Dominant Circulating Cell-free Mycobacterial Proteins in In-use Machining Fluid and their Antigenicity Potential

Background: Occupational exposure to industrial Metalworking Fluid (MWF) colonized by Mycobacterium immunogenum (MI) has been associated with immune lung disease hypersensitivity pneumonitis (HP) in machinists. This warrants regular fluid monitoring for early detection of mycobacterial proteins, especially those with antigenic potential. Objective: To detect and identify dominant MI proteins and antigens directly from the field-drawn in-use MWF using an integrated immunoproteomic-immunoinformatic approach. Methods: An MI-positive MWF selected by DNA-based screening of several field-drawn MWF samples was cultured to isolate the colonizing strain and profiled for dominant circulating cell-free (ccf) MI proteins, including antigens using an integrated immunoproteomic (1D- and 2Dgel fractionation of seroreactive proteins combined with shotgun proteomic analysis using LC-MS/MS) and immunoinformatic strategy. Results: A new MI strain (MJY-27) was identified. The gel fractionated MI protein bands (1Dgel) or spots (2D-gel) seroreactive with anti-MI sera probes (Rabbit and Patient sera) yielded 86 MI proteins, 29 of which showed peptide abundance. T-cell epitope analysis revealed high (90-100%) binding frequency for HLA-I & II alleles for 13 of the 29 proteins. Their antigenicity analysis revealed the presence of 6 to 37 antigenic determinants. Interestingly, one of the identified candidates corresponded to an experimentally validated strong B- and T-cell antigen (AgD) from our laboratory culture-based studies. Conclusion: This first report on dominant proteins, including putative antigens of M. immunogenum prevalent in field in-use MWF, is a significant step towards the overall goal of developing fluid monitoring for exposure and disease risk assessment for HP development in machining environments.