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1.
Microbiol Spectr ; 10(4): e0196922, 2022 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-35938806

RESUMO

Mycobacterium tuberculosis is a highly specialized human pathogen. The success of M. tuberculosis is due to its ability to replicate within host macrophages, resist host immune responses, and ultimately enter a persistent state during a latent tuberculosis infection. Understanding how M. tuberculosis adapts to and replicates in the intracellular environment of the host is crucial for the development of novel, targeted therapeutics. We report the characterization of an M. tuberculosis mutant lacking Rv3249c, a TetR transcriptional regulator. We show that Rv3249c directly represses the adjacent alkB-rubA-rubB operon encoding an alkane hydroxylase/rubredoxin system. For consistency with related systems, we have named the rv3249c gene alkX. The alkX mutant survived better than wild-type M. tuberculosis inside macrophages. This could be phenocopied by overexpression of the alkB-rubA-rubB locus. We hypothesized that the improved intracellular survival phenotype is a result of increased fitness of the mutant; however, we found that the alkX mutant had a defect when grown on some host-associated carbon sources in vitro. We also found that the alkX mutant had a defect in biofilm formation, also linked to the overexpression of the alkB-rubAB genes. Combined, these results define the primary role of AlkX as a transcriptional repressor of the alkB-rubAB operon and suggest the operon contributes to intracellular survival of the pathogen. IMPORTANCE Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is the leading cause of death worldwide due to a single infectious agent. It is important to understand how M. tuberculosis adapts to and replicates in the intracellular environment of the host. In this study, we characterized the TetR transcriptional regulator Rv3249c and show that it regulates a highly conserved alkane hydroxylase/rubredoxin system. Our data demonstrate that the AlkBRubAB system contributes to the success of the bacterium in host macrophages.


Assuntos
Mycobacterium tuberculosis , Tuberculose , Proteínas de Bactérias/genética , Biofilmes , Citocromo P-450 CYP4A/genética , Citocromo P-450 CYP4A/metabolismo , Humanos , Macrófagos/microbiologia , Mycobacterium tuberculosis/metabolismo , Rubredoxinas/genética , Tuberculose/microbiologia
2.
Biochemistry ; 60(5): 381-397, 2021 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-33491458

RESUMO

Polyacylated trehaloses in Mycobacterium tuberculosis play important roles in pathogenesis and structural roles in the cell envelope, promoting the intracellular survival of the bacterium, and are potential targets for drug development. Herein, we describe a linear ion-trap multiple-stage mass spectrometric approach (LIT MSn) with high-resolution mass spectrometry to the structural characterization of a glycolipid family that includes a 2,3-diacyltrehalose, 2,3,6-triacyltrehalose, 2,3,6,2',4'-petaacyltrehalose, and a novel 2,3,6,2'-tetraacyltrehalose (TetraAT) subfamily isolated from biofilm cultures of M. tuberculosis H37Rv. The LIT MSn spectra (n = 2, 3, or 4) provide structural information to unveil the location of the palmitoyl/stearoyl and one to four multiple methyl-branched fatty acyl substituents attached to the trehalose backbone, leading to the identification of hundreds of glycolipid species with many isomeric structures. We identified a new TetraAT subfamily whose structure has not been previously defined. We also developed a strategy for defining the structures of the multiple methyl-branched fatty acid substituents, leading to the identification of mycosanoic acid, mycolipenic acid, mycolipodienoic acid, mycolipanolic acid, and a new cyclopropyl-containing acid. The observation of the new TetraAT family, and the realization of the structural similarity between the various subfamilies, may have significant implications in the biosynthetic pathways of this glycolipid family.


Assuntos
Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/enzimologia , Trealose/química , Biofilmes , Parede Celular/química , Ácidos Graxos/química , Cromatografia Gasosa-Espectrometria de Massas/métodos , Glicolipídeos/química , Mycobacterium tuberculosis/metabolismo , Isoformas de Proteínas/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Trealose/metabolismo
3.
Mol Microbiol ; 115(2): 208-221, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-32985735

