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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 Kgp mutant is deficient in the induction of RIPK1 and RIPK2 proteolysis in HAEC.HAEC were untreated or treated with P. gingivalis strain 381, strain ATCC 33277, or isogenic mutants of 33277: YPP1 (rgpA−), RgpA/B (rgpA−, rgpB−), or with YPP2 (kgp−) (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 asterisks. MW ladder is indicated on the left in kDa. GAPDH was detected as a loading control.
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ppat-1002723-g009: P. gingivalis Kgp mutant is deficient in the induction of RIPK1 and RIPK2 proteolysis in HAEC.HAEC were untreated or treated with P. gingivalis strain 381, strain ATCC 33277, or isogenic mutants of 33277: YPP1 (rgpA−), RgpA/B (rgpA−, rgpB−), or with YPP2 (kgp−) (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 asterisks. MW ladder is indicated on the left in kDa. GAPDH was detected as a loading control.

Mentions: To further confirm the role of P. gingivalis gingipains in the proteolysis of RIPK1 and RIPK2 in the absence of chemical inhibitors, we examined previously characterized bacterial mutant strains deficient in kgp or rgp[57] for their ability to induce the proteolysis of RIPK1 and RIPK2. The genetic background of the mutant strains used was in P. gingivalis strain 33277, a similarly classified organism in terms of clonality (clonal type 1) [58] serogroup (A) [59] and invasiveness [60] as P. gingivalis strain 381. We observed that P. gingivalis wild type strain 33277 reduced full-length RIPK1 (Figure 9A) and RIPK2 (Figure 9B) levels and induced the detection of LMW immunoreactive bands. However, the degree of RIPK1 and RIPK2 proteolysis induced by 33277 was less than that observed with strain 381. Both P. gingivalis strains YPP1 and RgpA/B induced a significant proteolysis of full-length RIPK1 and RIPK2. In contrast, RIPK1 and RIPK2 protein levels in HAEC treated with P. gingivalis YPP2 were similar to untreated cells. These findings confirm the role of Kgp in P. gingivalis-induced 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)

P. gingivalis Kgp mutant is deficient in the induction of RIPK1 and RIPK2 proteolysis in HAEC.HAEC were untreated or treated with P. gingivalis strain 381, strain ATCC 33277, or isogenic mutants of 33277: YPP1 (rgpA−), RgpA/B (rgpA−, rgpB−), or with YPP2 (kgp−) (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 asterisks. MW ladder is indicated on the left in kDa. GAPDH was detected as a loading control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369954&req=5

ppat-1002723-g009: P. gingivalis Kgp mutant is deficient in the induction of RIPK1 and RIPK2 proteolysis in HAEC.HAEC were untreated or treated with P. gingivalis strain 381, strain ATCC 33277, or isogenic mutants of 33277: YPP1 (rgpA−), RgpA/B (rgpA−, rgpB−), or with YPP2 (kgp−) (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 asterisks. MW ladder is indicated on the left in kDa. GAPDH was detected as a loading control.
Mentions: To further confirm the role of P. gingivalis gingipains in the proteolysis of RIPK1 and RIPK2 in the absence of chemical inhibitors, we examined previously characterized bacterial mutant strains deficient in kgp or rgp[57] for their ability to induce the proteolysis of RIPK1 and RIPK2. The genetic background of the mutant strains used was in P. gingivalis strain 33277, a similarly classified organism in terms of clonality (clonal type 1) [58] serogroup (A) [59] and invasiveness [60] as P. gingivalis strain 381. We observed that P. gingivalis wild type strain 33277 reduced full-length RIPK1 (Figure 9A) and RIPK2 (Figure 9B) levels and induced the detection of LMW immunoreactive bands. However, the degree of RIPK1 and RIPK2 proteolysis induced by 33277 was less than that observed with strain 381. Both P. gingivalis strains YPP1 and RgpA/B induced a significant proteolysis of full-length RIPK1 and RIPK2. In contrast, RIPK1 and RIPK2 protein levels in HAEC treated with P. gingivalis YPP2 were similar to untreated cells. These findings confirm the role of Kgp in P. gingivalis-induced 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