Mycoplasma bovis (M. bovis) is a pathogenic bacterium causing untreatable mastitis, abortions, swollen joints, and severe arthritis in cattle of all ages. Though eradication efforts are ongoing, they are mostly limited by ineffective antibiotics and suboptimal diagnostic tests. In recent years, small extracellular vesicles (sEVs), and exosomes in particular, have gained significant interest. Exosomes are small extracellular vesicles of 30-150 nm known to have important functions in cellular communication, signal transduction, and immunomodulation. As these nanoparticles contain proteins, lipids and metabolites, representative of their cell of origin, their content may have potential to carry biomarkers for diagnosis of early or hidden infections.
In this work, we want to unlock the diagnostic potential of sEVs isolated from control cells vs M. Bovis infected cells. We hypothesize that comparing their exosomal proteome may contain information representative of infection. To this end, a control culture of a bovine endometrial epithelial cell line (bEEL cells) (n = 5) and a co–culture of bEEL cells and M. bovis (n=5) were established within bioreactor flasks. From each bioreactor harvest, sEVs were isolated using Size Exclusion Chromatography qEV10 columns (IZON, New Zealand). The isolated exosomal fraction was further heat treated for 5 min to inactive M. Bovis before proteins were extracted and digested.
A nanoElute UHPLC was coupled online to a Bruker timsTOF Pro 2. Peptides were separated on a reversed-phase column (25 cm x 75 μm i.d.), packed with 1.6 μm C18 silica beads (IonOpticks, Australia) using a 60 min linear gradient from 2 to 35% B (0.1% formic acid/acetonitrile/water). Data were acquired using diaPASEF, with data independent isolation of multiple precursor windows within a single TIMS separation (100 ms). For each sample, data were acquired in duplicate. Analysis of the 4D data space was performed using PEAKS Online, combining a spectral library and database search.
In total, over 4100 proteins representing 2816 protein groups were identified across the samples. As expected, hundreds of mycoplasma proteins have been identified in the sEVs of the co-cultures, but not in the control samples. Additional comparative proteomics profiling of the altered bovine proteome of sEVs in response to infection by M. bovis indicated separate grouping. Specific exosomal proteins were detected using ExoCarta. In conclusion, our data provides preliminary evidence that exosomal protein profiles are altered in the presence of M. bovis infection and can thus play a crucial role in early diagnosis of the disease.