Nitrate-Reducing Commensals Reshape Oral Biofilm Ecology and Reveal Hcp as a Critical Determinant of Porphyromonas gingivalis Persistence

Dietary nitrate (NO₃⁻) supplementation is emerging as a promising strategy for suppressing oral pathobionts through microbial generation of reactive nitrogen species (RNS), including nitrite (NO₂⁻) and nitric oxide (NO). However, the mechanisms that enable periodontal pathogens to survive nitrate-derived nitrosative stress within polymicrobial communities remain poorly understood. Previously, we identified the hybrid cluster protein (Hcp) as a major nitrosative stress defense factor in Porphyromonas gingivalis demonstrating ∼170-fold induction of hcp expression following nitrite exposure and as a requirement for survival at physiologically relevant nitrite concentrations. Here we investigated the role of Hcp in promoting P. gingivalis persistence within nitrate-reducing biofilms. Using human ex vivo plaque biofilms, we found that Hcp is essential for P. gingivalis survival under both basal and nitrate-supplemented conditions. In a defined nine-species biofilm model, nitrate reduction suppressed wild-type P. gingivalis , whereas deletion of hcp ( Δhcp ) resulted in complete population clearance. Metatranscriptomics revealed that nitrate-induced hcp expression was not restricted to P. gingivalis but was part of a coordinated nitrosative stress response shared among oral anaerobes, including Prevotella intermedia , Fusobacterium nucleatum , and Veillonella atypica . Moreover, nitrate reduction disrupted a previously synergistic interaction between Veillonella spp. and P. gingivalis , converting a supportive relationship into an inhibitory microenvironment that constrained pathogen survival. Collectively, these findings identify Hcp-mediated nitrosative stress resistance as a major determinant of fitness within nitrate-reducing biofilms and reveal RNS as key ecological force shaping interactions between commensal nitrate reducers and periodontal pathogens. These results provide a mechanistic framework linking dietary nitrate metabolism to oral microbiome homeostasis.