Cleavage of protease-activated receptors on an immortalized oral epithelial cell line by Porphyromonas gingivalis gingipains

Rodrigo A. Giacaman, Anil C. Asrani, Karen F Ross, Mark C Herzberg

Research output: Contribution to journalArticle

33 Citations (Scopus)

Abstract

Porphyromonas gingivalis activates protease-activated receptors (PARs) on oral keratinocytes, resulting in downstream signalling for an innate immune response. Activation depends on P. gingivalis gingipains, but could be confounded by lipopolysaccharide signalling through Toll-like receptors. We therefore hypothesized that P. gingivalis cleaves oral keratinocyte PARs in an Arg-(Rgp) or Lys- (Kgp) gingipain-specific manner to upregulate pro-inflammatory cytokines. Immortalized human oral keratinocytes (TERT-2) were incubated with wild-type P. gingivalis (ATCC 33277) or strains from a panel of isogenic gingipain deletion mutants: Kgp-deficient (KDP 129); Rgp-deficient (KDP 133); or Kgp- and Rgp-deficient (KDP 136). After incubation with P. gingivalis, keratinocytes were probed with specific antibodies against the N-terminus of PAR-1 and PAR-2. Using flow cytometry and immunofluorescence, receptor cleavage was marked by loss of specific antibody binding to the respective PARs. TERT-2 cells constitutively expressed high levels of PAR-1 and PAR-2, and lower levels of PAR-3. P. gingivalis ATCC 33277 cleaved PAR-1 and PAR-2 in a dose-dependent manner, while the receptors were unaffected by the protease-negative double mutant (KDP 136) at all m.o.i. tested. The single Kgp-negative mutant preferentially cleaved PAR-1, whereas the Rgp-negative mutant cleaved PAR-2. Wild-type or Kgp-negative mutant cleavage of PAR-1 upregulated expression of IL-1α, IL-1β, IL-6 and TNF-α; the Rgp-negative mutant did not modulate these cytokines. Selective cleavage of PAR-1 on oral epithelial cells by P. gingivalis Rgp therefore upregulates expression of pro-inflammatory cytokines.

Original languageEnglish (US)
Pages (from-to)3238-3246
Number of pages9
JournalMicrobiology
Volume155
Issue number10
DOIs
StatePublished - Nov 16 2009

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Proteinase-Activated Receptors
Porphyromonas gingivalis
PAR-1 Receptor
PAR-2 Receptor
Epithelial Cells
Cell Line
Keratinocytes
Cytokines
Interleukin-1
Up-Regulation
Antibodies
Toll-Like Receptors
Porphyromonas gingivalis argingipain
Innate Immunity
Fluorescent Antibody Technique
Lipopolysaccharides
Interleukin-6
Flow Cytometry
Peptide Hydrolases

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Cleavage of protease-activated receptors on an immortalized oral epithelial cell line by Porphyromonas gingivalis gingipains. / Giacaman, Rodrigo A.; Asrani, Anil C.; Ross, Karen F; Herzberg, Mark C.

In: Microbiology, Vol. 155, No. 10, 16.11.2009, p. 3238-3246.

Research output: Contribution to journalArticle

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