Background: Anti-citrullinated protein antibodies (ACPA) are a hallmark of rheumatoid arthritis (RA) and specifically recognize citrulline, a post-translational modification formed by protein-arginine deiminase (PAD) enzymes. PAD enzymes may originate either from human or microbial sources. Currently, Porphyromonas gingivalis is the only bacterium identified in humans that expresses a PAD enzyme. Despite the believed importance of mucosal surfaces in RA, it remains unknown whether additional bacterial species possess PAD activity and contribute to the formation of citrullinated antigens that form the starting point for autoantibody responses in RA.

Objectives: We sought to determine whether other bacteria present in humans harbor active PAD enzymes and can generate citrullinated proteins which are recognized by ACPA.

Methods: Utilizing a bio-informatics approach, protein sequences from the NCBI database matching the PAD from P. gingivalis (PPAD) were identified. The bacterium with the highest homology to PPAD and known to be present in humans was subsequently cultured. Utilizing western blot and tandem mass spectrometry (MS/MS) we investigated the expression and activity of the PAD enzyme, the presence of citrullination and the recognition by ACPA.

Results: A comprehensive bio-informatics approach identified homologs of PPAD in at least 5,000 bacterial species. Among human-associated bacteria, Clostridioides difficile showed the highest sequence similarity to PPAD. However, culturing C. difficile did not result in detectable citrullinating PAD activity. In contrast, expression of the C. difficile PAD gene in Escherichia coli led to evidence for protein citrullination. Despite this enzymatic activity, bacterial lysates were not recognized by ACPA.

Conclusions: Unexpectedly, several human-associated bacteria were found to harbor PAD enzymes, including C. difficile . Although it remains unclear under which conditions these enzymes are expressed and whether they generate citrullinated proteins recognized by ACPA, our findings suggest that microorganisms beyond P. gingivalis could play a role in the formation of autoantigenic targets relevant to autoimmunity.

REFERENCES: NIL.

Acknowledgments: NIL.

Disclosure of Interests: None declared.