Polybacterial intracellular coinfection of epithelial stem cells in periodontitis

Periodontitis affects billions of people worldwide. To address interkingdom relationships of microbes and niche on periodontitis, we generated the first sin-gle-cell meta-atlas of human periodontium (34-sample, 105918-cell), harmo-nizing 32 annotations across 4 studies[1][1]–[4][2]. Highly multiplexed immunofluores-cence (32-antibody; 113910-cell) revealed spatial innate and adaptive immune foci segregation around tooth-adjacent epithelial cells. Sulcular and junctional keratinocytes (SK/JKs) within epithelia skewed toward proinflammatory phe-notypes; diseased JK stem/progenitors displayed altered differentiation states and chemotactic cytokines for innate immune cells. Single-cell metagenomics utilizing unmapped reads revealed 37 bacterial species. 16S and rRNA probes detected polybacterial intracellular pathogenesis (“co-infection”) of 4 species within single cells for the first time in vivo. Challenging coinfected primary human SK/JKs with lipopolysaccharide revealed solitary and synergistic ef-fects. Coinfected single-cell analysis independently displayed proinflammatory phenotypes in situ. Here, we demonstrate the first evidence of polybacterial intracellular pathogenesis in human tissues and cells—potentially influencing chronic diseases at distant sites. ### Competing Interest Statement The authors had access to the study data and reviewed and approved the final manuscript. Although the authors view each of these as noncompeting financial interests, KMB, QTE, BFM, DP, TW, AH, BMW, IS, JL, and SAT are all active members of the Human Cell Atlas; furthermore, KMB is a scientific advisor at Arcato Laboratories and Orange Grove Bio; IS is a consultant for LOreal Research and Innovation; and SAT has consulted for Roche and Genentech and is a scientific advisor for Biogen, GlaxoSmithKline, and Foresite Labs. All other authors declare no competing interests. [1]: #ref-1 [2]: #ref-4