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Pathogen-mediated proteolysis of the cell death regulator RIPK1 and the host defense modulator RIPK2 in human aortic endothelial cells.

Madrigal AG, Barth K, Papadopoulos G, Genco CA - PLoS Pathog. (2012)

Bottom Line: RIPK1 and RIPK2 cleavage was not observed in HAEC treated with an isogenic mutant deficient in the lysine-specific gingipain, confirming a role for Kgp in the cleavage of RIPK1 and RIPK2.Similar proteolysis of poly (ADP-ribose) polymerase (PARP) was observed.We also demonstrated direct proteolysis of RIPK2 by P. gingivalis in a cell-free system which was abrogated in the presence of a Kgp-specific protease inhibitor.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Section of Infectious Diseases, Boston University School of Medicine, Boston, Massachusetts, United States of America.

ABSTRACT
Porphyromonas gingivalis is the primary etiologic agent of periodontal disease that is associated with other human chronic inflammatory diseases, including atherosclerosis. The ability of P. gingivalis to invade and persist within human aortic endothelial cells (HAEC) has been postulated to contribute to a low to moderate chronic state of inflammation, although how this is specifically achieved has not been well defined. In this study, we demonstrate that P. gingivalis infection of HAEC resulted in the rapid cleavage of receptor interacting protein 1 (RIPK1), a mediator of tumor necrosis factor (TNF) receptor-1 (TNF-R1)-induced cell activation or death, and RIPK2, a key mediator of both innate immune signaling and adaptive immunity. The cleavage of RIPK1 or RIPK2 was not observed in cells treated with apoptotic stimuli, or cells stimulated with agonists to TNF-R1, nucleotide oligomerization domain receptor 1(NOD1), NOD2, Toll-like receptor 2 (TLR2) or TLR4. P. gingivalis-induced cleavage of RIPK1 and RIPK2 was inhibited in the presence of a lysine-specific gingipain (Kgp) inhibitor. RIPK1 and RIPK2 cleavage was not observed in HAEC treated with an isogenic mutant deficient in the lysine-specific gingipain, confirming a role for Kgp in the cleavage of RIPK1 and RIPK2. Similar proteolysis of poly (ADP-ribose) polymerase (PARP) was observed. We also demonstrated direct proteolysis of RIPK2 by P. gingivalis in a cell-free system which was abrogated in the presence of a Kgp-specific protease inhibitor. Our studies thus reveal an important role for pathogen-mediated modification of cellular kinases as a potential strategy for bacterial persistence within target host cells, which is associated with low-grade chronic inflammation, a hallmark of pathogen-mediated chronic inflammatory disorders.

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Inhibition of Kgp activity alters P. gingivalis-mediated RIPK1 and RIPK2 cleavage in HAEC.P. gingivalis strain 381 was pretreated with 10 µM KYT-1, 10 µM KYT-36, 10 µM KYT-1 and 10 µM KYT-36, 1 mM TLCK, or vehicle controls (DMSO or acid water) for 45 min. HAEC were then immediately co-cultured with medium or with pretreated preparations of P. gingivalis 381 (MOI 100) for 2 h. Whole cell lysates were analyzed for A) RIPK1 or B) RIPK2. Full-length RIPK1 and RIPK2 are indicated with arrows. Prominent P. gingivalis-induced LMW bands are indicated with asterisk(s). MW ladder is indicated on the left in kDa. GAPDH was detected as a loading control.
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ppat-1002723-g008: Inhibition of Kgp activity alters P. gingivalis-mediated RIPK1 and RIPK2 cleavage in HAEC.P. gingivalis strain 381 was pretreated with 10 µM KYT-1, 10 µM KYT-36, 10 µM KYT-1 and 10 µM KYT-36, 1 mM TLCK, or vehicle controls (DMSO or acid water) for 45 min. HAEC were then immediately co-cultured with medium or with pretreated preparations of P. gingivalis 381 (MOI 100) for 2 h. Whole cell lysates were analyzed for A) RIPK1 or B) RIPK2. Full-length RIPK1 and RIPK2 are indicated with arrows. Prominent P. gingivalis-induced LMW bands are indicated with asterisk(s). MW ladder is indicated on the left in kDa. GAPDH was detected as a loading control.

