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1.
Molecules ; 26(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34361751

RESUMO

Species of Mycobacteriaceae cause disease in animals and humans, including tuberculosis and leprosy. Individuals infected with organisms in the Mycobacterium tuberculosis complex (MTBC) or non-tuberculous mycobacteria (NTM) may present identical symptoms, however the treatment for each can be different. Although the NTM infection is considered less vital due to the chronicity of the disease and the infrequency of occurrence in healthy populations, diagnosis and differentiation among Mycobacterium species currently require culture isolation, which can take several weeks. The use of volatile organic compounds (VOCs) is a promising approach for species identification and in recent years has shown promise for use in the rapid analysis of both in vitro cultures as well as ex vivo diagnosis using breath or sputum. The aim of this contribution is to analyze VOCs in the culture headspace of seven different species of mycobacteria and to define the volatilome profiles that are discriminant for each species. For the pre-concentration of VOCs, solid-phase micro-extraction (SPME) was employed and samples were subsequently analyzed using gas chromatography-quadrupole mass spectrometry (GC-qMS). A machine learning approach was applied for the selection of the 13 discriminatory features, which might represent clinically translatable bacterial biomarkers.


Assuntos
Metaboloma , Mycobacterium abscessus/química , Complexo Mycobacterium avium/química , Mycobacterium avium/química , Mycobacterium bovis/química , Mycobacterium/química , Compostos Orgânicos Voláteis/isolamento & purificação , Biomarcadores/análise , Cromatografia Gasosa-Espectrometria de Massas/métodos , Aprendizado de Máquina/estatística & dados numéricos , Mycobacterium/metabolismo , Mycobacterium abscessus/metabolismo , Mycobacterium avium/metabolismo , Complexo Mycobacterium avium/metabolismo , Mycobacterium bovis/metabolismo , Análise de Componente Principal , Microextração em Fase Sólida , Compostos Orgânicos Voláteis/classificação , Compostos Orgânicos Voláteis/metabolismo
2.
Viruses ; 13(7)2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34372584

RESUMO

Double-stranded DNA bacteriophages end their lytic cycle by disrupting the host cell envelope, which allows the release of the virion progeny. Each phage must synthesize lysis proteins that target each cell barrier to phage release. In addition to holins, which permeabilize the cytoplasmic membrane, and endolysins, which disrupt the peptidoglycan (PG), mycobacteriophages synthesize a specific lysis protein, LysB, capable of detaching the outer membrane from the complex cell wall of mycobacteria. The family of LysB proteins is highly diverse, with many members presenting an extended N-terminus. The N-terminal region of mycobacteriophage Ms6 LysB shows structural similarity to the PG-binding domain (PGBD) of the φKZ endolysin. A fusion of this region with enhanced green fluorescent protein (Ms6LysBPGBD-EGFP) was shown to bind to Mycobacterium smegmatis, Mycobacterium vaccae, Mycobacterium bovis BGC and Mycobacterium tuberculosis H37Ra cells pretreated with SDS or Ms6 LysB. In pulldown assays, we demonstrate that Ms6 LysB and Ms6LysBPGBD-EGFP bind to purified peptidoglycan of M. smegmatis, Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis, demonstrating affinity to PG of the A1γ chemotype. An infection assay with an Ms6 mutant producing a truncated version of LysB lacking the first 90 amino acids resulted in an abrupt lysis. These results clearly demonstrate that the N-terminus of Ms6 LysB binds to the PG.


Assuntos
Bacteriólise/fisiologia , Micobacteriófagos/metabolismo , Proteínas Virais/genética , Membrana Celular/metabolismo , Parede Celular/metabolismo , Endopeptidases , Hidrólise , Mycobacterium/metabolismo , Mycobacterium/virologia , Peptidoglicano/metabolismo , Ligação Proteica
3.
Int J Mol Sci ; 22(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34360967

