Abstract
Periodontitis, a chronic inflammation driven by dysbiotic subgingival bacterial flora, is linked on clinical levels to the development of a number of systemic diseases and to the development of oral and gastric tract tumors. A key pathogen, Porphyromonas gingivalis, secretes gingipains, cysteine proteases implicated as the main factors in the development of periodontitis. Here we hypothesize that gingipains may be linked to systemic pathologies through the deregulation of kallikrein-like proteinase (KLK) family members. KLKs are implicated in cancer development and are clinically utilized as tumor progression markers. In tissues, KLK activity is strictly controlled by a limited number of tissue-specific inhibitors, including SPINK6, an inhibitor of these proteases in skin and oral epithelium. Here we identify gingipains as the only P. gingivalis proteases responsible for SPINK6 degradation. We further show that gingipains, even at low nanomolar concentrations, cleaved SPINK6 in concentration- and time-dependent manner. The proteolysis was accompanied by loss of inhibition against KLK13. We also mapped the cleavage by Arg-specific gingipains to the reactive site loop of the SPINK6 inhibitor. Moreover, we identified a significant fraction of SPINK6-sensitive proteases in healthy saliva and confirmed the ability of gingipains to inactivate SPINK6 under ex vivo conditions. Finally, we demonstrate the double-edge action of gingipains, which, in addition, can activate KLKs because of gingipain K-mediated proteolytic processing of the zymogenic proform of KLK13. Altogether, the results indicate the potential of P. gingivalis to disrupt the control system of KLKs, providing a possible mechanistic link between periodontal disease and tumor development.
Highlights
Vate kallikrein-like proteinase (KLK) because of gingipain K-mediated proteolytic processing of the zymogenic proform of KLK13
SPINK6 Is Degraded by P. gingivalis Cultures—P. gingivalis produces many proteolytic enzymes, and, in addition to gingipains, it secretes several other proteases that may interact with SPINK6
To investigate the ability of gingipains and other proteases to degrade SPINK6, the inhibitor was preincubated with wild-type P. gingivalis W83 in the presence or absence of gingipain-specific inhibitors or with the triple gingipain knockout mutant W83⌬kgp⌬rgpA⌬rgpB, completely devoid of gingipain activity (Fig. 1)
Summary
Vate KLKs because of gingipain K-mediated proteolytic processing of the zymogenic proform of KLK13. Preincubation of the W83 strain with Kyt inhibitors that are gingipain-specific, reversible, and do not affect the activity of other proteases, at least at the concentration used [29], protected the majority of SPINK6 from degradation (Fig. 1).
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