RESUMO

The Mycobacterium tuberculosis cell envelope is a critical interface between the host and pathogen and provides a protective barrier against the immune response and antibiotics. Cell envelope lipids are also mycobacterial virulence factors that influence the host immune response. The mycobacterial membrane protein large (MmpL) proteins transport cell envelope lipids and siderophores that are important for the basic physiology and pathogenesis of M. tuberculosis. We recently identified MmpL11 as a conserved transporter of mycolic acid-containing lipids including monomeromycolyl diacylglycerol (MMDAG), mycolate wax ester (MWE), and long-chain triacylglycerols (LC-TAGs). These lipids contribute to biofilm formation in M. tuberculosis and M. smegmatis, and non-replicating persistence in M. tuberculosis. In this report, we identified domains and residues that are essential for MmpL11TB lipid transporter activity. Specifically, we show that the D1 periplasmic loop and a conserved tyrosine are essential for the MmpL11 function. Intriguingly, we found that MmpL11 levels are regulated by the phosphorylation of threonine in the cytoplasmic C-terminal domain, providing the first direct evidence of the phospho-regulation of MmpL11 transporter activity in M. tuberculosis and M. smegmatis. Our results offer further insight into the function of MmpL transporters and regulation of mycobacterial cell envelope biogenesis.


Assuntos
Proteínas de Membrana Transportadoras/metabolismo , Mycobacterium tuberculosis/metabolismo , Proteínas de Bactérias/metabolismo , Transporte Biológico , Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Parede Celular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/fisiologia , Ácidos Micólicos/metabolismo , Periplasma/metabolismo , Fosforilação , Sideróforos/metabolismo , Tuberculose/microbiologia , Fatores de Virulência/metabolismo
4.
Tuberculosis (Edinb) ; 125: 102007, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33035766

RESUMO

The success of Mycobacterium tuberculosis as a human pathogen depends on the bacterium's ability to persist in a quiescent form in oxygen and nutrient-poor host environments. In vitro studies have demonstrated that these restricting environments induce a shift from bacterial replication to non-replicating persistence (NRP). Entry into NRP involves changes in bacterial metabolism and remodeling of the cell envelope. Findings consistent with the phenotypes observed in vitro have been observed in patient and animal model samples. This review focuses on the cell envelope differences seen between replicating and NRP M. tuberculosis and summarizes the ways in which serine/threonine protein kinases (STPKs) may mediate this process.


Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Mycobacterium tuberculosis/genética , Tuberculose/microbiologia , Animais , Divisão Celular , Membrana Celular/metabolismo , Parede Celular/metabolismo , Humanos , Tuberculose/genética , Tuberculose/metabolismo
5.
J Biol Chem ; 294(43): 15711-15723, 2019 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-31471317

RESUMO

The mycobacterial cell envelope is crucial to host-pathogen interactions as a barrier against antibiotics and the host immune response. In addition, cell envelope lipids are mycobacterial virulence factors. Cell envelope lipid biosynthesis is the target of a number of frontline tuberculosis treatments and has been the focus of much research. However, the transport mechanisms by which these lipids reach the mycomembrane remain poorly understood. Many envelope lipids are exported from the cytoplasm to the periplasmic space via the mycobacterial membrane protein large (MmpL) family of proteins. In other bacteria, lipoproteins can contribute to outer membrane biogenesis through direct binding of substrates and/or protein-protein associations with extracytoplasmic biosynthetic enzymes. In this report, we investigate whether the lipoprotein LpqN plays a similar role in mycobacteria. Using a genetic two-hybrid approach, we demonstrate that LpqN interacts with periplasmic loop domains of the MmpL3 and MmpL11 transporters that export mycolic acid-containing cell envelope lipids. We observe that LpqN also interacts with secreted cell envelope biosynthetic enzymes such as Ag85A via pulldown assays. The X-ray crystal structures of LpqN and LpqN bound to dodecyl-trehalose suggest that LpqN directly binds trehalose monomycolate, the MmpL3 and Ag85A substrate. Finally, we observe altered lipid profiles of the ΔlpqN mutant during biofilm maturation, pointing toward a possible physiological role for the protein. The results of this study suggest that LpqN may act as a membrane fusion protein, connecting MmpL transporters with periplasmic proteins, and provide general insight into the role of lipoproteins in Mycobacterium tuberculosis cell envelope biogenesis.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Lipoproteínas/química , Lipoproteínas/metabolismo , Mycobacterium tuberculosis/metabolismo , Sítios de Ligação , Biofilmes , Transporte Biológico , Vias Biossintéticas , Ligantes , Modelos Moleculares , Ácidos Micólicos/metabolismo , Ligação Proteica
6.
Proc Natl Acad Sci U S A ; 116(23): 11241-11246, 2019 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-31113875

