PorKN Rings are Cogs of the Type IX Secretion Nanomachine

Objectives: The type IX secretion system (T9SS) is responsible for exporting proteins across the outer membrane (OM) of bacteria in the Bacteroidetes phylum and is essential for the pathogenicity of oral pathogens associated with severe periodontal disease. It is organized into a series of functional modules/sub-complexes that operate in a co-ordinated manner. The PorKN rings are essential for the function of the T9SS, but their precise role and the details of their assembly remain unclear. Additionally, the proteins that comprise the Attachment Complex have been identified, but the precise location of this complex in relation to the other T9SS components remains unknown.
Methods: Cryo-electron single particle, biochemical, molecular biology and mass spectrometry
Results: Here, we present a ~3.5Å cryo-electron microscopy structure of the PorKN rings which reveals that PorK multimers form a rim on the outer edge with a large, flat surface oriented towards the OM, while PorN folds to create cog-like projections on the periplasmic side providing functional insights into the rotation of the T9SS. The PorKN rings are known to associate with the PorG OM β-barrel, however, this component was not resolved in the cryo-EM structure. We show that the C-terminal extension of PorG lies within a shallow groove on the OM face of PorK and is stabilized by a disulfide bond. This disulfide bond was found to be essential for the function of the T9SS but not for the formation of the PorKN rings. We also show that the Attachment Complexes associate with the PorKN rings and are very likely located above the rings.
Conclusions: Based on these results, we propose that the PorKN rings are cogs of the T9SS nanomachine that have a rotational movement which translates to PorG and Attachment Complexes providing the energy for secretion and dispersion of virulence factors across the OM.