to proinflammatory effects of the capsule itself. Transcriptional profiling demonstrated relatively few differences in macrophage gene expression profiles between infections with encapsulated S. pneumoniae and those with unencapsulated S. pneumoniae, largely limited to reduced expression of proinflammatory genes in response to unencapsulated bacteria, predicted to be due to reduced activation of the NF-κB family of transcription factors. Blocking S. pneumoniae internalization using cytochalasin D had minimal effects on the inflammatory response to S. pneumoniae Experiments using murine macrophages indicated that the affected genes were dependent on Toll-like receptor 2 (TLR2) activation, although not through direct stimulation of TLR2 by capsule polysaccharide. Our data demonstrate that the early macrophage proinflammatory response to S. pneumoniae is mainly dependent on extracellular bacteria and reveal an unexpected proinflammatory effect of encapsulated S. pneumoniae that could contribute to disease pathogenesis.IMPORTANCE Multiple extra- and intracellular innate immune receptors have been identified that recognize Streptococcus pneumoniae, but the relative contributions of intra- versus extracellular bacteria to the inflammatory response were unknown. We have shown that intracellular S. pneumoniae contributes surprisingly little to the inflammatory responses, with production of important proinflammatory cytokines largely dependent on extracellular bacteria. Furthermore, although we expected the S. pneumoniae polysaccharide capsule to block activation of the host immune system by reducing bacterial internalization and therefore activation of intracellular innate immune receptors, there was an increased inflammatory response to encapsulated compared to unencapsulated bacteria, which is likely to contribute to disease pathogenesis.


Assuntos
Proteínas de Bactérias/fisiologia , Interações entre Hospedeiro e Microrganismos/fisiologia , Inflamação/fisiopatologia , Macrófagos/fisiologia , Transdução de Sinais/fisiologia , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/patogenicidade , Humanos
15.
J Med Chem ; 62(15): 7210-7232, 2019 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-31282680

RESUMO

Mycobacterium abscessus (Mab) is a rapidly growing species of multidrug-resistant nontuberculous mycobacteria that has emerged as a growing threat to individuals with cystic fibrosis and other pre-existing chronic lung diseases. Mab pulmonary infections are difficult, or sometimes impossible, to treat and result in accelerated lung function decline and premature death. There is therefore an urgent need to develop novel antibiotics with improved efficacy. tRNA (m1G37) methyltransferase (TrmD) is a promising target for novel antibiotics. It is essential in Mab and other mycobacteria, improving reading frame maintenance on the ribosome to prevent frameshift errors. In this work, a fragment-based approach was employed with the merging of two fragments bound to the active site, followed by structure-guided elaboration to design potent nanomolar inhibitors against Mab TrmD. Several of these compounds exhibit promising activity against mycobacterial species, including Mycobacterium tuberculosis and Mycobacterium leprae in addition to Mab, supporting the use of TrmD as a target for the development of antimycobacterial compounds.


Assuntos
Antibacterianos/química , Desenvolvimento de Medicamentos/métodos , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/metabolismo , Mycobacterium abscessus/efeitos dos fármacos , Mycobacterium abscessus/enzimologia , tRNA Metiltransferases/antagonistas & inibidores , tRNA Metiltransferases/metabolismo , Antibacterianos/farmacologia , Cristalografia por Raios X/métodos , Humanos , Estrutura Secundária de Proteína
16.
Science ; 364(6442)2019 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-31123110

RESUMO

Approximately 2.4% of the human mitochondrial DNA (mtDNA) genome exhibits common homoplasmic genetic variation. We analyzed 12,975 whole-genome sequences to show that 45.1% of individuals from 1526 mother-offspring pairs harbor a mixed population of mtDNA (heteroplasmy), but the propensity for maternal transmission differs across the mitochondrial genome. Over one generation, we observed selection both for and against variants in specific genomic regions; known variants were more likely to be transmitted than previously unknown variants. However, new heteroplasmies were more likely to match the nuclear genetic ancestry as opposed to the ancestry of the mitochondrial genome on which the mutations occurred, validating our findings in 40,325 individuals. Thus, human mtDNA at the population level is shaped by selective forces within the female germ line under nuclear genetic control, which ensures consistency between the two independent genetic lineages.


Assuntos
DNA Mitocondrial/genética , Genoma Mitocondrial , Herança Materna , Óvulo/crescimento & desenvolvimento , Seleção Genética , Feminino , Variação Genética , Humanos
17.
Cancer Cell ; 35(5): 767-781.e6, 2019 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-31085177

RESUMO

Although immune checkpoint blockers have yielded significant clinical benefits in patients with different malignancies, the efficacy of these therapies is still limited. Here, we show that disruption of transmembrane protein 176B (TMEM176B) contributes to CD8+ T cell-mediated tumor growth inhibition by unleashing inflammasome activation. Lack of Tmem176b enhances the antitumor activity of anti-CTLA-4 antibodies through mechanisms involving caspase-1/IL-1ß activation. Accordingly, patients responding to checkpoint blockade therapies display an activated inflammasome signature. Finally, we identify BayK8644 as a potent TMEM176B inhibitor that promotes CD8+ T cell-mediated tumor control and reinforces the antitumor activity of both anti-CTLA-4 and anti-PD-1 antibodies. Thus, pharmacologic de-repression of the inflammasome by targeting TMEM176B may enhance the therapeutic efficacy of immune checkpoint blockers.


