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. 2024 May 10;25(10):5192.
doi: 10.3390/ijms25105192.

Protein Citrullination by Peptidyl Arginine Deiminase/Arginine Deiminase Homologs in Members of the Human Microbiota and Its Recognition by Anti-Citrullinated Protein Antibodies

María-Elena Pérez-Pérez  1   2 , Enrique Nieto-Torres  3 , Juan-José Bollain-Y-Goytia  1   2 , Lucía Delgadillo-Ruíz  1
Affiliations

Protein Citrullination by Peptidyl Arginine Deiminase/Arginine Deiminase Homologs in Members of the Human Microbiota and Its Recognition by Anti-Citrullinated Protein Antibodies

María-Elena Pérez-Pérez et al. Int J Mol Sci. .

Abstract

The human microbiome exists throughout the body, and it is essential for maintaining various physiological processes, including immunity, and dysbiotic events, which are associated with autoimmunity. Peptidylarginine deiminase (PAD) enzymes can citrullinate self-proteins related to rheumatoid arthritis (RA) that induce the production of anti-citrullinated protein antibodies (ACPAs) and lead to inflammation and joint damage. The present investigation was carried out to demonstrate the expression of homologs of PADs or arginine deiminases (ADs) and citrullinated proteins in members of the human microbiota. To achieve the objective, we used 17 microbial strains and specific polyclonal antibodies (pAbs) of the synthetic peptide derived from residues 100-200 of human PAD2 (anti-PAD2 pAb), and the recombinant fragment of amino acids 326 and 611 of human PAD4 (anti-PAD4 pAb), a human anti-citrulline pAb, and affinity ACPAs of an RA patient. Western blot (WB), enzyme-linked immunosorbent assay (ELISA), elution, and a test with Griess reagent were used. This is a cross-sectional case-control study on patients diagnosed with RA and control subjects. Inferential statistics were applied using the non-parametric Kruskal-Wallis test and Mann-Whitney U test generated in the SPSS program. Some members of phyla Firmicutes and Proteobacteria harbor homologs of PADs/ADs and citrullinated antigens that are reactive to the ACPAs of RA patients. Microbial citrullinome and homolog enzymes of PADs/ADs are extensive in the human microbiome and are involved in the production of ACPAs. Our findings suggest a molecular link between microorganisms of a dysbiotic microbiota and RA pathogenesis.