RESUMO

Microbial biodegradation is one of the acceptable technologies to remediate and control the pollution by polycyclic aromatic hydrocarbon (PAH). Several bacteria, fungi, and cyanobacteria strains have been isolated and used for bioremediation purpose. This review paper is intended to provide key information on the various steps and actors involved in the bacterial and fungal aerobic and anaerobic degradation of pyrene, a high molecular weight PAH, including catabolic genes and enzymes, in order to expand our understanding on pyrene degradation. The aerobic degradation pathway by Mycobacterium vanbaalenii PRY-1 and Mycobactetrium sp. KMS and the anaerobic one, by the facultative bacteria anaerobe Pseudomonas sp. JP1 and Klebsiella sp. LZ6 are reviewed and presented, to describe the complete and integrated degradation mechanism pathway of pyrene. The different microbial strains with the ability to degrade pyrene are listed, and the degradation of pyrene by consortium is also discussed. The future studies on the anaerobic degradation of pyrene would be a great initiative to understand and address the degradation mechanism pathway, since, although some strains are identified to degrade pyrene in reduced or total absence of oxygen, the degradation pathway of more than 90% remains unclear and incomplete. Additionally, the present review recommends the use of the combination of various strains of anaerobic fungi and a fungi consortium and anaerobic bacteria to achieve maximum efficiency of the pyrene biodegradation mechanism.


Assuntos
Klebsiella/metabolismo , Mycobacterium/metabolismo , Pseudomonas/metabolismo , Pirenos/metabolismo , Klebsiella/genética , Consórcios Microbianos , Mycobacterium/genética , Oxigênio/metabolismo , Pseudomonas/genética
4.
Int J Mol Sci ; 22(14)2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-34299217

RESUMO

The mycobacterial cell wall is composed of large amounts of lipids with varying moieties. Some mycobacteria species hijack host cells and promote lipid droplet accumulation to build the cellular environment essential for their intracellular survival. Thus, lipids are thought to be important for mycobacteria survival as well as for the invasion, parasitization, and proliferation within host cells. However, their physiological roles have not been fully elucidated. Recent studies have revealed that mycobacteria modulate the peroxisome proliferator-activated receptor (PPAR) signaling and utilize host-derived triacylglycerol (TAG) and cholesterol as both nutrient sources and evasion from the host immune system. In this review, we discuss recent findings that describe the activation of PPARs by mycobacterial infections and their role in determining the fate of bacilli by inducing lipid metabolism, anti-inflammatory function, and autophagy.


Assuntos
Infecções por Mycobacterium/microbiologia , Mycobacterium/metabolismo , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Animais , Autofagia/fisiologia , Colesterol/metabolismo , Humanos , Metabolismo dos Lipídeos , Mycobacterium/crescimento & desenvolvimento , Mycobacterium/imunologia , Infecções por Mycobacterium/imunologia , Infecções por Mycobacterium/metabolismo , Receptores Ativados por Proliferador de Peroxissomo/genética , Transdução de Sinais
5.
Methods Mol Biol ; 2314: 109-150, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34235650

RESUMO

The very high content of structurally diverse and biologically active lipids of exotic structures is the hallmark of Mycobacteria. As such the lipid composition is commonly used to characterize mycobacterial strains at the species and type-species levels. The present chapter describes the methods that allow the purification of the most commonly isolated biologically active lipids and those used for analyzing extractable lipids and their constituents, cell wall-linked mycolic acids (MA), and lipoarabinomannan (LAM). These involve various chromatographic techniques and analytical procedures necessary for structural and metabolic studies of mycobacterial lipids. In addition, as the use of physical methods has brought important overhang on chemical structures of the very-long-chain MA, which typify mycobacteria, NMR and mass spectrometry data of these specific fatty acids are included.


