Capnocytophaga ochracea sialidase drives its biofilm maturation and host cell interactions.

INTRODUCTION

Capnocytophaga ochracea, an early colonizer of oral biofilms associated with gingivitis, plays an incompletely understood role in periodontal pathogenesis. While sialidases are established virulence factors in late-colonizing 'red-complex' pathogens, their function in early colonizers, such as C. ochracea, remains unknown.

METHODS

To address this knowledge gap, we identified and characterized Co-NanH, the sole sialidase of C. ochracea. We performed phylogenetic and structural analyses to compare it with known sialidases. Enzymatic assays were conducted using recombinant Co-NanH to determine its pH optimum, substrate specificity, and kinetic parameters. Furthermore, we utilized sialidase inhibitors (such as DANA) and generated a nanH genetic deletion mutant (ΔnanH) to assess the enzyme's role in planktonic growth, biofilm formation, and interactions with human gingival epithelial cells (adhesion and internalization).

RESULTS

Phylogenetic and structural analyses revealed that Co-NanH shares significant homology with a sialidase from Tannerella forsythia but possesses a distinct Sec/SPI signal peptide and a monomeric structure. Recombinant Co-NanH exhibited optimal activity at pH 5.5 and cleaved both α2,3- and α2,6-linked sialic acids, with kinetic parameters comparable to those of red-complex pathogen sialidases. Inhibitors like DANA potently suppressed its activity and reduced C. ochracea biofilm formation. Crucially, genetic deletion of nanH abolished sialidase activity without affecting planktonic growth. The ΔnanH mutant exhibited severely impaired biofilm formation, characterized by reduced biomass, thickness, and initial attachment. Furthermore, the mutant showed significantly reduced adhesion to and internalization by human gingival epithelial cells compared with wild-type and complemented strains.

DISCUSSION: These findings establish Co-NanH as a key mediator of C. ochracea in biofilm maturation and host cell interactions, revealing a functional parallel between early colonizers and classical periodontal pathogens. This work provides functional evidence that sialidase activity in an early colonizer contributes to oral dysbiosis by facilitating biofilm development and priming the host environment for disease progression.