Keywords: ACPAs; PADs; autoimmunity; citrullinome; microbiome; microbiota; rheumatoid arthritis.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Homologs of PAD/AD present in microbial extracts. The expression of homolog enzymes of PAD/AD shown in species belonging to the phyla Proteobacteria, Firmicutes, and Ascomycota. In the lower part of each blot, there are bands recognized by anti-HSP70 mAb that were used as the constitutive protein control of each microbial extract. Lines correspond to 1. P. mirabilis, 2. P. vulgaris, 3. E. coli, 4. K. pneumoniae, 5. K. oxytoca, 6. C. freundii, 7. M. morgannii, 8. P. aeruginosa, 9. A. baumannii, 10. Lactobacillus sp., 11. E. faecalis, 12. S. epidermidis, 13. S. aureus, 14. C. albicans, 15. C. glabrata, 16. C. tropicalis, and 17. S. cerevisiae.
Figure 2
Figure 2
Citrullinated antigens are present in microbial extracts. Expression of citrullinated antigens is shown by commercial anti-citrulline pAb. In the lower part of the panel is the constitutive protein HSP70 expressed in each microbial extract. The lines correspond to proteins from different species: 1. P. mirabilis, 2. P. vulgaris, 3. E. coli, 4. K. pneumoniae, 5. K. oxytoca, 6. C. freundii, 7. M. morgannii, 8. P. aeruginosa, 9. A. baumannii, 10. Lactobacillus sp, 11. E. faecalis, 12. S. epidermidis, 13. S. aureus, 14. C. albicans, 15. C. glabrata, 16. C. tropicalis, and 17. S. cerevisiae.
Figure 3
Figure 3
Expression of homologs of PAD2/PAD4 and citrullinated proteins in the phyla Proteobacteria, Firmicutes, and Ascomycota. A value of p ≤ 0.05 is considered statistically significant according to the Kruskal–Wallis test. Px = pixels. After normalization with the constitutive protein, the statistical analysis allowed us to differentiate the phyla with positive and negative values.
Figure 4
Figure 4
Comparative expression of homologs of PAD2, PAD4, and citrullinated antigens in microbial extracts. The stacked bar graph (A) represents the percentage of expression by species, while (B) shows the mean pixel intensity of the bands reactive to anti-PAD2 pAb, anti-PAD4 pAb, and anti-citrulline pAb, with values of p ≤ 0.05 that are considered significant according to the Kruskal–Wallis test. Px = pixels. The statistical analysis allowed us to represent only the species that had positive expression values after normalization with the constitutive protein HSP70.
Figure 5
Figure 5
Differences in reactivity to microbial extracts of 17 commensals between RA sera and controls. A representative panel of WB shows the immunological recognition of IgG in RA sera and lower reactivity in control sera. The reactivity to nonspecific bands that may only correspond to anti-microbial antibodies. The lines correspond to proteins from different species: 1. P. mirabilis, 2. P. vulgaris, 3. E. coli, 4. K. pneumoniae, 5. K. oxytoca, 6. C. freundii, 7. M. morgannii, 8. P. aeruginosa, 9. A. baumannii, 10. Lactobacillus sp., 11. E. faecalis, 12. S. epidermidis, 13. S. aureus, 14. C. albicans, 15. C. glabrata, 16. C. tropicalis, and 17. S. cerevisiae.
Figure 6
Figure 6
Isotype of immunoglobulins reactive to microbial extracts in RA and control sera. Note that the IgG isotype is mainly involved in the reactivity of microbial proteins, and the difference between the two groups is significant (p = 0.0007), while the reactivity of the IgA isotype is (p = 0.3023) according to the Mann–Whitney test. *** highly significant.
Figure 7
Figure 7
Coincident reactivity between eluted ACPAs against anti-citrulline pAb. The expression of citrullinated antigens is shown by reactivity with commercial anti-citrulline pAb and affinity ACPAs of RA patients. The lines correspond to proteins from different species: 1. P. mirabilis, 2. P. vulgaris, 3. E. coli, 4. K. pneumoniae, 5. K. oxytoca, 6. C. freundii, 7. M. morgannii, 8. P. aeruginosa, 9. A. baumannii, 10. Lactobacillus sp., 11. E. faecalis, 12. S. epidermidis, 13. S. aureus, 14. C. albicans, 15. C. glabrata, 16. C. tropicalis, and 17. S. cerevisiae.
Figure 8
Figure 8
Determination of citrullination activity in microbial cultures. The percentage of species within each microbial phylum that presented citrullination activity is shown, as well as the amount of L-citrulline produced by the synthesis of nitric oxide (NO) represented as intensity (pinkish-reddish color). Griess reagent (RG), Mueller–Hinton agar (MH), and MRS agar. Firmicutes: 1. Lactobacillus, 2. E. faecalis, 3. S. epidermidis, and 4. S. aureus; Ascomycota: 5. C. albicans, 6. C. glabrata, 7. C. tropicalis, and 8. S. cerevisiae; and Proteobacteria: 9. P. mirabilis, 10. P. vulgaris, 11. E. coli, 12. K. pneumoniae, 13. K. oxytoca (ND), 14. C. freundii, 15. M. morgannii, 16. P. aeruginosa (ND), and 17. A. baumannii. ND = Not determined.
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References

    1. Lederberg J., McCray A.T. ‘Ome sweet’Omics–A genealogical treasury of words. Scientist. 2001;15:7.
    1. Dekaboruah E., Vasant-Suryavanshi M., Chettri D., Kumar-Verma A. Human microbiome: An academic update on human body site specific surveillance and its possible role. Arch. Microbiol. 2020;202:2147–2167. doi: 10.1007/s00203-020-01931-x. - DOI - PMC - PubMed
    1. Sender R., Fuchs S., Milo R. Are we really vastly outnumbered? Revisiting the ratio of bacterial to host cells in humans. Cell. 2016;164:337–340. doi: 10.1016/j.cell.2016.01.013. - DOI - PubMed
    1. Kalbermatter C., Fernandez-Trigo N., Christensen S.C. Maternal microbiota, early life colonization and breast milk drive immune development in the newborn. Front. Immunol. 2021;12:683022. doi: 10.3389/fimmu.2021.683022. - DOI - PMC - PubMed
    1. Rooney C.M., Mankia K., Emery P. The role of the microbiome in driving RA-related autoimmunity. Front. Cell Dev. Biol. 2020;8:538130. doi: 10.3389/fcell.2020.538130. - DOI - PMC - PubMed

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