Identification of conserved Mycoplasma agalactiae surface antigens by immunoproteomics

Contagious agalactia represents one of the most relevant infectious diseases of dairy sheep, with Mycoplasma
agalactiae being the primary etiological agent. The early, sensitive, and specific identification of infected animals,
as well as the development of efficient prophylactic tools, remain challenging. Here, we present a comprehensive
characterization of M. agalactiae antigens focusing on those shared among different isolates. Leveraging on
previous proteomic data obtained on individual strains, we adopted a strategy entailing sample pooling to
optimize the identification of conserved proteins that induce an immune response. The liposoluble proteins from
previously characterized field isolates and the type strain PG2T were enriched by Triton X-114 fractionation,
pooled, analysed by one-dimensional (1D) and two-dimensional (2D) electrophoresis, and subjected to western
immunoblotting against sheep sera collected during natural infection with M. agalactiae. Immunodominant antigens
were identified by Matrix-Assisted Laser Desorption-Time-Of-Flight-Mass Spectrometry (MALDI-TOF-MS).
This combined immunoproteomic approach confirmed the role of several known immunogens, including P80,
P48, and P40, and most variable surface proteins (Vpmas), and unveiled novel immunodominant, conserved
antigens, including MAG_1000, MAG_2220, MAG_1980, phnD, MAG_4740, and MAG_2430. Genomic context,
functional prediction, subcellular localization, and invariable expression of these proteins in all isolates suggest
their possible involvement in bacterial pathogenicity and metabolism. Moreover, most of the identified antigens
elicit a host humoral response since the early stages of infection, persisting for at least 270 days. The immunodominant,
conserved antigen panel identified in this work supports the development of effective vaccines and
diagnostic tools with higher sensitivity and specificity in all the natural infection stages.