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1.
Braz. dent. j ; 27(3): 255-260, May-June 2016. tab, graf
Artículo en Inglés | LILACS | ID: lil-782824

RESUMEN

Abstract The periodontopathogen Aggregatibacter actinomycetemcomitans colonizes oral cavity by binding to and invading epithelial cells as well as by participating in biofilms formed on hard surfaces. Aae, an autotransporter protein, is implicated in bacterial adhesion to epithelial cells. Due to the multiple functions of bacterial autotransporter proteins, this study aimed to evaluate the role of aae in A. actinomycetemcomitans ability to adhere to both saliva-coated hydroxyapatite (SHA) and biofilm. An aae null mutant was constructed. Its hydrophobic properties as well as its ability to adhere to epithelial cells, SHA and to form biofilm were evaluated and compared with the parental strain, A. actinomycetemcomitans VT1169. The aae null mutant showed reduced hydrophobicity, as well as decreased binding to SHA and biofilm formation compared to the parental strain. These data suggest that aae mediates A. actinomycetemcomitans adhesion to epithelial cells and may be involved in biofilm formation and interaction with adsorbed salivary proteins.


Resumo O peridontopatógeno Aggregatibacter actinomycetemcomitans coloniza a cavidade oral aderindo e invadindo as células epiteliais e participando da formação de biofilme em superfícies duras. Aae, uma proteína autotransportadora está relacionada com a adesão bacteriana às células epiteliais. Devido às múltiplas funções desempenhadas por proteínas bacterianas autotransportadoras, este estudo teve como objetivo avaliar o papel de aae de A. actinomycetemcomitans tanto na capacidade de aderir à hidroxiapatita recoberta por saliva (SHA), quanto a de formar biofilme. Um mutante nulo aae foi construído. Suas propriedades hidrofóbicas, bem como a sia capacidade para aderir às células epiteliais, à SHA e para formar biofilme foram avaliadas e comparadas com a cepa -mãe, A. Actinomycetemcomitans VT1169. O mutante nulo aae apresentou redução de hidrofobicidade, assim como diminuição da adesão à SHA e na formação de biofilme, quando comparado à cepa parental. Estes dados sugerem que aae media a adesão de A. Actinomycetemcomitans às células epiteliais e pode também estar envolvida na formação de biofilme e na interação com proteínas salivares adsorvidas.


Asunto(s)
Aggregatibacter actinomycetemcomitans/fisiología , Adhesión Bacteriana/fisiología , Proteínas Bacterianas/fisiología , Proteínas de Transporte de Membrana/fisiología , Aggregatibacter actinomycetemcomitans/genética , Proteínas Bacterianas/genética , Biopelículas , Técnicas de Silenciamiento del Gen , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas de Transporte de Membrana/genética
2.
Int. j. morphol ; 30(2): 531-540, jun. 2012. ilus
Artículo en Español | LILACS | ID: lil-651825