RESUMO

The cell envelope of Mycobacterium tuberculosis is notable for the abundance of mycolic acids (MAs), essential to mycobacterial viability, and of other species-specific lipids. The mycobacterial cell envelope is extremely hydrophobic, which contributes to virulence and antibiotic resistance. However, exactly how fatty acids and lipidic elements are transported across the cell envelope for cell-wall biosynthesis is unclear. Mycobacterial membrane protein Large 3 (MmpL3) is essential and required for transport of trehalose monomycolates (TMMs), precursors of MA-containing trehalose dimycolates (TDM) and mycolyl arabinogalactan peptidoglycan, but the exact function of MmpL3 remains elusive. Here, we report a crystal structure of Mycobacterium smegmatis MmpL3 at a resolution of 2.59 Å, revealing a monomeric molecule that is structurally distinct from all known bacterial membrane proteins. A previously unknown MmpL3 ligand, phosphatidylethanolamine (PE), was discovered inside this transporter. We also show, via native mass spectrometry, that MmpL3 specifically binds both TMM and PE, but not TDM, in the micromolar range. These observations provide insight into the function of MmpL3 and suggest a possible role for this protein in shuttling a variety of lipids to strengthen the mycobacterial cell wall.


Assuntos
Proteínas de Bactérias/metabolismo , Fatores Corda/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Fosfatidiletanolaminas/metabolismo , Transporte Biológico/fisiologia , Membrana Celular/metabolismo , Parede Celular/metabolismo , Mycobacterium smegmatis/metabolismo , Ácidos Micólicos/metabolismo
7.
Microorganisms ; 7(3)2019 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-30841535

RESUMO

Mycobacterium tuberculosis (Mtb) remains an important human pathogen. The Mtb cell envelope is a critical bacterial structure that contributes to virulence and pathogenicity. Mycobacterial membrane protein large (MmpL) proteins export bulky, hydrophobic substrates that are essential for the unique structure of the cell envelope and directly support the ability of Mtb to infect and persist in the host. This review summarizes recent investigations that have enabled insight into the molecular mechanisms underlying MmpL substrate export and the role that these substrates play during Mtb infection.

9.
Microbiol Spectr ; 5(3)2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28597814

RESUMO

Tuberculosis is one of the most successful human diseases in our history due in large part to the multitude of virulence factors exhibited by the causative agent, Mycobacterium tuberculosis. Understanding the pathogenic nuances of this organism in the context of its human host is an ongoing topic of study facilitated by isolating cells from model organisms such as mice and non-human primates. However, M. tuberculosis is an obligate intracellular human pathogen, and disease progression and outcome in these model systems can differ from that of human disease. Current in vitro models of infection include primary macrophages and macrophage-like immortalized cell lines as well as the induced pluripotent stem cell-derived cell types. This article will discuss these in vitro model systems in general, what we have learned so far about utilizing them to answer questions about pathogenesis, the potential role of other cell types in innate control of M. tuberculosis infection, and the development of new coculture systems with multiple cell types. As we continue to expand current in vitro systems and institute new ones, the knowledge gained will improve our understanding of not only tuberculosis but all infectious diseases.


Assuntos
Técnicas In Vitro/métodos , Modelos Biológicos , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/patogenicidade , Tuberculose/imunologia , Animais , Antituberculosos/farmacologia , Morte Celular , Linhagem Celular , Humanos , Imunidade Inata , Macrófagos/microbiologia , Camundongos , Mycobacterium tuberculosis/metabolismo , Fagossomos , Tuberculose/microbiologia , Fatores de Virulência/metabolismo
10.
Infect Immun ; 85(8)2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28507063

