Interaction of Giant Unilamellar Vesicles with the Surface Nanostructures on Dragonfly Wings.

The waxy epicuticle of dragonfly wings contains a unique nanostructured pattern that exhibits bactericidal properties. In light of emerging concerns of antibiotic resistance, these mechano-bactericidal surfaces represent a particularly novel solution by which bacterial colonization and the formation of biofilms on biomedical devices can be prevented. Pathogenic bacterial biofilms on medical implant surfaces cause a significant number of human deaths every year. The proposed mechanism of bactericidal activity is through mechanical cell rupture; however, this is not yet well understood and has not been well characterized. In this study, we used giant unilamellar vesicles (GUVs) as a simplified cell membrane model to investigate the nature of their interaction with the surface of the wings of two dragonfly species, Austrothemis nigrescens and Trithemis annulata, sourced from Victoria, Australia, and the Baix Ebre and Terra Alta regions of Catalonia, Spain. Confocal laser scanning microscopy and cryo-scanning electron microscopy techniques were used to visualize the interactions between the GUVs and the wing surfaces. When exposed to both natural and gold-coated wing surfaces, the GUVs were adsorbed on the surface, exhibiting significant deformation, in the process of membrane rupture. Differences between the tensile rupture limit of GUVs composed of 1,2-dioleoyl- sn-glycero-3-phosphocholine and the isotropic tension generated from the internal osmotic pressure were used to indirectly determine the membrane tensions, generated by the nanostructures present on the wing surfaces. These were estimated as being in excess of 6.8 mN m-1, the first experimental estimate of such mechano-bactericidal surfaces. This simple model provides a convenient bottom-up approach toward understanding and characterizing the bactericidal properties of nanostructured surfaces.

Structural Insights into the PorK and PorN Components of the Porphyromonas gingivalis Type IX Secretion System.

The type IX secretion system (T9SS) has been recently discovered and is specific to Bacteroidetes species. Porphyromonas gingivalis, a keystone pathogen for periodontitis, utilizes the T9SS to transport many proteins including the gingipain virulence factors across the outer membrane and attach them to the cell surface via a sortase-like mechanism. At least 11 proteins have been identified as components of the T9SS including PorK, PorL, PorM, PorN and PorP, however the precise roles of most of these proteins have not been elucidated and the structural organization of these components is unknown. In this study, we purified PorK and PorN complexes from P. gingivalis and using electron microscopy we have shown that PorN and the PorK lipoprotein interact to form a 50 nm diameter ring-shaped structure containing approximately 32-36 subunits of each protein. The formation of these rings was dependent on both PorK and PorN, but was independent of PorL, PorM and PorP. PorL and PorM were found to form a separate stable complex. PorK and PorN were protected from proteinase K cleavage when present in undisrupted cells, but were rapidly degraded when the cells were lysed, which together with bioinformatic analyses suggests that these proteins are exposed in the periplasm and anchored to the outer membrane via the PorK lipid. Chemical cross-linking and mass spectrometry analyses confirmed the interaction between PorK and PorN and further revealed that they interact with the PG0189 outer membrane protein. Furthermore, we established that PorN was required for the stable expression of PorK, PorL and PorM. Collectively, these results suggest that the ring-shaped PorK/N complex may form part of the secretion channel of the T9SS. This is the first report showing the structural organization of any T9SS component.

Structural Characterization of the Type IX Secretion System in Porphyromonas gingivalis.

The type IX secretion system (T9SS) is the most recently discovered secretion system in the gram-negative bacteria and is specific to the Bacteroidetes phylum. It is comprised of at least 19 proteins, which together allows for the secretion and cell surface attachment of a specific group of proteins (T9SS substrates), that harbor a signal sequence at the C-terminus. Here we describe the structural characterization of the PorK, PorN and PorG components of the Porphyromonas gingivalis T9SS using electron microscopy and cross-linking mass spectrometry.