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

Area of Science:

• Occupational health and safety
• Immunology and infectious diseases
• Proteomics and bioinformatics

Background:

• Occupational exposure to Metalworking Fluid (MWF) colonized by *Mycobacterium immunogenum* (MI) is linked to hypersensitivity pneumonitis (HP) in machinists.
• Early detection of MI proteins, particularly antigenic ones, in MWF is crucial for monitoring and preventing HP.

Purpose of the Study:

• To identify dominant MI proteins and antigens directly from field-collected, in-use MWF.
• To develop an integrated immunoproteomic-immunoinformatic approach for detecting MI components in MWF.

Main Methods:

• DNA-based screening identified MI-positive MWF, followed by strain isolation and culture.
• Integrated immunoproteomics (1D/2D gel electrophoresis, LC-MS/MS) and immunoinformatics were used to profile cell-free MI proteins and antigens.
• Seroreactivity with anti-MI sera (rabbit and patient) guided protein identification.

Main Results:

• A novel MI strain (MJY-27) was identified.
• 86 MI proteins were detected, with 29 showing significant peptide abundance.
• T-cell epitope analysis identified 13 proteins with high binding frequency for HLA-I & II alleles, and antigenicity analysis revealed 6 to 37 antigenic determinants per protein. One candidate was a validated strong B- and T-cell antigen.

Conclusions:

• This study reports, for the first time, dominant proteins and putative antigens of *M. immunogenum* in field in-use MWF.
• This work represents a significant advancement towards developing MWF monitoring strategies for exposure and HP risk assessment in machining environments.