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1.
FASEB J ; 35(9): e21853, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34416038

RESUMO

We highlight the ability of the tuberculosis (TB) causing bacterial pathogen, Mycobacterium tuberculosis (Mtb), to induce key characteristics that are associated with established IARC classified Group 1 and Group 2A carcinogenic agents. There is sufficient evidence from epidemiological case-control, cohort and meta-analysis studies of increased lung cancer (LC) risk in pre-existing/active/old TB cases. Similar to carcinogens and other pathogenic infectious agents, exposure to aerosol-containing Mtb sprays in mice produce malignant transformation of cells that result in squamous cell carcinoma. Convincing, mechanistic data show several characteristics shared between TB and LC which include chronic inflammation, genomic instability and replicative immortality, just to name a few cancer hallmarks. These hallmarks of cancer may serve as precursors to malignant transformation. Together, these findings form the basis of our postulate that Mtb is a complete human pulmonary carcinogen. We also discuss how Mtb may act as both an initiating agent and promoter of tumor growth. Forthcoming experimental studies will not only serve as proof-of-concept but will also pivot our understanding of how to manage/treat TB cases as well as offer solutions to clinical conundrums of TB lesions masquerading as tumors. Clinical validation of our concept may also help pave the way for next generation personalized medicine for the management of pulmonary TB/cancer particularly for cases that are not responding well to conventional chemotherapy or TB drugs.


Assuntos
Transformação Celular Neoplásica , Neoplasias Pulmonares/etiologia , Neoplasias Pulmonares/microbiologia , Pulmão/microbiologia , Pulmão/patologia , Mycobacterium tuberculosis/patogenicidade , Tuberculose Pulmonar/complicações , Tuberculose Pulmonar/microbiologia , Adolescente , Adulto , Idoso , Células Epiteliais Alveolares/citologia , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/patologia , Animais , Carcinógenos , Transformação Celular Neoplásica/genética , Criança , Estudos de Coortes , Modelos Animais de Doenças , Progressão da Doença , Feminino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Modelos Biológicos , Mycobacterium tuberculosis/genética , Metástase Neoplásica/genética , Células-Tronco Neoplásicas/patologia , Fatores de Risco , Tuberculose Pulmonar/patologia , Adulto Jovem
2.
Methods Mol Biol ; 2314: 365-383, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34235663

RESUMO

The utility of fluorescent proteins in bacterial research has long been appreciated, with extensive use in the Mycobacterium tuberculosis field. In more recent years, a new generation of fluorescent tools has been developed for use in M. tuberculosis research. These new fluorescent reporters exploit the immense genetic and transcriptional knowledge now available, and enable the use of the bacteria as direct reporters of the local environment during infection, as well as provide insight into bacterial replication status in situ. Here we describe methods for the construction of such fluorescent reporter M. tuberculosis strains, and their use in combination with confocal microscopy and flow cytometry approaches for single bacterium-level analyses of M. tuberculosis physiology and M. tuberculosis-host interactions.


Assuntos
Proteínas Luminescentes/metabolismo , Pulmão/microbiologia , Mycobacterium tuberculosis/crescimento & desenvolvimento , Tuberculose Pulmonar/microbiologia , Animais , Citometria de Fluxo , Interações Hospedeiro-Patógeno , Proteínas Luminescentes/genética , Pulmão/metabolismo , Pulmão/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidade , Tuberculose Pulmonar/metabolismo , Tuberculose Pulmonar/patologia
3.
Methods Mol Biol ; 2314: 167-182, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34235652

RESUMO

Mycobacterium tuberculosis colonizes, survives, and grows inside macrophages. In vitro macrophage infection models, using both primary macrophages and cell lines, enable the characterization of the pathogen response to macrophage immune pressure and intracellular environmental cues. We describe methods to propagate and infect primary murine bone marrow-derived macrophages, HoxB8 conditionally immortalized myeloid cells, Max Planck Institute alveolar macrophage-like cells, and J774 and THP-1 macrophage-like cell lines. We also present methods on the characterization of M. tuberculosis intracellular survival and the preparation of infected macrophages for imaging.


Assuntos
Macrófagos Alveolares/microbiologia , Macrófagos/microbiologia , Imagem Molecular/métodos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Células Mieloides/microbiologia , Animais , Células Cultivadas , Humanos , Técnicas In Vitro , Macrófagos/patologia , Macrófagos Alveolares/patologia , Camundongos , Mycobacterium tuberculosis/patogenicidade , Células Mieloides/patologia
4.
Methods Mol Biol ; 2314: 261-271, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34235657

RESUMO

Flow cytometry enables the measurement of tens of features on individual cells from complex mixtures. Flow cytometry enables high-throughput quantification of cell size, gene and protein expression. In the case of studies of host-pathogen interactions, this tool provides a facile way of identifying cells that have been successfully infected by a pathogen. Several recent technological advances have greatly improved throughput and the number of features that can be simultaneously monitored by this technique. Here, we describe common workflows to study Mycobacterium tuberculosis heterogeneity and host-M. tuberculosis interactions using flow cytometry and related technologies.


