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1.
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
2.
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
3.
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
4.
Parasit Vectors ; 8: 123, 2015 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-25890064

RESUMO

BACKGROUND: Chagas disease is an anthropozoonosis caused by the protozoan parasite Trypanosoma cruzi that represents a major public health problem in Latin America. Although the United States is defined as non-endemic for Chagas disease due to the rarity of human cases, the presence of T. cruzi has now been amply demonstrated as enzootic in different regions of the south of the country from Georgia to California. In southeastern Louisiana, a high T. cruzi infection rate has been demonstrated in Triatoma sanguisuga, the local vector in this area. However, little is known about the role of small mammals in the wild and peridomestic transmission cycles. METHODS: This study focused on the molecular identification and genotyping of T. cruzi in both small rodents and T. sanguisuga from a rural area of New Orleans, Louisiana. DNA extractions were prepared from rodent heart, liver, spleen and skeletal muscle tissues and from cultures established from vector feces. T. cruzi infection was determined by standard PCR using primers specific for the minicircle variable region of the kinetoplastid DNA (kDNA) and the highly repetitive genomic satellite DNA (satDNA). Genotyping of discrete typing units (DTUs) was performed by amplification of mini-exon and 18S and 24Sα rRNA genes and subsequent sequence analysis. RESULTS: The DTUs TcI, TcIV and, for the first time, TcII, were identified in tissues of mice and rats naturally infected with T. cruzi captured in an area of New Orleans, close to the house where the first human case of Chagas disease was reported in Louisiana. The T. cruzi infection rate in 59 captured rodents was 76%. The frequencies of the detected DTUs in such mammals were TcI 82%, TcII 22% and TcIV 9%; 13% of all infections contained more than one DTU. CONCLUSIONS: Our results indicate a probable presence of a considerably greater diversity in T. cruzi DTUs circulating in the southeastern United States than previously reported. Understanding T. cruzi transmission dynamics in sylvatic and peridomestic cycles in mammals and insect vectors will be crucial to estimating the risk of local, vector-borne transmission of T. cruzi to humans in the United States.


Assuntos
Doença de Chagas/parasitologia , Variação Genética , Insetos Vetores/parasitologia , Triatoma/parasitologia , Trypanosoma cruzi/genética , Animais , Doença de Chagas/epidemiologia , DNA de Cinetoplasto/genética , DNA de Protozoário/genética , Genótipo , Humanos , Louisiana , Camundongos , Nova Orleans/epidemiologia , Ratos , Trypanosoma cruzi/classificação , Trypanosoma cruzi/isolamento & purificação
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