RESUMO

The mycobacterial cell wall is crucial to the host-pathogen interface, because it provides a barrier against antibiotics and the host immune response. In addition, cell wall lipids are mycobacterial virulence factors. The mycobacterial membrane protein large (MmpL) proteins are cell wall lipid transporters that are important for basic mycobacterial physiology and Mycobacterium tuberculosis pathogenesis. MmpL3 and MmpL11 are conserved across pathogenic and nonpathogenic mycobacteria, a feature consistent with an important role in the basic physiology of the bacterium. MmpL3 is essential and transports trehalose monomycolate to the mycobacterial surface. In this report, we characterize the role of MmpL11 in M. tuberculosis. M. tuberculosismmpL11 mutants have altered biofilms associated with lower levels of mycolic acid wax ester and long-chain triacylglycerols than those for wild-type bacteria. While the growth rate of the mmpL11 mutant is similar to that of wild-type M. tuberculosis in macrophages, the mutant exhibits impaired survival in an in vitro granuloma model. Finally, we show that the survival or recovery of the mmpL11 mutant is impaired when it is incubated under conditions of nutrient and oxygen starvation. Our results suggest that MmpL11 and its cell wall lipid substrates are important for survival in the context of adaptive immune pressure and for nonreplicating persistence, both of which are critically important aspects of M. tuberculosis pathogenicity.


Assuntos
Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Parede Celular/química , Citoplasma/microbiologia , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Mycobacterium tuberculosis/fisiologia , Proteínas de Bactérias/genética , Transporte Biológico , Parede Celular/metabolismo , Lipídeos/fisiologia , Proteínas de Membrana Transportadoras/genética , Mutação , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/patogenicidade , Fatores de Virulência
11.
ACS Infect Dis ; 2(7): 500-8, 2016 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-27626102

RESUMO

New treatments for tuberculosis infection are critical to combat the emergence of multidrug- and extensively drug-resistant Mycobacterium tuberculosis (Mtb). We report the characterization of a diphenylether-modified adamantyl 1,2-diamine that we refer to as TBL-140, which has a minimal inhibitory concentration (MIC99) of 1.2 µg/mL. TBL-140 is effective against drug-resistant Mtb and nonreplicating bacteria. In addition, TBL-140 eliminates expansion of Mtb in cell culture infection assays at its MIC. To define the mechanism of action of this compound, we performed a spontaneous mutant screen and biochemical assays. We determined that TBL-140 treatment affects the proton motive force (PMF) by perturbing the transmembrane potential (ΔΨ), consistent with a target in the electron transport chain (ETC). As a result, treated bacteria have reduced intracellular ATP levels. We show that TBL-140 exhibits greater metabolic stability than SQ109, a structurally similar compound in clinical trials for treatment of MDR-TB infections. Combined, these results suggest that TBL-140 should be investigated further to assess its potential as an improved therapeutic lead against Mtb.


Assuntos
Antituberculosos/química , Antituberculosos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Tuberculose/microbiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Diaminas/química , Desenho de Fármacos , Humanos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Éteres Fenílicos/química , Relação Estrutura-Atividade , Tuberculose/tratamento farmacológico
12.
Protein Sci ; 24(12): 1942-55, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26362239

RESUMO

Mycobacterium tuberculosis is a pathogenic bacterial species, which is neither Gram positive nor Gram negative. It has a unique cell wall, making it difficult to kill and conferring resistance to antibiotics that disrupt cell wall biosynthesis. Thus, the mycobacterial cell wall is critical to the virulence of these pathogens. Recent work shows that the mycobacterial membrane protein large (MmpL) family of transporters contributes to cell wall biosynthesis by exporting fatty acids and lipidic elements of the cell wall. The expression of the Mycobacterium tuberculosis MmpL proteins is controlled by a complicated regulatory network system. Here we report crystallographic structures of two forms of the TetR-family transcriptional regulator Rv0302, which participates in regulating the expression of MmpL proteins. The structures reveal a dimeric, two-domain molecule with architecture consistent with the TetR family of regulators. Comparison of the two Rv0302 crystal structures suggests that the conformational changes leading to derepression may be due to a rigid body rotational motion within the dimer interface of the regulator. Using fluorescence polarization and electrophoretic mobility shift assays, we demonstrate the recognition of promoter and intragenic regions of multiple mmpL genes by this protein. In addition, our isothermal titration calorimetry and electrophoretic mobility shift experiments indicate that fatty acids may be the natural ligand of this regulator. Taken together, these experiments provide new perspectives on the regulation of the MmpL family of transporters.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Membrana Transportadoras/genética , Mycobacterium tuberculosis/metabolismo , Proteínas de Bactérias/genética , Parede Celular/química , Parede Celular/metabolismo , Cristalografia por Raios X , Ácidos Graxos/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Mycobacterium tuberculosis/química , Regiões Promotoras Genéticas , Multimerização Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
13.
J Biol Chem ; 290(47): 28559-28574, 2015 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-26396194