Assuntos
Citometria de Fluxo/métodos , Interações Hospedeiro-Patógeno , Macrófagos/imunologia , Macrófagos/microbiologia , Infecções por Mycobacterium/microbiologia , Mycobacterium tuberculosis/patogenicidade , Humanos , Infecções por Mycobacterium/imunologia , Infecções por Mycobacterium/patologia
5.
Methods Mol Biol ; 2314: 649-702, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34235675

RESUMO

Mycobacterium tuberculosis is able to colonize, persist, and massively replicate in host cells, such as phagocytes and epithelial cells. The intracellular stage of the bacteria is critical to the development of tuberculosis pathogenesis. The detailed mechanisms of intracellular trafficking of the bacillus are not fully understood and require further investigations. Therefore, increasing the knowledge of this process will help to develop therapeutic tools that will lower the burden of tuberculosis. M. tuberculosis is genetically tractable and tolerates the expression of heterologous fluorescent proteins. Thus, the intracellular distribution of the bacteria expressing fluorescent tracers can be easily defined using confocal microscopy. Advances in imaging techniques and images-based analysis allow the rapid quantification of biological objects in complex environments. In this chapter, we detailed high-content / high-throughput imaging methods to track the bacillus within host cell settings.


Assuntos
Células Dendríticas/microbiologia , Células Epiteliais/microbiologia , Ensaios de Triagem em Larga Escala/métodos , Macrófagos/microbiologia , Mycobacterium tuberculosis/crescimento & desenvolvimento , Fagócitos/microbiologia , Tuberculose/microbiologia , Animais , Células Dendríticas/metabolismo , Testes Diagnósticos de Rotina , Células Epiteliais/metabolismo , Humanos , Macrófagos/metabolismo , Camundongos , Mycobacterium tuberculosis/patogenicidade , Estresse Oxidativo , Fagócitos/metabolismo , Espécies Reativas de Oxigênio , Tuberculose/metabolismo
6.
Int J Mol Sci ; 22(11)2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-34206086

RESUMO

Tuberculosis (TB) is an important infectious disease and a public health problem. The organs most frequently affected by TB are the lungs; despite this, it has been reported that TB patients suffer from depression and anxiety, which have been attributed to social factors. In previous experimental work, we observed that the extensive pulmonary inflammation characteristic of TB with high cytokine production induces neuroinflammation, neuronal death and behavioral abnormalities in the absence of brain infection. The objective of the present work was to reduce this neuroinflammation and avoid the psycho-affective disorders showed during pulmonary TB. Glucocorticoids (GCs) are the first-line treatment for neuroinflammation; however, their systemic administration generates various side effects, mostly aggravating pulmonary TB due to immunosuppression of cellular immunity. Intranasal administration is a route that allows drugs to be released directly in the brain through the olfactory nerve, reducing their doses and side effects. In the present work, dexamethasone's (DEX) intranasal administration was evaluated in TB BALB /c mice comparing three different doses (0.05, 0.25 and 2.5 mg/kg BW) on lung disease evolution, neuroinflammation and behavioral alterations. Low doses of dexamethasone significantly decreased neuroinflammation, improving behavioral status without aggravating lung disease.


Assuntos
Encéfalo/efeitos dos fármacos , Dexametasona/farmacologia , Inflamação/tratamento farmacológico , Tuberculose Pulmonar/tratamento farmacológico , Administração Intranasal , Animais , Ansiedade/complicações , Ansiedade/tratamento farmacológico , Ansiedade/patologia , Encéfalo/patologia , Depressão/complicações , Depressão/tratamento farmacológico , Depressão/patologia , Modelos Animais de Doenças , Glucocorticoides/farmacologia , Humanos , Inflamação/complicações , Inflamação/microbiologia , Inflamação/patologia , Camundongos , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/patogenicidade , Tuberculose Pulmonar/complicações , Tuberculose Pulmonar/microbiologia , Tuberculose Pulmonar/patologia
7.
Viruses ; 13(5)2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34067925

