Biofilm formation by Gallibacterium anatis depends on TolC-mediated initial attachment of cells

Gallibacterium anatis (G. anatis) is an important pathogen for poultry, mainly causing a decrease of egg production rate in laying hens and an increase in the mortality rate in broilers. TolC, an outer membrane channel protein, has been implicated in the formation of biofilms by various pathogenic bacteria. This study aimed to assess the role of TolC in mediating G. anatis biofilm formation by utilizing a ΔtolC mutant strain. Key findings revealed that TolC deletion reduced surface hydrophobicity and decreased biofilm biomass. Additionally, the mutant strain showed impaired secretion of extracellular polysaccharides and diminished autoaggregation capacity. Using both enzymatic treatments and confocal microscopy, biofilm composition and architecture were characterized. Compared against the wild-type (WT) strain，the ΔtolC mutant biofilm showed an increased relative content of DNA and protein, a significantly reduced polysaccharide content, and a higher proportion of dead bacteria during the early stages of biofilm development. The effect of tolC deletion on biofilm-associated gene expression were quantitatively analyzed using RT-qPCR, revealing altered expression of these genes at different stages of ΔtolC biofilm formation. Collectively, these findings preliminarily demonstrate that TolC is essential for G. anatis biofilm formation, particularly in early cell attachment. TolC positively regulates biofilm formation through multiple mechanisms, including the secretion of polysaccharides, quorum sensing, and two-component signaling systems. These insights provide a foundation for further exploration of TolC's role in G. anatis biofilm formation.