Cacciotto, Carla (2011) Characterization of Mycoplasma agalactiae membrane proteome. Doctoral Thesis.
Mycoplasma agalactiae is the etiological agent of contagious agalactia, a serious disease of small ruminants characterized by mastitis, polyarthritis, keratoconjunctivitis, and abortion. Mycoplasmas totally lack the cell wall, so membrane proteins are directly exposed to environment. To date, a limited number of M. agalactiae antigens have been described. The combination of 2-D PAGE and mass spectrometry is a well-established method for proteome studies; however, it is reported that standard 2-D PAGE lacks in resolution of hydrophobic and basic proteins, which are abundant in mycoplasmas membrane. Indeed, membrane proteins are poorly represented in total extracts maps. In this study, the membrane proteome of M. agalactiae PG2T was characterized. The Triton X-114 fractionation allowed the enrichment for M. agalactiae PG2T membrane proteins. Liposoluble proteins were subjected to 2-D PAGE-MS, leading to the identification of 40 unique proteins. The differential expression of liposoluble proteins, among PG2T strain and two field isolates, was revealed by 2D DIGE. The use of GeLC-MS/MS allowed to increase the coverage of the liposoluble proteome. A total of 194 unique proteins were identified, (26% of M. agalactiae PG2T genes) and subjected to gene ontology analysis for localization and function. Interestingly, the 11.5% of identified proteins derived from putative horizontal gene transfer events. This work paves the way for the development of diagnostic and prophylaxis tools.
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