The Gram-negative oral pathogen Porphyromonas gingivalis utilises the Type IX Secretion System (T9SS) to export surface-associated proteins across the outer membrane (OM). The abundant P. gingivalis virulence factors and T9SS substrates, the gingipains, were proposed to form the electron-dense surface layer (EDSL) surrounding the cell, observed via cryo-electron microscopy (cryo-EM). Recent cryo-EM of a mutant lacking all gingipains showed the presence of an EDSL of low intensity, which suggested that the EDSL is composed of material in addition to the gingipains, likely the other T9SS substrates. An lptO- mutant, deficient in a component of the T9SS, lacks the surface-associated T9SS substrates and the EDSL.
PG1058 is also considered a component of the T9SS. In this study, cryo-EM of a pg1058- mutant indicated that it too lacks the EDSL. Immunoblot and mass spectrometry analysis identified accumulation of T9SS substrates within the mutant periplasm indicating perturbed T9SS function. Furthermore, whole-cell ELISA and immunogold transmission EM of the mutant indicated that the T9SS substrate, the Kgp gingipain, was absent from the cell-surface.
The influence of the T9SS on interactions of P. gingivalis with the human host were assessed in-vitro, by comparing wild-type to the pg1058- and lptO- T9SS mutants. Susceptibility to macrophage phagocytosis, ability to bind to oral epithelial cells, ability to haemagglutinate red blood cells and ability to co-aggregate with oral pathogen Treponema denticola were in the order wild-type>pg1058->lptO-. This suggests that there are differences in the surface-associated molecules of the wild-type, pg1058- and lptO- T9SS mutants which can influence human host interactions and thereby the virulence of P. gingivalis.