RESUMO
In recent years, multidrug resistance of Shigella strains associated with genetic elements like pathogenicity islands, have become a public health problem. The Shigella resistance locus pathogenicity island (SRL PAI) of S. flexneri 2a harbors a 16Kbp region that contributes to the multidrug resistance phenotype. However, there is not much information about other functions such as metabolic, physiologic or ecological ones. For that, wild type S. flexneri YSH6000 strain, and its spontaneous SRL PAI mutant, 1363, were used to study the contribution of the island in different growth conditions. Interestingly, when both strains were compared by the Phenotype Microarrays, the ability to metabolize D-aspartic acid as a carbon source was detected in the wild type strain but not in the mutant. When D-aspartate was added to minimal medium with other carbon sources such as mannose or mannitol, the SRL PAI-positive strain was able to metabolize it, while the SRL PAI-negative strain did not. In order to identify the genetic elements responsible for this phenotype, a bioinformatic analysis was performed and two genes belonging to SRL PAI were found: orf8, coding for a putative aspartate racemase, and orf9, coding for a transporter. Thus, it was possible to measure, by an indirect analysis of racemization activity in minimal medium supplemented only with D-aspartate, that YSH6000 strain was able to transform the D-form into L-, while the mutant was impaired to do it. When the orf8-orf9 region from SRL island was transformed into S. flexneri and S. sonnei SRL PAI-negative strains, the phenotype was restored. Although, when single genes were cloned into plasmids, no complementation was observed. Our results strongly suggest that the aspartate racemase and the transporter encoded in the SRL pathogenicity island are important for bacterial survival in environments rich in D-aspartate.
Assuntos
Isomerases de Aminoácido/metabolismo , Ácido D-Aspártico/metabolismo , Farmacorresistência Bacteriana Múltipla/genética , Ilhas Genômicas , Shigella flexneri/genética , Isomerases de Aminoácido/genética , Proteínas de Bactérias/metabolismo , Ácido D-Aspártico/análise , Genes Bacterianos , Manose/metabolismo , Fases de Leitura Aberta/genética , Fenótipo , Shigella flexneri/enzimologia , Shigella flexneri/crescimento & desenvolvimento , Shigella sonnei/genéticaRESUMO
El objetivo de este trabajo fue comparar los niveles extracelulares de glutamato y aspartato en el fluido del surco gingival (GCF) de personas adultas, en la periodontitis crónica localizada inducida por placa (PCIP) y la gingivitis inducida por placa (GIP). La enfermedad periodontal produce cambios inflamatorios en los tejidos de sostén de las piezas dentales afectadas. El análisis químico del GCF, con diferentes métodos de colección y análisis, ha sido usado para determinar la presencia de algunos elementos inflamatorios que aparecen en la enfermedad periodontal, tales como diversas enzimas, aminoácidos, etc.Las muestras del GCF se tomaron con la técnica de microdiálisis en las zonas dentales con PCIP con una profundidad del surco > 3 mm; pérdida de soporte > 2mm y en las zonas dentales con GIP en el mismo paciente (n=10) Total de muestras: 100. Para medir el glutamato y aspartato en el GCF se usó la técnica de electroforesis capilar acoplada a laser con detección inducida por fluorescencia (CZE-LIFD). Los resultados mostraron que en los dientes con PCIP el glutamato disminuyó (p<0.05) y el aspartato aumentó (p< 0.02) en comparación con los dientes con GIP.
The objective of this work was to compare glutamate and aspartate levels in periodontal chronic localized disease (PCIP) and dental zones with gingivitis (GIP) in the gingival crevicular fluid (GCF). Periodontal inflammation produces histological changes, increase of blood irrigation and also increase of subgingival fluid. GCF was recognized as an inflammatory exudes derived from the periodontal tissue. Different methods to collect and analyze GCF samples had been used to identify some substances in the GCF, such as the proteinglycans metabolite, to be a possible marker of active periodontal disease. A combination of microdialysis in situ in dental zones with PCIP (probing depth > 3 mm; attachment level > 2 mm) and dental zones with GIP (n=10), total samples: 100, and capillary zone electrophoresis coupled to a laser induced fluorescence detection (CZE-LIFD) was used to measure extracellular concentrations of amino acids: glutamate and aspartate in the GCF in adult patients The results showed that glutamate decrease (p<0.05) and aspartate increase (p<0.02) in PCIP disease zones compared with dental zones with GIP. We observed chemical in vivo evidence that differentiate the GIP zones and PCIP zones.