Determination of multidrug resistance mechanisms in Clostridium perfringens type A isolates using RNA sequencing and 2D-electrophoresis

In this study, we screened differentially expressed genes in a multidrug-resistant isolate strain of Clostridium perfringens by RNA sequencing. We also separated and identified differentially expressed proteins (DEPs) in the isolate strain by two-dimensional electrophoresis (2-DE) and mass spectrometry (MS). The RNA sequencing results showed that, compared with the control strain, 1128 genes were differentially expressed in the isolate strain, and these included 227 up-regulated genes and 901 down-regulated genes. Bioinformatics analysis identified the following genes and gene categories that are potentially involved in multidrug resistance (MDR) in the isolate strain: drug transport, drug response, hydrolase activity, transmembrane transporter, transferase activity, amidase transmembrane transporter, efflux transmembrane transporter, bacterial chemotaxis, ABC transporter, and others. The results of the 2-DE showed that 70 proteins were differentially expressed in the isolate strain, 45 of which were up-regulated and 25 down-regulated. Twenty-seven DEPs were identified by MS and these included the following protein categories: ribosome, antimicrobial peptide resistance, and ABC transporter, all of which may be involved in MDR in the isolate strain of C. perfringens. The results provide reference data for further investigations on the drug resistant molecular mechanisms of C. perfringens.

2DSpotDB: A Database for the Annotated Two-dimensional Polyacrylamide Gel Electrophoresis of Pathogen Proteins

The biological interpretation of two-dimensional (2D) gel electrophoresis experiments is a key step toward understanding the functions of biological systems. We here present a web-based integrated database, called 2DSpotDB, for the management of proteome data derived from several pathogens. The 2DSpotDB was established as a part of the management of a pathogen proteome project at the Korea National Institute of Health. The goals of the 2DSpotDB implementation are to store and define important pathogen genes, retrieve information obtained by 2D polyacrylamide gel electrophoresis and mass spectrometry, and create an integrated system to provide pathogen proteome information for biological scientists. This database currently contains 14 gels and information on 387 protein spots, among which 329 proteins were identified and annotated.

Análisis proteóiico de cultivos primarios de tiroides / Proteomic nalysiis of primary cultures from thyroid / Análiseproteómicco de culturas preliminares do thyroid

En el presente estudio se identificaron proteínas de expresión constitutiva, como vimentina, actina, tubulina, proteína de choque térmico de 60 kDa, peroxirredoxina y la ATP sintasa mitocondrial, en cultivos primarios de tiroides normales y de carcinoma papilar de tiroides. Se establecieron las condiciones de extracción, solubilización, análisis cuantitativo y cualitativo de dichas proteínas, y se buscaron las mejores condiciones de isoelectroenfoque (IEF) en la electroforesis en dos dimensiones (2D). En la extracción y solubilización de las proteínas se evaluó la presencia o ausencia de anfolitos y sales, se obtuvo un mejor resultado empleando en el amortiguador de extracción sales como Tris-HCl y acetato de magnesio que incrementan la solubilidad de las proteínas. Para la cuantificación se recomienda el uso conjunto de técnicas colorimétricas con la electroforesis SDS-PAGE tiñendo con azul de Coomassie y corroborando los resultados mediante western blot, lo cual permite, además, verificar la integridad de las proteínas. Respecto a la electroforesis en dos dimensiones, se obtuvieron geles con un mayor número de manchas (spots),resueltos, enfocados y reproducibles empleando en el IEF gradientes inmovilizados de pH de 4-7 y voltaje final de 8.000 V. Las proteínas se identificaron mediante el análisis bioinformático de los geles 2D con el programa PDQuest (PDQuest 7.2, Bio-Rad®) y MALDI-TOF.

In this paper, proteins of constitutive expression such as vimentin, actin, tubulin, heat shock protein of 60 kDa, peroxiredoxin and the mitochondrial ATP sintase were identified in primary cultures of normal thyroid and papillary carcinoma. The extraction conditions, solubilization, quantitative and qualitative analysis of such proteins were established. In addition, the best conditions for isoelectrofocusing (IEF) in the two-dimensional electrophoresis (2D) were founded. In the extraction and solubilization of proteins, the presence or absence ofampholytes or salts was evaluated. The best result in the extraction was obtained using buffer salts such as Tris-HCl and magnesium acetate, which increase the solubility of these proteins. For quantification, the recommendation is to combine colorimetric techniques with SDS-PAGE electrophoresis stained with Coomasie blue and Western blot to confirm the results, which allows verifying the integrity of these proteins. In the two-dimensional electrophoresis, resolved, focused and reproducible gels with greater number of spots were obtained using in the IEF immobilized pH gradients of 4-7 and final voltage of 8000 V. The proteins were identified by the bioinformatic analysis of the 2DE gels with the PDQuest program (PDQuest 7.2 (BioRad®) and MALDI-TOF.