Assuntos
Parede Celular/metabolismo , Lipídeos/análise , Lipídeos/isolamento & purificação , Lipopolissacarídeos/análise , Lipopolissacarídeos/isolamento & purificação , Mycobacterium/metabolismo , Espectroscopia de Ressonância Magnética , Espectrometria de Massas
6.
Methods Mol Biol ; 2314: 205-229, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34235654

RESUMO

Studies on cell-to-cell phenotypic variation in microbial populations, with individuals sharing the same genetic background, provide insights not only on bacterial behavior but also on the adaptive spectrum of the population. Phenotypic variation is an innate property of microbial populations, and this can be further amplified under stressful conditions, providing a fitness advantage. Furthermore, phenotypic variation may also precede a latter step of genetic-based diversification, resulting in the transmission of the most beneficial phenotype to the progeny. While population-wide studies provide a measure of the collective average behavior, single-cell studies, which have expanded over the last decade, delve into the behavior of smaller subpopulations that would otherwise remain concealed. In this chapter, we describe approaches to carry out spatiotemporal analysis of individual mycobacterial cells using time-lapse microscopy. Our method encompasses the fabrication of a microfluidic device; the assembly of a microfluidic system suitable for long-term imaging of mycobacteria; and the quantitative analysis of single-cell behavior under varying growth conditions. Phenotypic variation is conceivably associated to the resilience and endurance of mycobacterial cells. Therefore, shedding light on the dynamics of this phenomenon, on the transience or stability of the given phenotype, on its molecular bases and its functional consequences, offers new scope for intervention.


Assuntos
Microfluídica/métodos , Mycobacterium/crescimento & desenvolvimento , Fenótipo , Análise de Célula Única/métodos , Imagem com Lapso de Tempo/métodos , Humanos , Microfluídica/instrumentação , Mycobacterium/genética , Mycobacterium/metabolismo , Análise Espaço-Temporal
7.
Methods Mol Biol ; 2314: 231-245, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34235655

RESUMO

Mycobacteria are intrinsically resistant to most antimicrobials, which is generally attributed to the impermeability of their cell wall that considerably limits drug uptake. Moreover, like in other pathogenic bacteria, active efflux systems have been widely characterized from diverse mycobacterial species in laboratory conditions, showing that they can promote resistance by extruding noxious compounds prior to their reaching their intended targets. Therefore, the intracellular concentration of a given compound is determined by the balance between permeability, influx, and efflux.Given the urgent need to discover and develop novel antimycobacterial compounds in order to design effective therapeutic strategies, the contributions to drug resistance made by the controlled permeability of the cell wall and the increased activity of efflux pumps must be determined. In this chapter, we will describe a method that allows (1) the measuring of permeability and the quantification of general efflux activity of mycobacteria, by the study of the transport (influx and efflux) of fluorescent compounds, such as ethidium bromide; and (2) the screening of compounds in search of agents that increase the permeability of the cell wall and efflux inhibitors that could restore the effectiveness of antimicrobials that are subject to efflux.


Assuntos
Proteínas de Bactérias/metabolismo , Permeabilidade da Membrana Celular , Etídio/metabolismo , Fluorometria/métodos , Mycobacterium/metabolismo , Antibacterianos/farmacologia , Transporte Biológico , Farmacorresistência Bacteriana Múltipla , Corantes Fluorescentes/metabolismo , Testes de Sensibilidade Microbiana , Mycobacterium/efeitos dos fármacos , Mycobacterium/crescimento & desenvolvimento
8.
Methods Mol Biol ; 2314: 533-548, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34235669

RESUMO

The use of proteomic technologies to characterize and study the proteome of mycobacteria has provided important information in terms of function, diversity, protein-protein interactions, and host-pathogen interactions in Mycobacterium spp. There are many different mass spectrometry methodologies that can be applied to proteomics studies of mycobacteria and microorganisms in general. Sample processing and appropriate study design are critical to generating high-quality data regardless of the mass spectrometry method applied. Appropriate study design relies on statistical rigor and data curation using bioinformatics approaches that are widely applicable regardless of the organism or system studied. Sample processing, on the other hand, is often a niched process specific to the physiology of the organism or system under investigation. Therefore, in this chapter, we will provide protocols for processing mycobacterial protein samples for the specific application of Top-down and Bottom-up proteomic analyses.


