Single-cell RNA sequencing using split-pool barcoding reveals transcriptional heterogeneity in Porphyromonas gingivalis with implications for periodontal pathogenesis

ABSTRACT Background Porphyromonas gingivalis is a keystone pathogen in periodontitis, associated with dysbiosis and chronic inflammation. While its virulence mechanisms are well characterized, its transcriptional heterogeneity at the single-cell level remains unexplored. Materials and Methods We applied split-pool barcoding-based single-cell RNA sequencing to profile gene expression in 1,942 individual P. gingivalis W83 cells cultured under anaerobic conditions. Clustering and differential expression analyses were conducted to identify distinct transcriptional subpopulations. Results We identified six transcriptionally distinct clusters, with the two largest accounting for 72.7% of the population. Minor clusters exhibited signatures related to stress responses, metabolism, membrane transport, and DNA regulation. Sub-clustering of major populations revealed rare subgroups, including one enriched for genes involved in iron acquisition, proteolysis, and transport. Conclusions This study presents the first single-cell transcriptomic map of P. gingivalis, revealing rare but functionally significant subpopulations. Such diversity may support bacterial adaptability, virulence, and immune evasion, informing future strategies for targeted periodontal therapy.