Mentions: A number of host cell surface proteins and proteins in the extracellular milieu are cleaved by the cysteine proteases of P. gingivalis[44], [52], [53]. To evaluate if P. gingivalis gingipains played a role in the proteolysis of RIPK1 and RIPK2, we first evaluated previously established gingipain-specific inhibitors KYT-1 (arginine-specific gingipain inhibitor) and KYT-36 (lysine-specific gingipain inhibitor) [54], [55] as well as TLCK, which inhibits both arginine-specific and lysine-specific protease activity [56] in our studies. We confirmed the ability of the protease inhibitors to reduce the arginine-specific activity by KYT-1, the lysine-specific activity by KYT-36 and both arginine- and lysine-specific protease activity by TLCK in a substrate-based assay (Figure 7A and B). Treatment of P. gingivalis 381 with KYT-1 or vehicle control (DMSO) did not alter the ability of P. gingivalis 381 to induce proteolysis of RIPK1 (Figure 8A) or RIPK2 (Figure 8B) in HAEC. However, treatment of P. gingivalis with KYT-36 alone or in combination with KYT-1 blocked P. gingivalis-induced proteolysis of RIPK1 and RIPK2. TLCK also inhibited the P. gingivalis-induced proteolysis of RIPK1 and RIPK2. Pretreatment of P. gingivalis with KYT-1, KYT-36, TLCK and vehicle controls did not alter bacterial viability (data not shown), nor did KYT-36 alter host caspase 3 activity (Figure 7C). These findings indicate that Kgp activity was responsible for P. gingivalis-mediated proteolysis of RIPK1 and RIPK2.


Pathogen-mediated proteolysis of the cell death regulator RIPK1 and the host defense modulator RIPK2 in human aortic endothelial cells.

Madrigal AG, Barth K, Papadopoulos G, Genco CA - PLoS Pathog. (2012)

Inhibition of Kgp activity alters P. gingivalis-mediated RIPK1 and RIPK2 cleavage in HAEC.P. gingivalis strain 381 was pretreated with 10 µM KYT-1, 10 µM KYT-36, 10 µM KYT-1 and 10 µM KYT-36, 1 mM TLCK, or vehicle controls (DMSO or acid water) for 45 min. HAEC were then immediately co-cultured with medium or with pretreated preparations of P. gingivalis 381 (MOI 100) for 2 h. Whole cell lysates were analyzed for A) RIPK1 or B) RIPK2. Full-length RIPK1 and RIPK2 are indicated with arrows. Prominent P. gingivalis-induced LMW bands are indicated with asterisk(s). MW ladder is indicated on the left in kDa. GAPDH was detected as a loading control.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3369954&req=5

