Identificação e caracterização do exoproteoma de duas linhagens de Corynebacterium pseudotuberculosis através das técnicas de 2D-DIGE e espectrometria de massa

Abstract

Corynebacterium pseudotuberculosis is the etiological agent of caseous lymphadenitis - a chronic infectious disease that affects small ruminants. The main virulence factors of this bacterium are exotoxin phospholipase D and toxic lipid on cell wall. Studies have shown that extracellular proteins are important for: nutrient acquisition, interaction with the host, survival of bacteria in different environments and targets for vaccine production and methods for diagnosis. The genomes of strains 1002 and C231 of C. pseudotuberculosis isolated from Brazil goat and sheep and from Australia, respectively, were sequenced and a list of the aforementioned proteins with their likely functions was generated by in silico predictions. In the advent of this work, a comparative analysis was performed between two strains of extracellular proteins for characterization, assessment of difference in expression and validation of the results predicted in silico using proteomic approaches. The protein samples were obtained by fractionation technique in three phases. To study the difference in expression, 2D-DIGE technique was used. After analysis by mass spectrometry resulted in the characterization of 10 proteins (5 from 1002 and 5 from strain C231 strain), related to virulence factors, stress response, structural components and unknown functions. Through the association of 2-DE/MALDI-TOF MS/MS it became posible to generate maps and characterize 55 and 45 extracellular proteins of strains 1002 and C231 respectively. The union of the present data with those obtained Pacheco et al. (2011), allowed experimentally characterized 104 proteins from the exoproteoma of the C. pseudotuberculosis. This work portrays the fact that even though the bacteria of the same species, there exist differences in expression and presence of specific proteins in each strain, as they are related to virulence factors and cellular physiology that may influence the mechanism of infection and adaptation to the host, which contribute to the progression and onset of illness. Furthermore, this work opens perspectives for specific studies of the proteins identified for possible application in the development of diagnostic methods and vaccines