RESUMO

By the middle of 2021, we are still immersed in the coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The concurrence of this new pandemic in regions where human immunodeficiency virus (HIV) and tuberculosis (TB) infections possess the same epidemiological consideration, has arisen concerns about the prognosis, clinical management, symptomatology, and treatment of patients with triple infection. At the same time, healthcare services previously devoted to diagnosis and treatment of TB and HIV are being jeopardized by the urgent need of resources and attention for COVID-19 patients. The aim of this review was to collect any article considering the three conditions (HIV, TB, and SARS-CoV-2), included in PubMed/Medline and published in the English language since the beginning of the COVID-19 pandemic. We focused on detailed descriptions of the unusual cases describing the three co-infections. Eighty-four out of 184 publications retrieved met our inclusion criteria, but only three of them reported cases (five in total) with the three concomitant infections. The clinical evolution, management, and therapy of all of them were not different from mild/severe cases with exclusive COVID-19; the outcome was not worse either, with recovery for the five patients. Cases of patients with COVID-19 besides HIV and TB infections are scarce in literature, but studies deliberately embracing the triple infection as a priori inclusion criterion should be carried out in order to provide a complete understanding of joint influence.


Assuntos
COVID-19/complicações , Coinfecção/epidemiologia , COVID-19/epidemiologia , Testes Diagnósticos de Rotina , HIV/patogenicidade , Infecções por HIV/complicações , Infecções por HIV/epidemiologia , Humanos , Mycobacterium tuberculosis/patogenicidade , Pandemias , SARS-CoV-2/patogenicidade , Tuberculose/complicações , Tuberculose/epidemiologia
8.
Nat Med ; 27(9): 1646-1654, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34183838

RESUMO

The pathophysiology of adverse events following programmed cell death protein 1 (PD-1) blockade, including tuberculosis (TB) and autoimmunity, remains poorly characterized. We studied a patient with inherited PD-1 deficiency and TB who died of pulmonary autoimmunity. The patient's leukocytes did not express PD-1 or respond to PD-1-mediated suppression. The patient's lymphocytes produced only small amounts of interferon (IFN)-γ upon mycobacterial stimuli, similarly to patients with inborn errors of IFN-γ production who are vulnerable to TB. This phenotype resulted from a combined depletion of Vδ2+ γδ T, mucosal-associated invariant T and CD56bright natural killer lymphocytes and dysfunction of other T lymphocyte subsets. Moreover, the patient displayed hepatosplenomegaly and an expansion of total, activated and RORγT+ CD4-CD8- double-negative αß T cells, similar to patients with STAT3 gain-of-function mutations who display lymphoproliferative autoimmunity. This phenotype resulted from excessive amounts of STAT3-activating cytokines interleukin (IL)-6 and IL-23 produced by activated T lymphocytes and monocytes, and the STAT3-dependent expression of RORγT by activated T lymphocytes. Our work highlights the indispensable role of human PD-1 in governing both antimycobacterial immunity and self-tolerance, while identifying potentially actionable molecular targets for the diagnostic and therapeutic management of TB and autoimmunity in patients on PD-1 blockade.


Assuntos
Autoimunidade/genética , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Receptor de Morte Celular Programada 1/genética , Fator de Transcrição STAT3/genética , Tuberculose/imunologia , Autoimunidade/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Antígeno CD56/genética , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Criança , Humanos , Inibidores de Checkpoint Imunológico/efeitos adversos , Inibidores de Checkpoint Imunológico/uso terapêutico , Imunoterapia/efeitos adversos , Interleucina-23/genética , Interleucina-6/genética , Linfócitos Intraepiteliais/imunologia , Linfócitos Intraepiteliais/patologia , Masculino , Mycobacterium tuberculosis/patogenicidade , Neoplasias/complicações , Neoplasias/tratamento farmacológico , Neoplasias/mortalidade , Receptor de Morte Celular Programada 1/deficiência , Tuberculose/genética , Tuberculose/mortalidade
9.
Front Immunol ; 12: 627638, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33936040

RESUMO

Background: Tuberculosis (TB) is still a major challenge for humankind. Because regions with the highest incidence also have a high prevalence of helminthiasis and nutritional scarcity, we wanted to understand the impact of these on TB progression. Methods: We have developed an experimental murine model for active TB in C3HeB/FeJ, coinfected with Trichuris muris and Heligmosomoides polygyrus nematodes, and exposed to an environmental mycobacterium (M. manresensis) and intermittent fasting. Cause-effect relationships among these factors were explored with Partial Least Squares Path modelling (PLSPM). Results: Previous parasitization had a major anti-inflammatory effect and reduced systemic levels of ADA, haptoglobin, local pulmonary levels of IL-1ß, IL-6, TNF-α, CXCL-1, CXCL-5 and IL-10. Oral administration of heat-killed M. manresensis resulted in a similar outcome. Both interventions diminished pulmonary pathology and bacillary load, but intermittent food deprivation reduced this protective effect increasing stress and inflammation. The PLSPM revealed nematodes might have protective effects against TB progression. Conclusions: Significantly higher cortisol levels in food-deprivation groups showed it is a stressful condition, which might explain its deleterious effect. This highlights the impact of food security on TB eradication policies and the need to prioritize food supply over deworming activities.


