Abstract
Porphyromonas gingivalis, the main etiologic agent of periodontitis, secretes cysteine proteases named gingipains. HRgpA and RgpB gingipains have Arg-specificity, while Kgp gingipain is Lys-specific. Together they can cleave an array of proteins and importantly contribute to the development of periodontitis. In this study we focused on gingipain-exerted proteolysis at the cell surface of human gingival epithelial cells [telomerase immortalized gingival keratinocytes (TIGK)] in order to better understand the molecular mechanisms behind tissue destruction in periodontitis. Using mass spectrometry, we investigated the whole sheddome/degradome of TIGK cell surface proteins by P. gingivalis strains differing in gingipain expression and by purified gingipains, and performed the first global proteomic analysis of gignpain proteolysis at the membrane. Incubation of TIGK cells with P. gingivalis resulted in massive degradation of proteins already at low multiplicity of infection, whereas incubating cells with purified gingipains resulted in more discrete patterns, indicative of a combination of complete degradation and shedding of membrane proteins. Most of the identified gingipain substrates were molecules involved in adhesion, suggesting that gingipains may cause tissue damage through cleavage of cell contacts, resulting in cell detachment and rounding, and consequently leading to anoikis. However, HRgpA and RgpB gingipains differ in their mechanism of action. While RgpB rapidly degraded the proteins, HRgpA exhibited a much slower proteolysis indicative of ectodomain shedding, as demonstrated for the transferrin receptor protein 1 (TFRC). These results reveal a molecular underpinning to P. gingivalis-induced tissue destruction and enhance our knowledge of the role of P. gingivalis proteases in the pathobiology of periodontitis. Proteomics data are available via ProteomeXchange with identifier PXD015679.
Highlights
Porphyromonas gingivalis is a gram-negative anaerobe, and the main causative agent of a chronic oral inflammatory disease – periodontitis (Lamont and Jenkinson, 1998)
To identify extracellular substrates of gingipains, we first performed a comparative evaluation of proteolysis of gingipain substrates on the surface of telomerase immortalized gingival keratinocytes (TIGK) cells, using P. gingivalis strains expressing: all gingipains, RgpAB, Kgp, or no gingipains
After the treatment of TIGK cells with P. gingivalis strains at two bacteria to cell ratios (MOI 100 and MOI 500), conditioned media were collected and analyzed by SDS-PAGE using a Coomassie Blue stain which showed that the concentration of proteins in the samples was very low at MOI 500 (Figure 1A) indicative of massive protein degradation
Summary
Porphyromonas gingivalis is a gram-negative anaerobe, and the main causative agent of a chronic oral inflammatory disease – periodontitis (Lamont and Jenkinson, 1998). Kgp contains HA domains that are capable of interacting with the related adhesion domains of HRgpA and both gingipains can form a very potent proteolytic HRgpA-Kgp complex with two active sites and specificity for both Arg-Xaa and Lys-Xaa peptide bonds (Bhogal et al, 1997; Takii et al, 2005). With their specific and very potent proteolytic activities, gingipains are important for bacterial survival at inflamed sites of periodontal pockets, and for the pathological outcome of the disease. Gingipains can degrade specific host cell-surface proteins, which can result in imbalanced signaling, cell detachment and anoikis, a form of cell death due to loss of intercellular connections (reviewed in Kuramitsu, 1998; Chiarugi and Giannoni, 2008; Fitzpatrick et al, 2009; Guo et al, 2010)
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