RESUMEN

Las proteínas NPC1L1, ABCG5 y ABCG8 participan en la absorción intestinal de colesterol. Ezetimiba inhibe este proceso bloqueando a NPC1L1, sin embargo, su efecto sobre ABCG5 y ABCG8 aún no está claro. Así, el objetivo del presente trabajo fue evaluar en ratones C57BL/6 con hipercolesterolemia inducida por dieta y tratados con ezetimiba, la expresión de NPC1L1, ABCG5 y ABCG8 mediante PCR en tiempo real y Western blot, en 3 grupos de animales: 1, dieta hipercolesterolémica D12336; 2, dieta D12336 más 5 mg/kg/día de ezetimiba; 3, dieta control. El nivel sérico de colesterol total fue significativamente diferente entre los grupos estudiados (control: 1,85 +/- 0,49 mmol/L; dieta D12336: 3,11 +/- 0,73 mmol/L; ezetimiba: 2,11 +/- 0,50 mmol/L, P = 0,001). La expresión génica de NPC1L1 aumentó 5,4 veces en el grupo que recibió la dieta D12336 (P = 0,003). Por otro lado, la expresión génica de ABCG5 y ABCG8 no fue diferente en el grupo con hipercolesterolemia (P = 0,239 y P = 0,201, respectivamente). Después del tratamiento con ezetimiba, la expresión génica de ABCG5 se incrementó 15,6 veces (P = 0.038). No hubo diferencias significativas en la expresión génica de NPC1L1 (P = 0,134) y ABCG8 (P = 0,067). En relación a la expresión proteica, la dieta D12336 incrementó los niveles de expresión de NPC1L1 (P = 0,022) y ABCG5 (P = 0,008); el tratamiento con ezetimiba incrementó los niveles de NPC1L1 (P = 0,048) y redujo los niveles de ABCG5 (P = 0,036) y ABCG8 (P = 0,016). En conclusión, nuestros resultados sugieren que tanto la dieta hipercolesterolémica como el tratamiento con ezetimiba, en un modelo experimental, afectan los niveles de expresión de NPC1L1, ABCG5 y ABCG8, sugiriendo que ABCG5 y ABCG8 están involucrados en la respuesta hipolipemiante a este fármaco. No obstante, el mecanismo mediante el cual se explica esta interacción requiere de un futuro estudio.


Proteins NPC1L1, ABCG5 and ABCG8 are involved in the intestinal absorption of cholesterol. Ezetimibe inhibits this process by blocking NPC1L1, however, its effect on ABCG5 and ABCG8 is not yet clear. Thus, the objective of this study was to evaluate in C57BL / 6 mice with diet-induced hypercholesterolemia treated with ezetimibe, the expression of NPC1L1, ABCG5 and ABCG8 by real time PCR and Western blot, in 3 groups of animals: 1, diet hypercholesterolemic D12336, 2, D12336 diet plus 5 mg/kg/ day of ezetimibe, 3, diet control. The serum level of total cholesterol was significantly different between groups (control: 1.85 +/- 0.49 mmol / L; diet D12336: 3.11 +/- 0.73 mmol / L; ezetimibe: 2.11 +/- 0.50 mmol / L, P = 0.001). NPC1L1 gene expression increased 5.4-fold in the group receiving the diet D12336 (P = 0.003). Furthermore, the gene expression of ABCG5 and ABCG8 was not different in the group with hypercholesterolemia (P = 0.239 and P = 0.201, respectively). After treatment with ezetimibe, ABCG5 gene expression was increased 15.6 times (P = 0.038). No significant differences in gene expression of NPC1L1 (P = 0.134) and ABCG8 (P = 0.067). Regarding protein expression, the D12336 diet increased the levels of expression of NPC1L1 (P = 0.022) and ABCG5 (P = 0.008), treatment with ezetimibe increased the levels of NPC1L1 (P = 0.048) and reduced levels of ABCG5 (P = 0.036) and ABCG8 (P = 0.016). In conclusion, our results suggest that both hypercholesterolemic diet as treatment with ezetimibe, in an experimental model, affect the expression levels of NPC1L1, ABCG5 and ABCG8, suggesting that ABCG5 and ABCG8 are involved in lipid-lowering response to this drug. However, the mechanism by which this interaction is explained requires further study.


Asunto(s)
Animales , Ratas , Anticolesterolemiantes/administración & dosificación , Azetidinas/administración & dosificación , Hipercolesterolemia/tratamiento farmacológico , Lipoproteínas/fisiología , Proteínas de Transporte de Membrana/fisiología , Transportadoras de Casetes de Unión a ATP/fisiología , Western Blotting , Colesterol en la Dieta , Modelos Animales de Enfermedad , Expresión Génica , Lipoproteínas/genética , Proteínas de Transporte de Membrana/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Transportadoras de Casetes de Unión a ATP/genética
3.
Artículo en Español | LILACS | ID: lil-612947

