Shotgun Metagenomic Analysis of Subgingival Biofilms in Rheumatoid Arthritis Patients

Objectives: It is believed that the periodontitis-associated microbiome may be involved in the induction of pathogenic autoimmune responses linked with the development of rheumatoid arthritis (RA). Porphyromonas gingivalis produces a peptidyl arginine deiminase (PPAD) enzyme capable of citrullinating proteins, and is considered a key organism in inducing the production of antibodies against these citrullinated proteins systemically. The aim of this study was to use a shotgun metagenomic approach to observe the microbial profiles and functional capability of subgingival biofilms in patients with and without periodontitis, with and without RA, and in those with autoantibodies against citrullinated peptides (CCP+) and at risk of developing RA.
Methods: CCP+ patients with no evidence of synovitis (n=48), healthy controls (HC, n=32) and RA patients (n=36, of which 26 were new onset RA) were recruited. Subgingival plaque was collected from healthy and deep pocket sites using paper points. DNA was isolated, sheared and libraries prepared for paired-end DNA sequencing on the Illumina HiSeq3000 machine. Fastq files were quality trimmed, reads de novo assembled, and aligned to the non-redundant (NCBI) protein database using blastx in Diamond, which outputs were analysed using megan and DESeq2.
Results: Taxonomic profiling indicated that the most abundant phyla in all groups of patients were Actinobacteria (34%), Bacteroidetes (21%), Firmicutes (19%), Proteobacteria (11%), Fusobacteria (7%) and Spirochaetes (6%). The relative abundance of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans were high in the samples from the CCP+ group. Members of the Prevotella genus harbour genes annotated as PPAD-like.
Conclusions: Shotgun metagenomic analyses of the subgingival biofilms provided a detailed characterisation of the microbiota in a large group of HC, CCP+ and RA patients. Several members of the microbiota (including A. actinomycetemcomitans, Porphyromonas and Prevotella spp.) harbour genes that may be involved in citrullination, and therefore in the link between the oral microbiota and RA.