Assuntos
Coinfecção , Privação de Alimentos , Helmintíase/parasitologia , Enteropatias Parasitárias/parasitologia , Pulmão/microbiologia , Mycobacterium tuberculosis/patogenicidade , Nematospiroides dubius/patogenicidade , Infecções por Strongylida/parasitologia , Tricuríase/parasitologia , Trichuris/patogenicidade , Tuberculose Pulmonar/microbiologia , Fenômenos Fisiológicos da Nutrição Animal , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Feminino , Helmintíase/imunologia , Helmintíase/metabolismo , Interações Hospedeiro-Parasita , Mediadores da Inflamação/metabolismo , Enteropatias Parasitárias/imunologia , Enteropatias Parasitárias/metabolismo , Pulmão/imunologia , Pulmão/metabolismo , Masculino , Camundongos Endogâmicos C3H , Mycobacterium tuberculosis/imunologia , Nematospiroides dubius/imunologia , Estado Nutricional , Infecções por Strongylida/imunologia , Infecções por Strongylida/metabolismo , Tricuríase/imunologia , Tricuríase/metabolismo , Trichuris/imunologia , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/metabolismo
10.
FASEB J ; 35(6): e21543, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34046950

RESUMO

Clinically, bone destruction caused by Mycobacterium tuberculosis was serious especially in patients with vitamin D (VD) deficiency. However, the role of VD in M. tuberculosis-induced bone destruction remains clear. In this context, we investigate the role of VD and vitamin D receptor (VDR) in the M. tuberculosis-induced bone destruction. First, we infected RAW264.7 and bone marrow-derived macrophages (BMMs) with Mycobacterium bovis Bacillus Calmette-Guérin (M. bovis BCG) in vitro. Then, we activated VDR through VD administration. TRAP and FAK staining, bone resorption assays, immunofluorescence staining, qPCR, and western blot were carried out. In vivo, the M. tuberculosis-induced osteolytic model on the murine skull was established and the µCT and histological analyses were performed. We found that VDR and TRAP were upregulated in bone tuberculosis tissue and proved that M. tuberculosis infection promoted osteoclastogenesis in RAW264.7 and BMMs. VD could inhibit osteoclasts differentiation, fusion, and bone resorption dose-dependently. However, when VDR was knocked down, the inhibitory effect of VD on osteoclasts disappeared. In mechanism, activation of VDR inhibits the phosphorylation of IκB α, thereby inhibiting NFκB signaling pathway and alleviating osteoclastogenesis. Furthermore, in the skull osteolysis model, VD administration reduced osteolysis, but not in VDR-/- mice. Our study, for the first time, demonstrates that activation of VDR by VD administration inhibits M. tuberculosis-induced bone destruction. Our results reveal that VD and VDR are potential therapeutic targets for M. tuberculosis-induced bone destruction, and are of great clinical significance for the development of new therapeutic strategies.


Assuntos
Macrófagos/efeitos dos fármacos , Mycobacterium tuberculosis/patogenicidade , NF-kappa B/antagonistas & inibidores , Osteólise/prevenção & controle , Receptores de Calcitriol/metabolismo , Tuberculose/complicações , Vitamina D/administração & dosagem , Animais , Macrófagos/metabolismo , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/genética , NF-kappa B/metabolismo , Osteólise/etiologia , Osteólise/metabolismo , Osteólise/patologia , Receptores de Calcitriol/genética , Tuberculose/microbiologia , Vitaminas/administração & dosagem
11.
PLoS Pathog ; 17(5): e1009570, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33989345

