A protein secretion system linked to bacteroidete gliding motility and pathogenesis

Abstract

Porphyromonas gingivalis secretes strong proteases called gingipains that are implicated in periodontal pathogenesis. Protein secretion systems common to other Gram-negative bacteria are lacking in P. gingivalis, but several proteins, including PorT, have been linked to gingipain secretion. Comparative genome analysis and genetic experiments revealed 11 additional proteins involved in gingipain secretion. Six of these (PorK, PorL, PorM, PorN, PorW, and Sov) were similar in sequence to Flavobacterium johnsoniae gliding motility proteins, and two others (PorX and PorY) were putative two-component system regulatory proteins. Real-time RT-PCR analysis revealed that porK, porL, porM, porN, porP, porT, and sov were down-regulated in P. gingivalis porX and porY mutants. Disruption of the F. johnsoniae porT ortholog resulted in defects in motility, chitinase secretion, and translocation of a gliding motility protein, SprB adhesin, to the cell surface, providing a link between a unique protein translocation system and a motility apparatus in members of the Bacteroidetes phylum.

Fig. 1.

Kgp and Rgp proteins and activities in wild-type and mutant P. gingivalis. (A) Rgp and Kgp activities in intact cells and in culture supernatants. P. gingivalis cells were grown anaerobically in enriched brain heart infusion medium at 37 °C for 24 h. All cultures had similar cell densities of OD₆₀₀ of ∼1.0. Kgp and Rgp activities were measured at units per milliliter of cell suspensions or culture supernatants and are indicated as percent of activity relative to that of the wild type. (B) Accumulation of gingipain proproteins with high molecular masses in mutant cells. P. gingivalis cell lysates were subjected to SDS/PAGE and immunoblot analysis with anti-Kgp and anti-Hgp44. (C) Plasmid-mediated complementation. Rgp and Kgp activities in intact cells and in culture supernatants were measured for wild-type, mutant, and complemented cells.

Fig. 2.

Subcellular location of PorP, PorK, PorL, PorM, and PorN proteins and formation of a large complex. (A) Subcellular location of the Por proteins. The cell lysates of P. gingivalis strains were subjected to detergent fractionation followed by SDS/PAGE and immunoblot analysis with anti-PorK antibody for the wild-type strain, and anti-Myc antibody for the porP/porP’-‘myc, porL/porL’-‘myc, porM/porM’-‘myc, and porN/porN’-‘myc strains. As a control, a major outer membrane protein, RagA, which was identified by mass spectrometry, is indicated by an asterisk (*). CBB, Coomassie Brilliant Blue staining. (B) Detection of protein complexes by Blue Native PAGE. Cell lysates of P. gingivalis strains were subjected to 3–12% gradient Blue Native PAGE and immunoblot analysis with anti-Myc antibody and anti-PorK antibody.

Fig. 3.

Response regulator PorX- and histidine kinase PorY-mediated regulation of the expression of porT, sov, porP, porK, porL, porM, and porN. (A) Real-time RT-PCR analysis of gene expression of porP, porK, porL, porM, porN, porT, and sov in the wild-type, porX, and porX porX⁺ (complemented) strains. (B) Real-time RT-PCR analysis of gene expression of porP, porK, porL, porM, porN, porT, sov, and porX in the wild-type, porY, and porY porY⁺ (complemented) strains.

Fig. 4.

Effect of disruption of F. johnsoniae sprT on gliding motility, surface localization of SprB protein, and extracellular chitinase activity. (A) Colony morphology of wild-type, mutant, and complemented strains. Cells were incubated on PY2 agar at 25 °C for 45 h. (Scale bar: 0.5 mm.) (B) Detection of surface localized SprB protein by immunofluorescence microscopy. Cells of wild-type and mutant F. johnsoniae were exposed to DAPI and to anti-SprB antibodies followed by secondary antibodies conjugated to Alexa 488. Arrows indicate InSpeck relative-intensity fluorescence beads. (Scale bar: 10 μm.) (C) Chitinase activities of intact cells and culture supernatants of F. johnsoniae strains with three synthetic substrates for chitinase, 4-MU-GlcNAc, 4-MU-(GlcNAc)₂, and 4-MU-(GlcNAc)₃.