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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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P. gingivalis modifies RIPK2 in wild type and caspase-deficient murine bone marrow-derived macrophages.A) C57BL/6 (wt) or casp1-deficient (casp1−/−) BMDM were untreated (M) or treated with 100 ng/ml E. coli LPS (LPS), live P. gingivalis 381 (MOI 100, Live) or heat-killed (60°C, 60 min) P. gingivalis 381 (MOI 100 equivalency, HK) for 2 h. B) C57BL/6 (wt), casp2-deficient (casp2−/−), casp3-deficient (casp3−/−), or casp7-deficient (casp7−/−) BMDM were untreated (−) or treated with P. gingivalis 381 (MOI 100) (+) for 2 h. Whole cell lysates were analyzed for RIPK2. Full-length RIPK2 is indicated with an arrow. A prominent P. gingivalis-induced LMW band is indicated with an asterisk. MW ladder is indicated on the left in kDa.
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ppat-1002723-g006: P. gingivalis modifies RIPK2 in wild type and caspase-deficient murine bone marrow-derived macrophages.A) C57BL/6 (wt) or casp1-deficient (casp1−/−) BMDM were untreated (M) or treated with 100 ng/ml E. coli LPS (LPS), live P. gingivalis 381 (MOI 100, Live) or heat-killed (60°C, 60 min) P. gingivalis 381 (MOI 100 equivalency, HK) for 2 h. B) C57BL/6 (wt), casp2-deficient (casp2−/−), casp3-deficient (casp3−/−), or casp7-deficient (casp7−/−) BMDM were untreated (−) or treated with P. gingivalis 381 (MOI 100) (+) for 2 h. Whole cell lysates were analyzed for RIPK2. Full-length RIPK2 is indicated with an arrow. A prominent P. gingivalis-induced LMW band is indicated with an asterisk. MW ladder is indicated on the left in kDa.

Mentions: To examine the role of cellular caspase activity in P. gingivalis-induced proteolysis of RIPK2 directly, murine bone marrow derived macrophages (BMDM) from casp1-, casp2-, casp3-, or casp7-deficient mice were co-cultured with P. gingivalis. P. gingivalis induced a similar proteolysis of RIPK2 in cells deficient in casp1 (Figure 6A), casp2, casp3, or casp7 (Figure 6B) as that observed in wild type cells. These findings suggest that caspase 1, caspase 2, caspase 3 and caspase 7 singly do not play a role in P. gingivalis-induced RIPK2 proteolysis. However, they do not exclude the possibility that non-redundant host caspase activity could contribute to RIPK2 cleavage induced by P. gingivalis stimulation.


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)

P. gingivalis modifies RIPK2 in wild type and caspase-deficient murine bone marrow-derived macrophages.A) C57BL/6 (wt) or casp1-deficient (casp1−/−) BMDM were untreated (M) or treated with 100 ng/ml E. coli LPS (LPS), live P. gingivalis 381 (MOI 100, Live) or heat-killed (60°C, 60 min) P. gingivalis 381 (MOI 100 equivalency, HK) for 2 h. B) C57BL/6 (wt), casp2-deficient (casp2−/−), casp3-deficient (casp3−/−), or casp7-deficient (casp7−/−) BMDM were untreated (−) or treated with P. gingivalis 381 (MOI 100) (+) for 2 h. Whole cell lysates were analyzed for RIPK2. Full-length RIPK2 is indicated with an arrow. A prominent P. gingivalis-induced LMW band is indicated with an asterisk. MW ladder is indicated on the left in kDa.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369954&req=5

ppat-1002723-g006: P. gingivalis modifies RIPK2 in wild type and caspase-deficient murine bone marrow-derived macrophages.A) C57BL/6 (wt) or casp1-deficient (casp1−/−) BMDM were untreated (M) or treated with 100 ng/ml E. coli LPS (LPS), live P. gingivalis 381 (MOI 100, Live) or heat-killed (60°C, 60 min) P. gingivalis 381 (MOI 100 equivalency, HK) for 2 h. B) C57BL/6 (wt), casp2-deficient (casp2−/−), casp3-deficient (casp3−/−), or casp7-deficient (casp7−/−) BMDM were untreated (−) or treated with P. gingivalis 381 (MOI 100) (+) for 2 h. Whole cell lysates were analyzed for RIPK2. Full-length RIPK2 is indicated with an arrow. A prominent P. gingivalis-induced LMW band is indicated with an asterisk. MW ladder is indicated on the left in kDa.
Mentions: To examine the role of cellular caspase activity in P. gingivalis-induced proteolysis of RIPK2 directly, murine bone marrow derived macrophages (BMDM) from casp1-, casp2-, casp3-, or casp7-deficient mice were co-cultured with P. gingivalis. P. gingivalis induced a similar proteolysis of RIPK2 in cells deficient in casp1 (Figure 6A), casp2, casp3, or casp7 (Figure 6B) as that observed in wild type cells. These findings suggest that caspase 1, caspase 2, caspase 3 and caspase 7 singly do not play a role in P. gingivalis-induced RIPK2 proteolysis. However, they do not exclude the possibility that non-redundant host caspase activity could contribute to RIPK2 cleavage induced by P. gingivalis stimulation.

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