ppat-1002723-g008: Inhibition of Kgp activity alters P. gingivalis-mediated RIPK1 and RIPK2 cleavage in HAEC.P. gingivalis strain 381 was pretreated with 10 µM KYT-1, 10 µM KYT-36, 10 µM KYT-1 and 10 µM KYT-36, 1 mM TLCK, or vehicle controls (DMSO or acid water) for 45 min. HAEC were then immediately co-cultured with medium or with pretreated preparations of P. gingivalis 381 (MOI 100) for 2 h. Whole cell lysates were analyzed for A) RIPK1 or B) RIPK2. Full-length RIPK1 and RIPK2 are indicated with arrows. Prominent P. gingivalis-induced LMW bands are indicated with asterisk(s). MW ladder is indicated on the left in kDa. GAPDH was detected as a loading control.
Mentions: A number of host cell surface proteins and proteins in the extracellular milieu are cleaved by the cysteine proteases of P. gingivalis[44], [52], [53]. To evaluate if P. gingivalis gingipains played a role in the proteolysis of RIPK1 and RIPK2, we first evaluated previously established gingipain-specific inhibitors KYT-1 (arginine-specific gingipain inhibitor) and KYT-36 (lysine-specific gingipain inhibitor) [54], [55] as well as TLCK, which inhibits both arginine-specific and lysine-specific protease activity [56] in our studies. We confirmed the ability of the protease inhibitors to reduce the arginine-specific activity by KYT-1, the lysine-specific activity by KYT-36 and both arginine- and lysine-specific protease activity by TLCK in a substrate-based assay (Figure 7A and B). Treatment of P. gingivalis 381 with KYT-1 or vehicle control (DMSO) did not alter the ability of P. gingivalis 381 to induce proteolysis of RIPK1 (Figure 8A) or RIPK2 (Figure 8B) in HAEC. However, treatment of P. gingivalis with KYT-36 alone or in combination with KYT-1 blocked P. gingivalis-induced proteolysis of RIPK1 and RIPK2. TLCK also inhibited the P. gingivalis-induced proteolysis of RIPK1 and RIPK2. Pretreatment of P. gingivalis with KYT-1, KYT-36, TLCK and vehicle controls did not alter bacterial viability (data not shown), nor did KYT-36 alter host caspase 3 activity (Figure 7C). These findings indicate that Kgp activity was responsible for P. gingivalis-mediated proteolysis of RIPK1 and RIPK2.

Bottom Line: RIPK1 and RIPK2 cleavage was not observed in HAEC treated with an isogenic mutant deficient in the lysine-specific gingipain, confirming a role for Kgp in the cleavage of RIPK1 and RIPK2.Similar proteolysis of poly (ADP-ribose) polymerase (PARP) was observed.We also demonstrated direct proteolysis of RIPK2 by P. gingivalis in a cell-free system which was abrogated in the presence of a Kgp-specific protease inhibitor.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Section of Infectious Diseases, Boston University School of Medicine, Boston, Massachusetts, United States of America.

ABSTRACT
Porphyromonas gingivalis is the primary etiologic agent of periodontal disease that is associated with other human chronic inflammatory diseases, including atherosclerosis. The ability of P. gingivalis to invade and persist within human aortic endothelial cells (HAEC) has been postulated to contribute to a low to moderate chronic state of inflammation, although how this is specifically achieved has not been well defined. In this study, we demonstrate that P. gingivalis infection of HAEC resulted in the rapid cleavage of receptor interacting protein 1 (RIPK1), a mediator of tumor necrosis factor (TNF) receptor-1 (TNF-R1)-induced cell activation or death, and RIPK2, a key mediator of both innate immune signaling and adaptive immunity. The cleavage of RIPK1 or RIPK2 was not observed in cells treated with apoptotic stimuli, or cells stimulated with agonists to TNF-R1, nucleotide oligomerization domain receptor 1(NOD1), NOD2, Toll-like receptor 2 (TLR2) or TLR4. P. gingivalis-induced cleavage of RIPK1 and RIPK2 was inhibited in the presence of a lysine-specific gingipain (Kgp) inhibitor. RIPK1 and RIPK2 cleavage was not observed in HAEC treated with an isogenic mutant deficient in the lysine-specific gingipain, confirming a role for Kgp in the cleavage of RIPK1 and RIPK2. Similar proteolysis of poly (ADP-ribose) polymerase (PARP) was observed. We also demonstrated direct proteolysis of RIPK2 by P. gingivalis in a cell-free system which was abrogated in the presence of a Kgp-specific protease inhibitor. Our studies thus reveal an important role for pathogen-mediated modification of cellular kinases as a potential strategy for bacterial persistence within target host cells, which is associated with low-grade chronic inflammation, a hallmark of pathogen-mediated chronic inflammatory disorders.

Show MeSH
Related in: MedlinePlus