RESUMO

The mycobacterial cell wall is critical to the virulence of these pathogens. Recent work shows that the MmpL (mycobacterial membrane protein large) family of transporters contributes to cell wall biosynthesis by exporting fatty acids and lipidic elements of the cell wall. The expression of the Mycobacterium tuberculosis MmpL proteins is controlled by a complex regulatory network, including the TetR family transcriptional regulators Rv3249c and Rv1816. Here we report the crystal structures of these two regulators, revealing dimeric, two-domain molecules with architecture consistent with the TetR family of regulators. Buried extensively within the C-terminal regulatory domains of Rv3249c and Rv1816, we found fortuitous bound ligands, which were identified as palmitic acid (a fatty acid) and isopropyl laurate (a fatty acid ester), respectively. Our results suggest that fatty acids may be the natural ligands of these regulatory proteins. Using fluorescence polarization and electrophoretic mobility shift assays, we demonstrate the recognition of promoter and intragenic regions of multiple mmpL genes by these proteins. Binding of palmitic acid renders these regulators incapable of interacting with their respective operator DNAs, which will result in derepression of the corresponding mmpL genes. Taken together, these experiments provide new perspectives on the regulation of the MmpL family of transporters.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Mycobacterium tuberculosis/metabolismo , Proteínas de Bactérias/química , Cristalografia por Raios X , Proteínas de Membrana Transportadoras/química , Conformação Proteica
14.
PLoS One ; 10(8): e0136231, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26295942

RESUMO

Tuberculosis (TB) is the world's deadliest curable disease, responsible for an estimated 1.5 million deaths annually. A considerable challenge in controlling this disease is the prolonged multidrug chemotherapy (6 to 9 months) required to overcome drug-tolerant mycobacteria that persist in human tissues, although the same drugs can sterilize genetically identical mycobacteria growing in axenic culture within days. An essential component of TB infection involves intracellular Mycobacterium tuberculosis bacteria that multiply within macrophages and are significantly more tolerant to antibiotics compared to extracellular mycobacteria. To investigate this aspect of human TB, we created a physical cell culture system that mimics confinement of replicating mycobacteria, such as in a macrophage during infection. Using this system, we uncovered an epigenetic drug-tolerance phenotype that appears when mycobacteria are cultured in space-confined bioreactors and disappears in larger volume growth contexts. Efflux mechanisms that are induced in space-confined growth environments contribute to this drug-tolerance phenotype. Therefore, macrophage-induced drug tolerance by mycobacteria may be an effect of confined growth among other macrophage-specific mechanisms.


Assuntos
Antituberculosos/farmacologia , Tolerância a Medicamentos/genética , Dispositivos Lab-On-A-Chip , Modelos Biológicos , Mycobacterium smegmatis/efeitos dos fármacos , Antituberculosos/metabolismo , Carga Bacteriana , Transporte Biológico , Cinamatos/metabolismo , Cinamatos/farmacologia , Difusão , Dimetilpolisiloxanos , Epigênese Genética , Humanos , Higromicina B/análogos & derivados , Higromicina B/metabolismo , Higromicina B/farmacologia , Isoniazida/metabolismo , Isoniazida/farmacologia , Macrófagos/microbiologia , Microdiálise , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/crescimento & desenvolvimento , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crescimento & desenvolvimento , Ofloxacino/metabolismo , Ofloxacino/farmacologia , Fenótipo , Rifampina/metabolismo , Rifampina/farmacologia
15.
J Bacteriol ; 196(14): 2578-86, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24794563

RESUMO

Shigella flexneri two-component regulatory systems (TCRS) are responsible for sensing changes in environmental conditions and regulating gene expression accordingly. We examined 12 TCRS that were previously uncharacterized for potential roles in S. flexneri growth within the eukaryotic intracellular environment. We demonstrate that the TCRS EvgSA, NtrBC, and RstBA systems are required for wild-type plaque formation in cultured epithelial cells. The phenotype of the NtrBC mutant depended in part on the Nac transcriptional regulator. Microarray analysis was performed to identify S. flexneri genes differentially regulated by the NtrBC system or Nac in the intracellular environment. This study contributes to our understanding of the transcriptional regulation necessary for Shigella to effectively adapt to the mammalian host cell.


Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Shigella flexneri/citologia , Shigella flexneri/metabolismo , Proteínas de Bactérias/genética , Linhagem Celular , Humanos , Intestinos/citologia , Intestinos/microbiologia , Análise Serial de Proteínas , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Elementos Reguladores de Transcrição/genética , Shigella flexneri/fisiologia
16.
J Biol Chem ; 289(23): 16526-40, 2014 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-24737322

RESUMO

Recent work demonstrates that the MmpL (mycobacterial membrane protein large) transporters are dedicated to the export of mycobacterial lipids for cell wall biosynthesis. An MmpL transporter frequently works with an accessory protein, belonging to the MmpS (mycobacterial membrane protein small) family, to transport these key virulence factors. One such efflux system in Mycobacterium tuberculosis is the MmpS5-MmpL5 transporter. The expression of MmpS5-MmpL5 is controlled by the MarR-like transcriptional regulator Rv0678, whose open reading frame is located downstream of the mmpS5-mmpL5 operon. To elucidate the structural basis of Rv0678 regulation, we have determined the crystal structure of this regulator, to 1.64 Å resolution, revealing a dimeric two-domain molecule with an architecture similar to members of the MarR family of transcriptional regulators. Rv0678 is distinct from other MarR regulators in that its DNA-binding and dimerization domains are clustered together. These two domains seemingly cooperate to bind an inducing ligand that we identified as 2-stearoylglycerol, which is a fatty acid glycerol ester. The structure also suggests that the conformational change leading to substrate-mediated derepression is primarily caused by a rigid body rotational motion of the entire DNA-binding domain of the regulator toward the dimerization domain. This movement results in a conformational state that is incompatible with DNA binding. We demonstrate using electrophoretic mobility shift assays that Rv0678 binds to the mmpS5-mmpL5, mmpS4-mmpL4, and the mmpS2-mmpL2 promoters. Binding by Rv0678 was reversed upon the addition of the ligand. These findings provide new insight into the mechanisms of gene regulation in the MarR family of regulators.


Assuntos
Mycobacterium tuberculosis/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Cristalografia por Raios X , Primers do DNA , Dimerização , Dados de Sequência Molecular , Mycobacterium tuberculosis/química , Reação em Cadeia da Polimerase , Homologia de Sequência de Aminoácidos
17.
Protein Sci ; 23(4): 423-32, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24424575

RESUMO

The Rv1217c-Rv1218c multidrug efflux system, which belongs to the ATP-binding cassette superfamily, recognizes and actively extrudes a variety of structurally unrelated toxic chemicals and mediates the intrinsic resistance to these antimicrobials in Mycobacterium tuberculosis. The expression of Rv1217c-Rv1218c is controlled by the TetR-like transcriptional regulator Rv1219c, which is encoded by a gene immediately upstream of rv1218c. To elucidate the structural basis of Rv1219c regulation, we have determined the crystal structure of Rv1219c, which reveals a dimeric two-domain molecule with an entirely helical architecture similar to members of the TetR family of transcriptional regulators. The N-terminal domains of the Rv1219c dimer are separated by a large center-to-center distance of 64 Å. The C-terminal domain of each protomer possesses a large cavity. Docking of small compounds to Rv1219c suggests that this large cavity forms a multidrug binding pocket, which can accommodate a variety of structurally unrelated antimicrobial agents. The internal wall of the multidrug binding site is surrounded by seven aromatic residues, indicating that drug binding may be governed by aromatic stacking interactions. In addition, fluorescence polarization reveals that Rv1219c binds drugs in the micromolar range.


Assuntos
Proteínas de Bactérias/química , Mycobacterium tuberculosis/química , Fatores de Transcrição/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Cristalização , Cristalografia por Raios X , Modelos Moleculares , Conformação Proteica , Fatores de Transcrição/isolamento & purificação , Fatores de Transcrição/metabolismo
18.
PLoS One ; 8(6): e66985, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23825603

RESUMO

Non-human primates, notably rhesus macaques (Macaca mulatta, RM), provide a robust experimental model to investigate the immune response to and effective control of Mycobacterium tuberculosis infections. Changes in the function of immune cells and immunosenescence may contribute to the increased susceptibility of the elderly to tuberculosis. The goal of this study was to examine the impact of age on M. tuberculosis host-pathogen interactions following infection of primary alveolar macrophages derived from young and aged rhesus macaques. Of specific interest to us was whether the mycobactericidal capacity of autophagic macrophages was reduced in older animals since decreased autophagosome formation and autophagolysosomal fusion has been observed in other cells types of aged animals. Our data demonstrate that alveolar macrophages from old RM are as competent as those from young animals for autophagic clearance of M. tuberculosis infection and controlling mycobacterial replication. While our data do not reveal significant differences between alveolar macrophage responses to M. tuberculosis by young and old animals, these studies are the first to functionally characterize autophagic clearance of M. tuberculosis by alveolar macrophages from RM.