RESUMO

Mycobacterium tuberculosis (Mtb) has complex and dynamic interactions with the human host, and subpopulations of Mtb that emerge during infection can influence disease outcomes. This study implicates zinc ion (Zn2+) availability as a likely driver of bacterial phenotypic heterogeneity in vivo. Zn2+ sequestration is part of "nutritional immunity", where the immune system limits micronutrients to control pathogen growth, but this defense mechanism seems to be ineffective in controlling Mtb infection. Nonetheless, Zn2+-limitation is an environmental cue sensed by Mtb, as calprotectin triggers the zinc uptake regulator (Zur) regulon response in vitro and co-localizes with Zn2+-limited Mtb in vivo. Prolonged Zn2+ limitation leads to numerous physiological changes in vitro, including differential expression of certain antigens, alterations in lipid metabolism and distinct cell surface morphology. Furthermore, Mtb enduring limited Zn2+ employ defensive measures to fight oxidative stress, by increasing expression of proteins involved in DNA repair and antioxidant activity, including well described virulence factors KatG and AhpC, along with altered utilization of redox cofactors. Here, we propose a model in which prolonged Zn2+ limitation defines a population of Mtb with anticipatory adaptations against impending immune attack, based on the evidence that Zn2+-limited Mtb are more resistant to oxidative stress and exhibit increased survival and induce more severe pulmonary granulomas in mice. Considering that extracellular Mtb may transit through the Zn2+-limited caseum before infecting naïve immune cells or upon host-to-host transmission, the resulting phenotypic heterogeneity driven by varied Zn2+ availability likely plays a key role during early interactions with host cells.


Assuntos
Granuloma/microbiologia , Lipidômica , Mycobacterium tuberculosis/fisiologia , Proteoma , Transcriptoma , Zinco/deficiência , Adaptação Fisiológica , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Granuloma/imunologia , Homeostase , Interações Hospedeiro-Patógeno , Humanos , Pulmão/microbiologia , Camundongos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/patogenicidade , Oxirredução , Estresse Oxidativo , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
12.
Nat Med ; 27(7): 1171-1177, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34031604

RESUMO

Multidrug-resistant tuberculosis (MDR-TB) accounts for one third of the annual deaths due to antimicrobial resistance1. Drug resistance-conferring mutations frequently cause fitness costs in bacteria2-5. Experimental work indicates that these drug resistance-related fitness costs might be mitigated by compensatory mutations6-10. However, the clinical relevance of compensatory evolution remains poorly understood. Here we show that, in the country of Georgia, during a 6-year nationwide study, 63% of MDR-TB was due to patient-to-patient transmission. Compensatory mutations and patient incarceration were independently associated with transmission. Furthermore, compensatory mutations were overrepresented among isolates from incarcerated individuals that also frequently spilled over into the non-incarcerated population. As a result, up to 31% of MDR-TB in Georgia was directly or indirectly linked to prisons. We conclude that prisons fuel the epidemic of MDR-TB in Georgia by acting as ecological drivers of fitness-compensated strains with high transmission potential.


Assuntos
Mycobacterium tuberculosis/patogenicidade , Prisões , Tuberculose Resistente a Múltiplos Medicamentos/transmissão , Antituberculosos/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Humanos , Mutação/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Prisioneiros , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologia , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia
13.
Am J Respir Crit Care Med ; 204(5): 583-595, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34015247

RESUMO

Rationale: Our current understanding of tuberculosis (TB) pathophysiology is limited by a reliance on animal models, the paucity of human TB lung tissue, and traditional histopathological analysis, a destructive two-dimensional approach that provides limited spatial insight. Determining the three-dimensional (3D) structure of the necrotic granuloma, a characteristic feature of TB, will more accurately inform preventive TB strategies.Objectives: To ascertain the 3D shape of the human tuberculous granuloma and its spatial relationship with airways and vasculature within large lung tissues.Methods: We characterized the 3D microanatomical environment of human tuberculous lungs by using micro computed tomography, histopathology, and immunohistochemistry. By using 3D segmentation software, we accurately reconstructed TB granulomas, vasculature, and airways in three dimensions and confirmed our findings by using histopathology and immunohistochemistry.Measurements and Main Results: We observed marked heterogeneity in the morphology, volume, and number of TB granulomas in human lung sections. Unlike depictions of granulomas as simple spherical structures, human necrotic granulomas exhibit complex, cylindrical, branched morphologies that are connected to the airways and shaped by the bronchi. The use of 3D imaging of human TB lung sections provides unanticipated insight into the spatial organization of TB granulomas in relation to the airways and vasculature.Conclusions: Our findings highlight the likelihood that a single, structurally complex lesion could be mistakenly viewed as multiple independent lesions when evaluated in two dimensions. In addition, the lack of vascularization within obstructed bronchi establishes a paradigm for antimycobacterial drug tolerance. Lastly, our results suggest that bronchogenic spread of Mycobacterium tuberculosis reseeds the lung.


