Identifying immunogenic proteins of Erysipelothrix rhusiopathiae C43065 by MALDI-TOF mass spectrometry and cloning their encoding genes

Abstract:

[Objective] To identify immunogenic proteins of Erysipelothrix rhusiopathiae C43065. [Methods] Antigens were extracted from E. rhusiopathiae C43065 by the alkaline extraction method. Proteins in the NaOH-extracted antigen were separated by SDS-PAGE and transferred to nitrocellulose membranes, and then Western blotting was performed with rabbit antiserum against the NaOH-extracted antigens. The immunogenic protein bands were identified by MALDI-TOF mass spectrometry. The genes encoding 5 major immunogenic proteins was amplified by PCR from the genomic DNA of E. rhusiopathiae C43065, and inserted into the pMD18-T vector and then sequenced. [Results] A total of 9 immunogenic surface proteins in the NaOH-extracted antigen from E. rhusiopathiae C43065 were successfully identified by MALDI-TOF mass spectrometry. Four of the proteins were putative virulence-associated proteins: enolase, ATP-binding cassette transporter, glyceraldehyde-3-phosphate dehydrogenase and fructose-bisphosphate aldolase class-II. The genes encoding the chaperone protein GroEL, enolase, ATP-binding cassette transporter, glyceraldehyde-3-phosphate dehydrogenase and fructose-bisphosphate aldolase class-II were 1614, 1296, 1260, 1005 and 867 bp in length, and the nucleotide sequences homologies of the genes between the C43065 strain and the previously reported E. rhusiopathiae Fujisawa strain was more than 98%. [Conclusion] Several putative virulence-associated proteins in the NaOH-extracted antigen of E. rhusiopathiae C43065 will be useful for elucidating the roles of these proteins in the pathogenesis of the organism.