Many bacterial pathogens deploy specialized secretion systems to deliver virulence factors to eukaryotic host
cells or the environment. Type IX secretion system (T9SS) of Porphyromonas gingivalis, a periodontal
pathogen, mediates secretion of extracellular and cell-surface-bound substrates including gingipain proteases.
Our knowledge of T9SS structures has largely derived from X-ray crystallography and single-particle electron
microscopy approaches, which by definition is limited to solving structures of subcomplexes amenable to
isolation and purification. However, revolutionary advances in in situ cryo-electron tomography (Cryo-ET) are
now enabling visualization of T9SS in the native context of the bacterial cell envelope. Drs. Hu and Lamont
have been at the forefront of this work, and we have now visualized the intact T9SS of P. gingivalis in situ.
The T9SS is unique, presenting not as a single functional entity, as do all other known bacterial and eukaryotic
protein transport systems, but rather as a composite of seemingly independent translocation motors and Sov
translocons, arranged symmetrically as distinct rings in the cytoplasm, periplasm, and cell surface.
The long-term objective of our work is to comprehensively define structure & function of the T9SS in
the periodontal pathogen P. gingivalis. In this application, we will continue to exploit in situ Cryo-ET
approaches to solve structures of T9SS machines from wild-type and mutant strains, and we will integrate
complementary biochemical and other electron and fluorescence microscopy approaches to add depth to the
Cryo-ET studies and to evaluate the spatial organization of subunits and function of T9SS. In Aim 1, we will
refine the in situ structures at high resolution (10-15 Å) and integrate analyses of mutant machines to map
individual machine subunits. We will seek to determine the PorK/N complex structure at near-atomic
resolution by Cryo-EM. We will also determine the stoichiometry of the Sov translocon by fluorescence
microscopy. Aim 2 will complete ongoing work aimed at identifying structural changes accompanying
activation of the T9SS upon sensing of PMF. We will inhibit energy transduction via mutagenesis of key
residues of the molecular motor PorM/L to detect any structural changes within the T9SS, and define the
interaction domains between PorM and PorKN, which will shed light on the mechanism of energy transduction
from PorM to the PorKN complex. We anticipate that our studies will provide a structural basis for T9SS
mediated translocation of virulence factors and a foundation for development of intervention strategies to
suppress P. gingivalis virulence.