Assuntos
Proteínas de Bactérias/metabolismo , Cromatografia Líquida/métodos , Mycobacterium/metabolismo , Proteoma/análise , Proteoma/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Humanos , Processamento de Proteína Pós-Traducional
9.
Methods Mol Biol ; 2314: 549-577, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34235670

RESUMO

Decades of study have highlighted the richness and uniqueness of the repertoire of lipid and glycolipid families produced by mycobacteria. Many of these families potently regulate host immune responses, in stimulatory or suppressive ways. Thus, the global study of this repertoire in different genetic backgrounds or under model conditions of infection is gaining interest. Despite the difficulties associated with the specificities of this repertoire, the field of mass spectrometry-based lipidomics of mycobacteria has recently made considerable progress, particularly at the analytical level. There is still considerable scope for further progress, especially with regard to the development of an efficient bioinfomatics pipeline for the analysis of the large datasets generated. This chapter describes an HPLC-MS methodology allowing the simultaneous screening of more than 20 of the lipid families produced by mycobacteria and provides recommendations to analyze the generated data given the state-of-the-art.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Glicolipídeos/metabolismo , Lipidômica/métodos , Lipídeos/análise , Espectrometria de Massas/métodos , Mycobacterium/metabolismo , Glicolipídeos/análise , Humanos
10.
Nat Rev Microbiol ; 19(9): 567-584, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34040228

RESUMO

Type VII secretion systems (T7SSs) have a key role in the secretion of effector proteins in non-pathogenic mycobacteria and pathogenic mycobacteria such as Mycobacterium tuberculosis, the main causative agent of tuberculosis. Tuberculosis-causing mycobacteria, still accounting for 1.4 million deaths annually, rely on paralogous T7SSs to survive in the host and efficiently evade its immune response. Although it is still unknown how effector proteins of T7SSs cross the outer membrane of the diderm mycobacterial cell envelope, recent advances in the structural characterization of these secretion systems have revealed the intricate network of interactions of conserved components in the plasma membrane. This structural information, added to recent advances in the molecular biology and regulation of mycobacterial T7SSs as well as progress in our understanding of their secreted effector proteins, is shedding light on the inner working of the T7SS machinery. In this Review, we highlight the implications of these studies and the derived transport models, which provide new scenarios for targeting the deathly human pathogen M. tuberculosis.


Assuntos
Mycobacterium/metabolismo , Sistemas de Secreção Tipo VII/fisiologia , Transporte Biológico , Membrana Celular , Conformação Proteica
11.
Genes (Basel) ; 12(4)2021 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-33918798

RESUMO

The mycobacterial nonhomologous end-joining pathway (NHEJ) involved in double-strand break (DSB) repair consists of the multifunctional ATP-dependent ligase LigD and the DNA bridging protein Ku. The other ATP-dependent ligases LigC and AEP-primase PrimC are considered as backup in this process. The engagement of LigD, LigC, and PrimC in the base excision repair (BER) process in mycobacteria has also been postulated. Here, we evaluated the sensitivity of Mycolicibacterium smegmatis mutants defective in the synthesis of Ku, Ku-LigD, and LigC1-LigC2-PrimC, as well as mutants deprived of all these proteins to oxidative and nitrosative stresses, with the most prominent effect observed in mutants defective in the synthesis of Ku protein. Mutants defective in the synthesis of LigD or PrimC/LigC presented a lower frequency of spontaneous mutations than the wild-type strain or the strain defective in the synthesis of Ku protein. As identified by whole-genome sequencing, the most frequent substitutions in all investigated strains were T→G and A→C. Double substitutions, as well as insertions of T or CG, were exclusively identified in the strains carrying functional Ku and LigD proteins. On the other hand, the inactivation of Ku/LigD increased the efficiency of the deletion of G in the mutant strain.


Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , DNA Primase/metabolismo , Ligases/metabolismo , Taxa de Mutação , Mycobacterium/genética , Estresse Oxidativo , Proteínas de Bactérias/genética , DNA Primase/genética , Ligases/genética , Mycobacterium/crescimento & desenvolvimento , Mycobacterium/metabolismo
12.
Proc Natl Acad Sci U S A ; 118(12)2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33723035

RESUMO

GTPase high frequency of lysogenization X (HflX) is highly conserved in prokaryotes and acts as a ribosome-splitting factor as part of the heat shock response in Escherichia coli. Here we report that HflX produced by slow-growing Mycobacterium bovis bacillus Calmette-Guérin (BCG) is a GTPase that plays a critical role in the pathogen's transition to a nonreplicating, drug-tolerant state in response to hypoxia. Indeed, HflX-deficient M. bovis BCG (KO) replicated markedly faster in the microaerophilic phase of a hypoxia model that resulted in premature entry into dormancy. The KO mutant displayed hallmarks of nonreplicating mycobacteria, including phenotypic drug resistance, altered morphology, low intracellular ATP levels, and overexpression of Dormancy (Dos) regulon proteins. Mice nasally infected with HflX KO mutant displayed increased bacterial burden in the lungs, spleen, and lymph nodes during the chronic phase of infection, consistent with the higher replication rate observed in vitro in microaerophilic conditions. Unlike fast growing mycobacteria, M. bovis BCG HlfX was not involved in antibiotic resistance under aerobic growth. Proteomics, pull-down, and ribo-sequencing approaches supported that mycobacterial HflX is a ribosome-binding protein that controls translational activity of the cell. With HflX fully conserved between M. bovis BCG and M. tuberculosis, our work provides further insights into the molecular mechanisms deployed by pathogenic mycobacteria to adapt to their hypoxic microenvironment.


Assuntos
Replicação do DNA , GTP Fosfo-Hidrolases/metabolismo , Hipóxia/genética , Hipóxia/metabolismo , Mycobacterium/genética , Mycobacterium/metabolismo , Animais , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , GTP Fosfo-Hidrolases/genética , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Camundongos , Mutação , Mycobacterium bovis/genética , Mycobacterium bovis/metabolismo , Ribossomos/metabolismo
13.
Org Biomol Chem ; 19(13): 2856-2870, 2021 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-33725048

RESUMO

Bacterial infections are still one of the leading causes of death worldwide; despite the near-ubiquitous availability of antibiotics. With antibiotic resistance on the rise, there is an urgent need for novel classes of antibiotic drugs. One particularly troublesome class of bacteria are those that have evolved highly efficacious mechanisms for surviving inside the host. These contribute to their virulence by immune evasion, and make them harder to treat with antibiotics due to their residence inside intracellular membrane-limited compartments. This has sparked the development of new chemical reporter molecules and bioorthogonal probes that can be metabolically incorporated into bacteria to provide insights into their activity status. In this review, we provide an overview of several classes of metabolic labeling probes capable of targeting either the peptidoglycan cell wall, the mycomembrane of mycobacteria and corynebacteria, or specific bacterial proteins. In addition, we highlight several important insights that have been made using these metabolic labeling probes.


Assuntos
Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Corynebacterium/metabolismo , Mycobacterium/metabolismo , Peptidoglicano/metabolismo , Proteínas de Bactérias/química , Parede Celular/química , Corynebacterium/química , Interações Hospedeiro-Patógeno , Humanos , Conformação Molecular , Mycobacterium/química , Peptidoglicano/química
14.
Life Sci Alliance ; 4(6)2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33771876

RESUMO

The mycobacterial cell wall glycolipid trehalose-6,6-dimycolate (TDM) activates macrophages through the C-type lectin receptor MINCLE. Regulation of innate immune cells relies on miRNAs, which may be exploited by mycobacteria to survive and replicate in macrophages. Here, we have used macrophages deficient in the microprocessor component DGCR8 to investigate the impact of miRNA on the response to TDM. Deletion of DGCR8 in bone marrow progenitors reduced macrophage yield, but did not block macrophage differentiation. DGCR8-deficient macrophages showed reduced constitutive and TDM-inducible miRNA expression. RNAseq analysis revealed that they accumulated primary miRNA transcripts and displayed a modest type I IFN signature at baseline. Stimulation with TDM in the absence of DGCR8 induced overshooting expression of IFNß and IFN-induced genes, which was blocked by antibodies to type I IFN. In contrast, signaling and transcriptional responses to recombinant IFNß were unaltered. Infection with live Mycobacterium bovis Bacille Calmette-Guerin replicated the enhanced IFN response. Together, our results reveal an essential role for DGCR8 in curbing IFNß expression macrophage reprogramming by mycobacteria.