Assuntos
Granuloma/diagnóstico por imagem , Pulmão/diagnóstico por imagem , Pulmão/patologia , Pulmão/ultraestrutura , Tuberculose Pulmonar/diagnóstico por imagem , Tuberculose Pulmonar/microbiologia , Tuberculose Pulmonar/fisiopatologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Imageamento Tridimensional/métodos , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/patogenicidade , África do Sul , Microtomografia por Raio-X/métodos
14.
Front Immunol ; 12: 666293, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34017340

RESUMO

Although Mycobacterium tuberculosis (Mtb) is an intracellular pathogen in phagocytic cells, the factors and mechanisms by which they invade and persist in host cells are still not well understood. Characterization of the bacterial proteins modulating macrophage function is essential for understanding tuberculosis pathogenesis and bacterial virulence. Here we investigated the pathogenic role of the Rv2145c protein in stimulating IL-10 production. We first found that recombinant Rv2145c stimulated bone marrow-derived macrophages (BMDMs) to secrete IL-10, IL-6 and TNF-α but not IL-12p70 and to increase the expression of surface molecules through the MAPK, NF-κB, and TLR4 pathways and enhanced STAT3 activation and the expression of IL-10 receptor in Mtb-infected BMDMs. Rv2145c significantly enhanced intracellular Mtb growth in BMDMs compared with that in untreated cells, which was abrogated by STAT3 inhibition and IL-10 receptor (IL-10R) blockade. Expression of Rv2145c in Mycobacterium smegmatis (M. smegmatis) led to STAT3-dependent IL-10 production and enhancement of intracellular growth in BMDMs. Furthermore, the clearance of Rv2145c-expressing M. smegmatis in the lungs and spleens of mice was delayed, and these effects were abrogated by administration of anti-IL-10R antibodies. Finally, all mice infected with Rv2145c-expressing M. smegmatis died, but those infected with the vector control strain did not. Our data suggest that Rv2145c plays a role in creating a favorable environment for bacterial survival by modulating host signals.


Assuntos
Proteínas de Bactérias/imunologia , Mycobacterium tuberculosis/patogenicidade , Receptores de Interleucina-10/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Proteínas de Bactérias/genética , Interleucina-10/metabolismo , Ativação de Macrófagos , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Viabilidade Microbiana/genética , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/crescimento & desenvolvimento , Mycobacterium smegmatis/imunologia , Mycobacterium smegmatis/patogenicidade , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/imunologia , Receptores de Interleucina-10/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Fator de Transcrição STAT3/antagonistas & inibidores , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo , Virulência
15.
Elife ; 102021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-34003742

RESUMO

Bacterial pathogens that infect phagocytic cells must deploy mechanisms that sense and neutralize host microbicidal effectors. For Mycobacterium tuberculosis, the causative agent of tuberculosis, these mechanisms allow the bacterium to rapidly adapt from aerosol transmission to initial growth in the lung alveolar macrophage. Here, we identify a branched signaling circuit in M. tuberculosis that controls growth in the lung through integrated direct sensing of copper ions and nitric oxide by coupled activity of the Rip1 intramembrane protease and the PdtaS/R two-component system. This circuit uses a two-signal mechanism to inactivate the PdtaS/PdtaR two-component system, which constitutively represses virulence gene expression. Cu and NO inhibit the PdtaS sensor kinase through a dicysteine motif in the N-terminal GAF domain. The NO arm of the pathway is further controlled by sequestration of the PdtaR RNA binding response regulator by an NO-induced small RNA, controlled by the Rip1 intramembrane protease. This coupled Rip1/PdtaS/PdtaR circuit controls NO resistance and acute lung infection in mice by relieving PdtaS/R-mediated repression of isonitrile chalkophore biosynthesis. These studies identify an integrated mechanism by which M. tuberculosis senses and resists macrophage chemical effectors to achieve pathogenesis.


Assuntos
Pulmão/microbiologia , Macrófagos/microbiologia , Mycobacterium tuberculosis/patogenicidade , Tuberculose Pulmonar/microbiologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cobre/metabolismo , Modelos Animais de Doenças , Regulação Bacteriana da Expressão Gênica , Histidina Quinase/genética , Histidina Quinase/metabolismo , Interações Hospedeiro-Patógeno , Pulmão/imunologia , Pulmão/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Tuberculose Pulmonar/metabolismo , Virulência
16.
Nat Commun ; 12(1): 2716, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976135

RESUMO

Polyclonal infections occur when at least two unrelated strains of the same pathogen are detected in an individual. This has been linked to worse clinical outcomes in tuberculosis, as undetected strains with different antibiotic resistance profiles can lead to treatment failure. Here, we examine the amount of polyclonal infections in sputum and surgical resections from patients with tuberculosis in the country of Georgia. For this purpose, we sequence and analyse the genomes of Mycobacterium tuberculosis isolated from the samples, acquired through an observational clinical study (NCT02715271). Access to the lung enhanced the detection of multiple strains (40% of surgery cases) as opposed to just using a sputum sample (0-5% in the general population). We show that polyclonal infections often involve genetically distant strains and can be associated with reversion of the patient's drug susceptibility profile over time. In addition, we find different patterns of genetic diversity within lesions and across patients, including mutational signatures known to be associated with oxidative damage; this suggests that reactive oxygen species may be acting as a selective pressure in the granuloma environment. Our results support the idea that the magnitude of polyclonal infections in high-burden tuberculosis settings is underestimated when only testing sputum samples.


Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Genoma Bacteriano , Granuloma/patologia , Mycobacterium tuberculosis/genética , Tuberculose Resistente a Múltiplos Medicamentos/patologia , Tuberculose Pulmonar/patologia , Antituberculosos/uso terapêutico , Biópsia , Células Clonais , Estudos de Coortes , Variação Genética , República da Geórgia , Granuloma/tratamento farmacológico , Granuloma/microbiologia , Granuloma/cirurgia , Humanos , Pulmão/microbiologia , Pulmão/patologia , Pulmão/cirurgia , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/patogenicidade , Espécies Reativas de Oxigênio/metabolismo , Escarro/microbiologia , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Tuberculose Resistente a Múltiplos Medicamentos/cirurgia , Tuberculose Pulmonar/tratamento farmacológico , Tuberculose Pulmonar/microbiologia , Tuberculose Pulmonar/cirurgia
17.
Front Immunol ; 12: 628973, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33868247

RESUMO

Exosomes are cell-derived nanovesicles carrying protein, lipid, and nucleic acid for secreting cells, and act as significant signal transport vectors for cell-cell communication and immune modulation. Immune-cell-derived exosomes have been found to contain molecules involved in immunological pathways, such as MHCII, cytokines, and pathogenic antigens. Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), remains one of the most fatal infectious diseases. The pathogen for tuberculosis escapes the immune defense and continues to replicate despite rigorous and complicate host cell mechanisms. The infected-cell-derived exosomes under this circumstance are found to trigger different immune responses, such as inflammation, antigen presentation, and activate subsequent pathways, highlighting the critical role of exosomes in anti-MTB immune response. Additionally, as a novel kind of delivery system, exosomes show potential in developing new vaccination and treatment of tuberculosis. We here summarize recent research progress regarding exosomes in the immune environment during MTB infection, and further discuss the potential of exosomes as delivery system for novel anti-MTB vaccines and therapies.


Assuntos
Exossomos/transplante , Imunoterapia , Mycobacterium tuberculosis/patogenicidade , Vacinas contra a Tuberculose/uso terapêutico , Tuberculose/terapia , Imunidade Adaptativa , Animais , Autofagia , Exossomos/imunologia , Exossomos/microbiologia , Humanos , Evasão da Resposta Imune , Mycobacterium tuberculosis/imunologia , Tuberculose/imunologia , Tuberculose/microbiologia
18.
J Biol Chem ; 296: 100257, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33837735

RESUMO

Mycobacterium tuberculosis (Mtb) remains the deadliest pathogenic bacteria worldwide. The search for new antibiotics to treat drug-sensitive as well as drug-resistant tuberculosis has become a priority. The essential enzyme phenylalanyl-tRNA synthetase (PheRS) is an antibacterial drug target because of the large differences between bacterial and human PheRS counterparts. In a high-throughput screening of 2148 bioactive compounds, PF-3845, which is a known inhibitor of human fatty acid amide hydrolase, was identified inhibiting Mtb PheRS at Ki ∼ 0.73 ± 0.06 µM. The inhibition mechanism was studied with enzyme kinetics, protein structural modeling, and crystallography, in comparison to a PheRS inhibitor of the noted phenyl-thiazolylurea-sulfonamide class. The 2.3-Å crystal structure of Mtb PheRS in complex with PF-3845 revealed its novel binding mode, in which a trifluoromethyl-pyridinylphenyl group occupies the phenylalanine pocket, whereas a piperidine-piperazine urea group binds into the ATP pocket through an interaction network enforced by a sulfate ion. It represents the first non-nucleoside bisubstrate competitive inhibitor of bacterial PheRS. PF-3845 inhibits the in vitro growth of Mtb H37Rv at ∼24 µM, and the potency of PF-3845 increased against an engineered strain Mtb pheS-FDAS, suggesting on target activity in mycobacterial whole cells. PF-3845 does not inhibit human cytoplasmic or mitochondrial PheRS in biochemical assay, which can be explained from the crystal structures. Further medicinal chemistry efforts focused on the piperidine-piperazine urea moiety may result in the identification of a selective antibacterial lead compound.