Assuntos
Antineoplásicos/farmacologia , Inflamassomos/efeitos dos fármacos , Inflamassomos/imunologia , Proteínas de Membrana/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Animais , Anticorpos Monoclonais/farmacologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Células CHO , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cricetulus , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neoplasias/metabolismo , Xenopus laevis/metabolismo
18.
Philos Trans A Math Phys Eng Sci ; 377(2147): 20180422, 2019 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-31030650

RESUMO

Structure-guided drug discovery emerged in the 1970s and 1980s, stimulated by the three-dimensional structures of protein targets that became available, mainly through X-ray crystal structure analysis, assisted by the development of synchrotron radiation sources. Structures of known drugs or inhibitors were used to guide the development of leads. The growth of high-throughput screening during the late 1980s and the early 1990s in the pharmaceutical industry of chemical libraries of hundreds of thousands of compounds of molecular weight of approximately 500 Da was impressive but still explored only a tiny fraction of the chemical space of the predicted 1040 drug-like compounds. The use of fragments with molecular weights less than 300 Da in drug discovery not only decreased the chemical space needing exploration but also increased promiscuity in binding targets. Here we discuss advances in X-ray fragment screening and the challenge of identifying sites where fragments not only bind but can be chemically elaborated while retaining their positions and binding modes. We first describe the analysis of fragment binding using conventional X-ray difference Fourier techniques, with Mycobacterium abscessus SAICAR synthetase (PurC) as an example. We observe that all fragments occupy positions predicted by computational hotspot mapping. We compare this with fragment screening at Diamond Synchrotron Light Source XChem facility using PanDDA software, which identifies many more fragment hits, only some of which bind to the predicted hotspots. Many low occupancy sites identified may not support elaboration to give adequate ligand affinity, although they will likely be useful in drug discovery as 'warm spots' for guiding elaboration of fragments bound at hotspots. We discuss implications of these observations for fragment screening at the synchrotron sources. This article is part of the theme issue 'Fifty years of synchrotron science: achievements and opportunities'.


Assuntos
Descoberta de Drogas/história , Síncrotrons/história , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Descoberta de Drogas/métodos , Descoberta de Drogas/tendências , Ensaios de Triagem em Larga Escala/história , Ensaios de Triagem em Larga Escala/métodos , Ensaios de Triagem em Larga Escala/tendências , História do Século XX , História do Século XXI , Humanos , Modelos Moleculares , Mycobacterium abscessus/efeitos dos fármacos , Mycobacterium abscessus/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Peptídeo Sintases/química , Peptídeo Sintases/metabolismo
19.
Cell Rep ; 26(7): 1828-1840.e4, 2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30759393

RESUMO

Infection by rapidly growing Mycobacterium abscessus is increasingly prevalent in cystic fibrosis (CF), a genetic disease caused by a defective CF transmembrane conductance regulator (CFTR). However, the potential link between a dysfunctional CFTR and vulnerability to M. abscessus infection remains unknown. Herein, we exploit a CFTR-depleted zebrafish model, recapitulating CF immuno-pathogenesis, to study the contribution of CFTR in innate immunity against M. abscessus infection. Loss of CFTR increases susceptibility to infection through impaired NADPH oxidase-dependent restriction of intracellular growth and reduced neutrophil chemotaxis, which together compromise granuloma formation and integrity. As a consequence, extracellular multiplication of M. abscessus expands rapidly, inducing abscess formation and causing lethal infections. Because these phenotypes are not observed with other mycobacteria, our findings highlight the crucial and specific role of CFTR in the immune control of M. abscessus by mounting effective oxidative responses.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/imunologia , Infecções por Mycobacterium não Tuberculosas/imunologia , Mycobacterium abscessus/imunologia , Estresse Oxidativo/imunologia , Proteínas de Peixe-Zebra/imunologia , Animais , Animais Geneticamente Modificados , Modelos Animais de Doenças , Infecções por Mycobacterium não Tuberculosas/microbiologia , Infecções por Mycobacterium não Tuberculosas/prevenção & controle , Mycobacterium abscessus/isolamento & purificação , Espécies Reativas de Oxigênio/imunologia , Peixe-Zebra
20.
Respirology ; 24(11): 1053-1062, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-30801930

RESUMO

Bronchiectasis has historically been considered to be irreversible dilatation of the airways, but with modern imaging techniques it has been proposed that 'irreversible' be dropped from the definition. The upper limit of normal for the ratio of airway to arterial development increases with age, and a developmental perspective is essential. Bronchiectasis (and persistent bacterial bronchitis, PBB) is a descriptive term and not a diagnosis, and should be the start not the end of the patient's diagnostic journey. PBB, characterized by airway infection and neutrophilic inflammation but without significant airway dilatation may be a precursor of bronchiectasis, and there are many commonalities in the microbiology and the pathology, which are reviewed in this article. A high index of suspicion is essential, and a history of chronic wet or productive cough for more than 4-8 weeks should prompt investigation. There are numerous underlying causes of bronchiectasis, although in many cases no cause is found. Causes include post-infectious, especially after tuberculosis, adenoviral or pertussis infection; aspiration syndromes; defects in host defence, which may solely affect the airways (cystic fibrosis, not considered in this review, and primary ciliary dyskinesia); and primary ciliary dyskinesia or be systemic, such as common variable immunodeficiency; genetic syndromes; and anatomical defects such as intraluminal airway obstruction (e.g. foreign body), intramural obstruction (e.g. complete cartilage rings) and external airway compression (e.g. by tuberculous lymph nodes). Identification of the underlying cause is important, because some of these conditions have specific treatments and others genetic implications for the family.


Assuntos
Bronquiectasia , Sistema Respiratório , Adulto , Bronquiectasia/diagnóstico , Bronquiectasia/etiologia , Criança , Diagnóstico Diferencial , Humanos , Sistema Respiratório/diagnóstico por imagem , Sistema Respiratório/crescimento & desenvolvimento , Sistema Respiratório/imunologia , Sistema Respiratório/fisiopatologia
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