Assuntos
Macrófagos/metabolismo , Mycobacterium/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Citocinas/metabolismo , Feminino , Interferons/imunologia , Interferons/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Mycobacterium/genética , Mycobacterium/patogenicidade , Proteínas de Ligação a RNA/genética , Fosfatos Açúcares/metabolismo , Trealose/análogos & derivados , Trealose/metabolismo
15.
FASEB J ; 35(4): e21475, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33772870

RESUMO

Cell signaling relies on second messengers to transduce signals from the sensory apparatus to downstream signaling pathway components. In bacteria, one of the most important and ubiquitous second messenger is the small molecule cyclic diguanosine monophosphate (c-di-GMP). While the biosynthesis, degradation, and regulatory pathways controlled by c-di-GMP are well characterized, the mechanisms through which c-di-GMP controls these processes are not entirely understood. Herein we present the report of a c-di-GMP sensing sensor histidine kinase PdtaS (Rv3220c), which binds to c-di-GMP at submicromolar concentrations, subsequently perturbing signaling of the PdtaS-PdtaR (Rv1626) two-component system. Aided by biochemical analysis, genetics, molecular docking, FRET microscopy, and structural modelling, we have characterized the binding of c-di-GMP in the GAF domain of PdtaS. We show that a pdtaS knockout in Mycobacterium smegmatis is severely compromised in growth on amino acid deficient media and exhibits global transcriptional dysregulation. The perturbation of the c-di-GMP-PdtaS-PdtaR axis results in a cascade of cellular changes recorded by a multiparametric systems' approach of transcriptomics, unbiased metabolomics, and lipid analyses.


Assuntos
Carbono/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Histidina Quinase/metabolismo , Bactérias , Proteínas de Bactérias/metabolismo , Simulação de Acoplamento Molecular/métodos , Mycobacterium/metabolismo , Mycobacterium smegmatis/crescimento & desenvolvimento , Mycobacterium smegmatis/metabolismo , Sistemas do Segundo Mensageiro/fisiologia , Transdução de Sinais/fisiologia
16.
Microbiology (Reading) ; 167(2)2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33555244

RESUMO

Treatment of tuberculosis requires a multi-drug regimen administered for at least 6 months. The long-term chemotherapy is attributed in part to a minor subpopulation of nonreplicating Mycobacterium tuberculosis cells that exhibit phenotypic tolerance to antibiotics. The origins of these cells in infected hosts remain unclear. Here we discuss some recent evidence supporting the hypothesis that hibernation of ribosomes in M. tuberculosis, induced by zinc starvation, could be one of the primary mechanisms driving the development of nonreplicating persisters in hosts. We further analyse inconsistencies in previously reported studies to clarify the molecular principles underlying mycobacterial ribosome hibernation.


Assuntos
Mycobacterium/fisiologia , Tuberculose/microbiologia , Antituberculosos/metabolismo , Antituberculosos/uso terapêutico , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana , Humanos , Mycobacterium/efeitos dos fármacos , Mycobacterium/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Tuberculose/tratamento farmacológico , Zinco/deficiência
17.
Molecules ; 26(2)2021 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-33435591

RESUMO

Mycobacterium avium complex (MAC) is the most common non-tuberculous mycobacterium (NTM) and causes different types of pulmonary diseases. While genomic and transcriptomic analysis of Mycobacterium avium 104 (M. avium 104) has been extensive, little is known about the proteomics of M. avium 104. We utilized proteomics technology to analyze the changes in the whole proteome of M. avium 104 during exponential and stationary growth phases. We found 12 dys-regulated proteins; the up-regulated protein hits in the stationary phase were involved in aminopeptidase, choline dehydrogenase, oxidoreductase, and ATP binding, while the down-regulated proteins in the stationary phase were acetyl-CoA acetyltransferase, universal stress protein, catalase peroxidase, and elongation factor (Tu). The differently expressed proteins between exponential and stationary phases were implicated in metabolism and stress response, pointing to the functional adaptation of the cells to the environment. Proteomic analysis in different growth phases could participate in understanding the course of infection, the mechanisms of virulence, the means of survival, and the possible targets for treatment.