Assuntos
Mycobacterium tuberculosis/enzimologia , Fenilalanina-tRNA Ligase/ultraestrutura , Conformação Proteica , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Amidoidrolases/antagonistas & inibidores , Amidoidrolases/química , Sequência de Aminoácidos/genética , Antibacterianos/química , Sítios de Ligação/efeitos dos fármacos , Cristalografia por Raios X , Humanos , Cinética , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/patogenicidade , Fenilalanina-tRNA Ligase/antagonistas & inibidores , Fenilalanina-tRNA Ligase/química , Piperidinas/química , Piperidinas/farmacologia , Piridinas/química , Piridinas/farmacologia , Tuberculose Resistente a Múltiplos Medicamentos/enzimologia , Tuberculose Resistente a Múltiplos Medicamentos/genética
19.
Methods Mol Biol ; 2285: 375-384, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33928565

RESUMO

Experimental autoimmune encephalomyelitis, originally experimental allergic encephalomyelitis, is the well-known animal model of multiple sclerosis, an immune- mediated, demyelinating, inflammatory chronic disease of the central nervous system. The experimental disease is widely utilized to test new therapies in preclinical studies, to investigate new hypothesis on the possible pathogenic mechanisms of autoimmune reaction directed against the central nervous system or more generally to investigate the interactions between the immune system and the central nervous system that lead to neuroinflammation. The experimental autoimmune encephalomyelitis may be induced following different protocols in mammals, including nonhuman primates, and autoreactive CD4+ T-lymphocytes directed against myelin antigens are the main factors. Here, after introducing the model, we describe the protocol to induce active EAE in inbred mice, we report on a table the different clinical courses of EAE depending on the combination of antigen /mouse strain and we provide indications on how to evaluate the clinics and pathology of this induced disease.


Assuntos
Encefalomielite Autoimune Experimental/imunologia , Linfócitos T/imunologia , Animais , Antígenos , Encefalomielite Autoimune Experimental/metabolismo , Feminino , Adjuvante de Freund/imunologia , Lipídeos/imunologia , Masculino , Camundongos Endogâmicos , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/patogenicidade , Proteína Básica da Mielina/imunologia , Proteína Proteolipídica de Mielina/imunologia , Glicoproteína Mielina-Oligodendrócito/imunologia , Fragmentos de Peptídeos/imunologia , Fenótipo , Projetos de Pesquisa , Fatores Sexuais , Especificidade da Espécie , Linfócitos T/metabolismo , Fluxo de Trabalho
20.
PLoS One ; 16(4): e0249012, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33793612

RESUMO

INTRODUCTION: Preventing tuberculosis (TB) disease requires treatment of latent TB infection (LTBI) as well as prevention of person-to-person transmission. We estimated the LTBI prevalence for the entire United States and for each state by medical risk factors, age, and race/ethnicity, both in the total population and stratified by nativity. METHODS: We created a mathematical model using all incident TB disease cases during 2013-2017 reported to the National Tuberculosis Surveillance System that were classified using genotype-based methods or imputation as not attributed to recent TB transmission. Using the annual average number of TB cases among US-born and non-US-born persons by medical risk factor, age group, and race/ethnicity, we applied population-specific reactivation rates (and corresponding 95% confidence intervals [CI]) to back-calculate the estimated prevalence of untreated LTBI in each population for the United States and for each of the 50 states and the District of Columbia in 2015. RESULTS: We estimated that 2.7% (CI: 2.6%-2.8%) of the U.S. population, or 8.6 (CI: 8.3-8.8) million people, were living with LTBI in 2015. Estimated LTBI prevalence among US-born persons was 1.0% (CI: 1.0%-1.1%) and among non-US-born persons was 13.9% (CI: 13.5%-14.3%). Among US-born persons, the highest LTBI prevalence was in persons aged ≥65 years (2.1%) and in persons of non-Hispanic Black race/ethnicity (3.1%). Among non-US-born persons, the highest LTBI prevalence was estimated in persons aged 45-64 years (16.3%) and persons of Asian and other racial/ethnic groups (19.1%). CONCLUSIONS: Our estimations of the prevalence of LTBI by medical risk factors and demographic characteristics for each state could facilitate planning for testing and treatment interventions to eliminate TB in the United States. Our back-calculation method feasibly estimates untreated LTBI prevalence and can be updated using future TB disease case counts at the state or national level.


Assuntos
Tuberculose Latente/epidemiologia , Modelos Teóricos , Tuberculose/epidemiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Grupos Étnicos , Feminino , Humanos , Lactente , Tuberculose Latente/microbiologia , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/patogenicidade , Fatores de Risco , Teste Tuberculínico , Tuberculose/microbiologia , Estados Unidos , Adulto Jovem
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