RESUMEN

Objetivo. Identificar la proteína de membrana externa ausente en los aislamientos resistentes y determinar tanto las causas de su ausencia en la membrana, como la presencia de otros mecanismos de resistencia a carbapenemes en aislamientos clínicos de Pseudomonas aeruginosa. Métodos. Se estudió un brote de 20 aislamientos de P. aeruginosa previamente caracterizados como productores de la metalobetalactamasa IMP-13. Estos aislamientos presentaron igual expresión de la enzima IMP-13, pero solo cinco de ellos fueron resistentes acarbapenemes. En esos cinco aislamientos resistentes se confirmó la ausencia de una proteína de membrana externa. Se secuenciaron oprD y ampC; se identificaron las proteínas de membrana externa por desorción/ionización láser asistida por matriz/espectometría de masa tiempo de vuelo (MALDI-TOF); se determinó el nivel de expresión de OprD, de AmpC y de los sistemas de eflujo tipo Mex, por reacción en cadena de polimerasa en tiempo real, y por último, se determinó la contribución del déficit de OprD a la resistencia a carbapenemes. Resultados. La proteína de la membrana externa ausente en el grupo R (resistentes a ambos carbapenemes) fue identificada como OprD-TS, pero no se observaron variaciones en suexpresión. El gen oprD presentó mutaciones en los cinco aislamientos resistentes. Se observó la misma producción de la enzima tipo AmpC PDC-5 y del sistema de eflujo Mex AB-OprM entre los aislamientos sensibles y resistentes a carbapenemes. Se analizó cómo la presencia conjunta de IMP-13 y el déficit de OprD contribuyen al aumento de la resistencia.Conclusiones. Distintos mecanismos contribuyen a la resistencia de aislamientos productores de IMP-13 a carbapenemes. La posibilidad de no detectar estos aislamientos productores de IMP-13 representa un riesgo latente de selección de mutantes con mecanismos de resistencia que se suman para aumentar la resistencia a carbapenemes.


Objective. To identify the outer membrane protein absent in the resistant isolates and to determine both the causes of its absence in the membrane and the presence of othermechanisms of carbapenem resistance in clinical isolates of Pseudomonas aeruginosa. Methods. Twenty isolates from an outbreak of P. aeruginosa previously characterized as metallo-beta-lactamase IMP-13 producers were studied. All the isolates exhibitedequal expression of the IMP-13 enzyme, but only five of them were carbapenemresistant. It was found that the five resistant isolates lacked a outer membrane protein. The oprD and ampC genes were sequenced; the outer membrane proteins were identifiedusing matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry; the OprD and AmpC expressions, as well as the Mex efflux system, were assessed by real-time polymerase chain reaction; and finally, the contribution of reduced OprD to carbapenem resistance was determined. Results. The absent outer membrane protein in group R was identified as OprD-TS; however, no variations in its expression were observed. The oprD gene presentedmutations in the five resistant isolates. The production of AmpC PDC-5-type enzyme and the MexAB-OprM efflux system was the same in both carbapenem-sensitive and‑resistant isolates. The contribution of the combined presence of IMP-13 and reducedOprD to increased resistance was examined. Conclusions. Different mechanisms contribute to carbapenem resistance in IMP-13-producing isolates. The possibility that these IMP-13-producing isolates could go undetected poses a latent risk when selecting mutants with added resistancemechanisms in order to enhance carbapenem resistance.


Asunto(s)
Humanos , Proteínas Bacterianas/fisiología , Carbapenémicos/farmacología , Farmacorresistencia Bacteriana Múltiple/fisiología , Porinas/genética , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/efectos de los fármacos , Resistencia betalactámica/fisiología , beta-Lactamasas/fisiología , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/fisiología , Proteínas Bacterianas/genética , Análisis Mutacional de ADN , ADN Bacteriano/genética , Farmacorresistencia Bacteriana Múltiple/genética , Electroforesis en Gel de Campo Pulsado , Genes Bacterianos , Imipenem/metabolismo , Imipenem/farmacología , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/fisiología , Mutación , Porinas/deficiencia , Porinas/fisiología , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/genética , Estudios Retrospectivos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Tienamicinas/metabolismo , Tienamicinas/farmacología , Resistencia betalactámica/genética , beta-Lactamasas/genética
4.
Artículo en Inglés | IMSEAR | ID: sea-135571