Neste estudo, se identificaram proteínas da expressão constitutiva, como vimentina, actina, tubulina, proteína de choque do calor do kDa 60, peroxiredoxinaeasintase mitocondrial do ATP, em cultivos primários de tireóide normal e de carcinoma papilar de tireóide. Se estabeleceram as condições de extração e de solubilization, assim como a análise quantitativaeaqualitativa de tais proteínas, e procuraram-se asmelhores condições para isoeletroenfoque (IEF) na eletroforese bidimensional (2D). Na extração e no solubilization das proteínas, se avaliou a presença ou a ausência de anfolitos ou de sais. O melhor resultado foi obtido usando sais como Tris-HCl e acetato do magnésio no amortecedor da extração, as quais aumentam a solubilida-de das proteínas. Na quantificação, se recomenda o uso de técnicas colorimétricas com eletroforese SDS-PAGE, tingindo com azul de Coomasie. Os resultados se confirmam com Western blot, o qual permite também verificar a integridade das proteínas. Quanto à eletroforese bidimensional, se obtiveram géis com um maior número de pontos (spots) determinados, enfocados e reprodutíveis no IEF usando gradientes imobilizados de pH 4-7 e tensão final de 8000 V. As proteínas foram identificadas por análise bioinformática de géis 2D com o programa PDQuest (PDQuest 7.2 (Bio-Rad®) e MALDITOF.

La proteomica n la era postgenómica / The Proteomics In The Postgenomic Era

El principal desafío de la biología moderna es entender la expresión, función y regulación del conjunto completo de proteínas codificadas por un organismo, lo cual describe el objetivo del nuevo campo de la proteómica. Las proteínas son las efectoras del trabajo celular, por ello el estudio de sus perfiles globales de expresión y de sus cambios bajo determinadas condiciones fisiológicas o patológicas, permite entender la red compleja de interacciones en que se basa el funcionamiento de una célula. La electroforesis en dos dimensiones (2D-PAGE) es la técnica central de la proteómica. En la actualidad no existe otro método con la capacidad para resolver simultáneamente miles de proteínas en un solo procedimiento y para detectar modificaciones post y co-traduccionales imposibles de predecir a partir de la secuencia genómica. Sus aplicaciones incluyen el análisis de proteomas, señalización, detección de marcadores de enfermedades y cáncer.

The main challenge of modern biology is to understand the expression, function and regulation of the whole set of proteins codified by an organism, which is the objective of the new field of proteomics. Proteins are the effectors of cellular work and the knowledge of their global expression profiles and changes under physiological and pathological conditions can help us to understand the complex network of interactions involved in cellular function. Two-dimensional electrophoresis (2-DE) is the central technology in proteomics. At present no other technique has the throughput and high resolution of 2-DE for the separation of thousands of proteins in one procedure and for the analysis of post-and co-translation modifications, not predictable from the genome sequence. The scope of applications extends from proteome analysis, to cell signaling, disease markers and cancer.

Analysis of the Cell Lysate Proteome of a Korean Mycobacterium tuberculosis Isolate K01 with H37Rv and H37Ra Strains

Despite recent economic prosperity, Korea still has high prevalence of tuberculosis. Molecular biologic characterization of Korean Mycobacterium tuberculosis strains might provide a deeper understanding of the forces contributing to the spread of tuberculosis in Korea. Therefore, we analyzed the cell lysate proteome of a representative Korean Mycobacterium tuberculosis isolate (K01) in comparison with laboratory reference strains H37Rv and H37Ra. Seven spots were strongly expressed only in K01 strain compared with M. tuberculosis H37Rv and H37Ra. Through continuous MALDI-MS analysis, these spots were identified as hypothetical protein Rv3849, secreted immunogenic protein Mpt64, Acetyl/propionyl-CoA Carbpxylase (AccD1), alkyl hydroperoxide reductase C (AhpC), N-acetylmuramyl-L-alanine amidase, a putative UDP glucose epimerase, and a transposase. A deeper study of these proteins may provide a clue in the development of effective new anti-tuberculosis vaccines against Korean M. tuberculosis isolates.