Assuntos
Autofagia , Macrófagos Alveolares/imunologia , Mycobacterium tuberculosis/imunologia , Fatores Etários , Animais , Macaca mulatta , Macrófagos Alveolares/microbiologia
19.
J Biol Chem ; 288(33): 24213-22, 2013 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-23836904

RESUMO

A growing body of evidence indicates that MmpL (mycobacterial membrane protein large) transporters are dedicated to cell wall biosynthesis and transport mycobacterial lipids. How MmpL transporters function and the identities of their substrates have not been fully elucidated. We report the characterization of Mycobacterium smegmatis MmpL11. We showed previously that M. smegmatis lacking MmpL11 has reduced membrane permeability that results in resistance to host antimicrobial peptides. We report herein the further characterization of the M. smegmatis mmpL11 mutant and identification of the MmpL11 substrates. We found that biofilm formation by the M. smegmatis mmpL11 mutant was distinct from that by wild-type M. smegmatis. Analysis of cell wall lipids revealed that the mmpL11 mutant failed to export the mycolic acid-containing lipids monomeromycolyl diacylglycerol and mycolate ester wax to the bacterial surface. In addition, analysis of total lipids indicated that the mycolic acid-containing precursor molecule mycolyl phospholipid accumulated in the mmpL11 mutant compared with wild-type mycobacteria. MmpL11 is encoded at a chromosomal locus that is conserved across pathogenic and nonpathogenic mycobacteria. Phenotypes of the M. smegmatis mmpL11 mutant are complemented by the expression of M. smegmatis or M. tuberculosis MmpL11, suggesting that MmpL11 plays a conserved role in mycobacterial cell wall biogenesis.


Assuntos
Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Parede Celular/metabolismo , Mycobacterium smegmatis/citologia , Mycobacterium smegmatis/fisiologia , Ácidos Micólicos/metabolismo , Transporte Biológico , Fatores Corda/metabolismo , Ésteres/química , Galactanos/biossíntese , Modelos Biológicos , Mutação/genética , Mycobacterium smegmatis/crescimento & desenvolvimento , Mycobacterium smegmatis/ultraestrutura , Plâncton/crescimento & desenvolvimento , Espectrometria de Massas por Ionização por Electrospray , Ceras/química
20.
Anal Bioanal Chem ; 405(23): 7415-26, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23852148

RESUMO

The storage of triacylglycerols (TAGs) is essential for non-replicating persistence relevant to survival and the re-growth of mycobacteria during their exit from non-replicating state stress conditions. However, the detailed structures of this lipid family in mycobacteria largely remain unexplored. In this contribution, we describe a multiple-stage linear ion-trap mass spectrometric approach with high resolution mass spectrometry toward direct structural analysis of the TAGs, including a novel lipid subclass previously defined as monomeromycolyl diacylglycerol (MMDAG) isolated from biofilm of Mycobacterium smegmatis, a rapidly growing, non-pathogenic mycobacterium that has been used as a tool for molecular analysis of mycobacteria. Our results demonstrate that the major isomer in each of the molecular species of TAGs and MMDAGs consists of the common structure in which Δ(9)18:1- and 16:0-fatty acyl substituents are exclusively located at sn-1 and sn-2, respectively. Several isomers were found for most of the molecular species, and thus hundreds of structures are present in this lipid family. More importantly, this study revealed the structures of MMDAG, a novel subclass of TAG that has not been previously reported by direct mass spectrometric approaches.


Assuntos
Biofilmes/crescimento & desenvolvimento , Diglicerídeos/química , Mycobacterium smegmatis/química , Triglicerídeos/química , Diglicerídeos/isolamento & purificação , Isomerismo , Mycobacterium smegmatis/crescimento & desenvolvimento , Espectrometria de Massas por Ionização por Electrospray , Triglicerídeos/isolamento & purificação
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