RESUMEN

Background & objectives: Drug efflux pumps have been contributing factor(s) in the development of multidrug resistance in various clinically relevant bacteria. During efflux pump gene expression studies on mycobacteria, we have found a previously uncharacterized open reading frame (ORF) Rv2459 to be overexpressed in drug stressed conditions. The objective of the present study was to investigate the role of this ORF as a drug efflux pump, which might add new information in our understanding about the alternative mechanisms of drug resistance in mycobacteria. Methods: The open reading frame Rv2459 of Mycobacterium tuberculosis encoding a probable drug efflux protein has been cloned using pSD5 E.coli-Mycobacterium shuttle vector and overexpressed in M. tuberculosis H37Rv. This ORF was named as jefA. Overexpression of this gene in clones has been verified by real-time reverse transcription PCR. Minimum inhibitory concentrations (MICs) of recombinant as well as non-recombinant clones were determined by resazurin microtitre assay plate method (REMA) with and without efflux pump inhibitors carbonyl cyanide m-chlorophenylhydrazone (CCCP) and verapamil. Results: In recombinant strains of M. tuberculosis, the overexpression of this gene led to an increase in MIC of anti-tubercular drugs isoniazid and ethambutol when tested by REMA. In the presence of CCCP and verapamil, the recombinant strains showed decrease in MIC for these drugs. Bioinformatic analysis has shown a close relation of JefA protein with drug efflux pumps of other clinically relevant bacteria. In homology derived structure prepared from nearest available model, it was observed that amino acids forming TMH 1, 8 and 11 participated in ethambutol specificity and those forming TMH 2, 7 and 10 participated in isoniazid specificity in JefA. Interpretation & conclusion: The increased transcription of jefA leads to increased resistance to ethambutol and isoniazid in M. tuberculosis via efflux pump like mechanism and contributes in the development of resistance to these drugs. JefA amino acid sequence is well conserved among clinically important bacterial genera, which further provides evidence of being a potent drug efflux pump. The involvement in drug resistance and very little homology with any of the human proteins makes JefA important to be included in the list of potential drug targets.


Asunto(s)
Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Secuencia de Bases , Clonación Molecular , Análisis por Conglomerados , Biología Computacional , Cartilla de ADN/genética , Farmacorresistencia Microbiana/genética , Etambutol , Isoniazida , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/fisiología , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Datos de Secuencia Molecular , Mycobacterium tuberculosis/genética , Sistemas de Lectura Abierta/genética , Filogenia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN
5.
J Postgrad Med ; 2006 Oct-Dec; 52(4): 271-6
Artículo en Inglés | IMSEAR | ID: sea-116286

RESUMEN

Chloroquine (CQ) is the most successful antimalarial drug ever discovered. Unfortunately, parasites resistant to the drug eventually emerged after its large scale use and are now widespread. Although great progress in our understanding of the mechanisms of CQ action and CQ resistance (CQR) has been achieved over the past two decades, including the identification of the molecules responsible for CQR (e.g., Plasmodium falciparum chloroquine resistant transporter, PfCRT) many questions remain unanswered. Here we highlight recent advances in our understanding of the genetics and molecular mechanisms of CQR, with particular emphasis on the role of genes such as pfcrt and pfmdr1 in the resistance to CQ and other drugs. New drug development and applications will undoubtedly benefit from a better understanding of CQR, eventually leading to more effective malaria control measures.