Assuntos
Adaptação Fisiológica , Proteínas de Bactérias/metabolismo , Meio Ambiente , Mycobacterium/crescimento & desenvolvimento , Mycobacterium/metabolismo , Proteoma/metabolismo , Humanos , Mycobacterium/isolamento & purificação , Proteoma/análise
18.
PLoS Pathog ; 17(1): e1009124, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33411813

RESUMO

Mycobacterial pathogens pose a sustained threat to human health. There is a critical need for new diagnostics, therapeutics, and vaccines targeting both tuberculous and nontuberculous mycobacterial species. Understanding the basic mechanisms used by diverse mycobacterial species to cause disease will facilitate efforts to design new approaches toward detection, treatment, and prevention of mycobacterial disease. Molecular, genetic, and biochemical approaches have been widely employed to define fundamental aspects of mycobacterial physiology and virulence. The recent expansion of genetic tools in mycobacteria has further increased the accessibility of forward genetic approaches. Proteomics has also emerged as a powerful approach to further our understanding of diverse mycobacterial species. Detection of large numbers of proteins and their modifications from complex mixtures of mycobacterial proteins is now routine, with efforts of quantification of these datasets becoming more robust. In this review, we discuss the "genetic proteome," how the power of genetics, molecular biology, and biochemistry informs and amplifies the quality of subsequent analytical approaches and maximizes the potential of hypothesis-driven mycobacterial research. Published proteomics datasets can be used for hypothesis generation and effective post hoc supplementation to experimental data. Overall, we highlight how the integration of proteomics, genetic, molecular, and biochemical approaches can be employed successfully to define fundamental aspects of mycobacterial pathobiology.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Genômica , Infecções por Mycobacterium/metabolismo , Mycobacterium/metabolismo , Proteoma/metabolismo , Humanos , Mycobacterium/genética , Mycobacterium/patogenicidade , Infecções por Mycobacterium/genética , Infecções por Mycobacterium/microbiologia , Proteoma/análise
19.
J Biochem ; 169(1): 43-53, 2021 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-32706888

RESUMO

It is urgent to understand the regulatory mechanism of drug resistance in widespread bacterial pathogens. In Mycobacterium tuberculosis, several transcriptional regulators have been found to play essential roles in regulating its drug resistance. In this study, we found that an ArsR family transcription regulator encoded by Rv2642 (CdiR) responds to isoniazid (INH), a widely used anti-tuberculosis (TB) drug. CdiR negatively regulates self and adjacent genes, including arsC (arsenic-transport integral membrane protein ArsC). CdiR directly interacts with INH and Cd(II). The binding of INH and Cd(II) both reduce its DNA-binding activity. Disrupting cdiR increased the drug susceptibility to INH, whereas overexpressing cdiR decreased the susceptibility. Strikingly, overexpressing arsC increased the drug susceptibility as well as cdiR. Additionally, both changes in cdiR and arsC expression caused sensitivity to other drugs such as rifamycin and ethambutol, where the minimal inhibitory concentrations in the cdiR deletion strain were equal to those of the arsC-overexpressing strain, suggesting that the function of CdiR in regulating drug resistance primarily depends on arsC. Furthermore, we found that Cd(II) enhances bacterial resistance to INH in a CdiR-dependent manner. As a conclusion, CdiR has a critical role in directing the interplay between Cd(II) metal ions and drug susceptibility in mycobacteria.


Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/metabolismo , Cádmio/metabolismo , Isoniazida/farmacologia , Mycobacterium tuberculosis/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana/efeitos dos fármacos , Etambutol/farmacologia , Regulação Bacteriana da Expressão Gênica , Humanos , Testes de Sensibilidade Microbiana/métodos , Mycobacterium/genética , Mycobacterium/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Rifamicinas/farmacologia , Fatores de Transcrição/genética
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