Asunto(s)
Animales , Antimaláricos/farmacología , Cloroquina/farmacología , Resistencia a Medicamentos/genética , Proteínas de Transporte de Membrana/fisiología , Plasmodium falciparum/efectos de los fármacos , Proteínas Protozoarias/fisiología
6.
Genet. mol. res. (Online) ; 4(2): 390-408, 30 jun. 2005. ilus, tab
Artículo en Inglés | LILACS | ID: lil-445280

RESUMEN

In the struggle for life, the capacity of microorganisms to synthesize and secrete toxic compounds (inhibiting competitors) plays an important role in successful survival of these species. This ability must come together with the capability of being unaffected by these same compounds. Several mechanisms are thought to avoid the toxic effects. One of them is toxin extrusion from the intracellular environment to the outside vicinity, using special transmembrane proteins, referred to as transporters. These proteins are also important for other reasons, since most of them are involved in nutrient uptake and cellular excretion. In cancer cells and in pathogens, and particularly in fungi, some of these proteins have been pointed out as responsible for an important phenotype known as multidrug resistance (MDR). In the present study, we tried to identify in the Paracoccidioides brasiliensis transcriptome, transporter-ortholog genes from the two major classes: ATP binding cassette and major facilitator superfamily transporter. We found 22 groups with good similarity with other fungal ATP binding cassette transporters, and four Paracoccidioides brasilienses assembled expressed sequence tags that probably code for major facilitator superfamily proteins. We also focused on fungicide resistance orthologs already characterized in other pathogenic fungi. We were able to find homologs to C. albicans CDR1, CDR2, and MDR1, Saccharomyces cerevisiae PDR5 and Aspergillus AtrF genes, all of them related to azole resistance. As current treatment for paracoccidioidomycosis mainly uses azole derivatives, the presence of these genes can be postulated to play a similar role in P. brasiliensis, warning us for the possibility of resistant isolate emergence.


Asunto(s)
Humanos , Antifúngicos/farmacología , Etiquetas de Secuencia Expresada/metabolismo , Paracoccidioides/efectos de los fármacos , Farmacorresistencia Fúngica Múltiple/genética , Transcripción Genética , Transportadoras de Casetes de Unión a ATP/genética , Paracoccidioides/genética , Paracoccidioides/metabolismo , Proteínas de Transporte de Membrana/efectos de los fármacos , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/fisiología , Farmacorresistencia Fúngica Múltiple/fisiología , Transportadoras de Casetes de Unión a ATP/efectos de los fármacos , Transportadoras de Casetes de Unión a ATP/metabolismo
7.
Genet. mol. res. (Online) ; 2(1): 48-62, Mar. 2003.
Artículo en Inglés | LILACS | ID: lil-417624

RESUMEN

Pseudomonas aeruginosa is an opportunistic human pathogen exhibiting innate resistance to multiple antimicrobial agents. This intrinsic multidrug resistance is caused by synergy between a low-permeability outer membrane and expression of a number of broadly-specific multidrug efflux (Mex) systems, including MexAB-OprM and MexXY-OprM. In addition to this intrinsic resistance, these and three additional systems, MexCD-OprJ, MexEF-OprN and MexJK-OprM promote acquired multidrug resistance as a consequence of hyper-expression of the efflux genes by mutational events. In addition to antibiotics, these pumps export biocides, dyes, detergents, metabolic inhibitors, organic solvents and molecules involved in bacterial cell-cell communication. Homologues of the resistance-nodulation-division systems of P. aeruginosa have been found in Burkholderia cepacia, B. pseudomallei, Stenotrophomonas maltophilia, and the nonpathogen P. putida, where they play roles in resistance to antimicrobials and/or organic solvents. Despite intensive studies of these multidrug efflux systems over the past several years, their precise molecular architectures, their modes of regulation of expression and their natural functions remain largely unknown


Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Pseudomonas aeruginosa/metabolismo , Farmacorresistencia Bacteriana Múltiple , Secuencia de Aminoácidos , Secuencia de Bases , Transporte Biológico , Regulación Bacteriana de la Expresión Génica , Datos de Secuencia Molecular , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/fisiología , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/fisiología , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/genética
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