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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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Related in: MedlinePlus

Model of P. gingivalis innate immune activation/invasion in endothelial cells.Outcome of innate immune responses is representative as a balance of functional, intact pathways (TLR-left panel) and dysregulated pathways (NLR-right panel). P. gingivalis represents a human pathogen that utilizes fimbriae for attachment and invasion of endothelial cells. Fimbriae are not only expressed on whole bacteria (A), but within outer membrane vesicles (OMV) that are released from the cell (B), as occurs with all Gram-negative bacteria identified to date. Fimbriae bind to TLR2 and MD2 to activate TLR2 and TLR4, resulting in the induction of NF-κB, leading to inflammation, and de novo protein synthesis (C) of cell adhesion molecules (CAM) and TLR (TLR) expressed at the cell surface (D). Many studies have demonstrated invasion of endothelial cells by P. gingivalis (E). However, recent studies have shown that OMV (containing fimbriae and active gingipain activity) gain entry into host cells rapidly in a gingipain-dependent manner (E), independent of whole organism. Upon entry, intracellular gingipain activity degrades RIPK1 and RIPK2 (abrogated by protease inhibitors) (F), resulting in a variety of possible consequences as a function of selective targeting of intracellular NOD1 or NOD2 (NOD1/2) signaling pathways and/or disruption of RIPK1 or RIPK2-mediated cell signaling. Alteration of immune signaling responses may result in decreased host cell death, decreased inflammatory mediator expression, and subsequent enhancement of intracellular bacterial cell survival, all of which contributes to an intracellular niche for P. gingivalis (G).
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ppat-1002723-g011: Model of P. gingivalis innate immune activation/invasion in endothelial cells.Outcome of innate immune responses is representative as a balance of functional, intact pathways (TLR-left panel) and dysregulated pathways (NLR-right panel). P. gingivalis represents a human pathogen that utilizes fimbriae for attachment and invasion of endothelial cells. Fimbriae are not only expressed on whole bacteria (A), but within outer membrane vesicles (OMV) that are released from the cell (B), as occurs with all Gram-negative bacteria identified to date. Fimbriae bind to TLR2 and MD2 to activate TLR2 and TLR4, resulting in the induction of NF-κB, leading to inflammation, and de novo protein synthesis (C) of cell adhesion molecules (CAM) and TLR (TLR) expressed at the cell surface (D). Many studies have demonstrated invasion of endothelial cells by P. gingivalis (E). However, recent studies have shown that OMV (containing fimbriae and active gingipain activity) gain entry into host cells rapidly in a gingipain-dependent manner (E), independent of whole organism. Upon entry, intracellular gingipain activity degrades RIPK1 and RIPK2 (abrogated by protease inhibitors) (F), resulting in a variety of possible consequences as a function of selective targeting of intracellular NOD1 or NOD2 (NOD1/2) signaling pathways and/or disruption of RIPK1 or RIPK2-mediated cell signaling. Alteration of immune signaling responses may result in decreased host cell death, decreased inflammatory mediator expression, and subsequent enhancement of intracellular bacterial cell survival, all of which contributes to an intracellular niche for P. gingivalis (G).

Mentions: The interactions between bacteria with host defense mechanisms are dynamic cellular processes involving pathogen-mediated factors and host signaling cascades, microenvironments and appropriate immune responses. We and others have demonstrated the role of membrane-bound TLR in the recognition and host defense against P. gingivalis. However, the roles of intracellular PRR in P. gingivalis infection and persistence are poorly understood. Our results demonstrate that P. gingivalis Kgp activity induces the proteolysis of key signaling proteins involved in TNF-mediated cell death and NOD-mediated host defense pathways. Further studies will be required to determine the functional consequence of these observations, in a manner that can distinguish between RIPK1 and RIPK2-mediated processes. In addition, studies will examine the internalization of OMV and the extent of gingipain colocalization with PRR, RIPK1, and RIPK2. We propose that the disruption of intracellular signaling facilitates the persistence of P. gingivalis in endothelial cells by evading host defense mechanisms and altering cell death pathways (Figure 11).


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)

Model of P. gingivalis innate immune activation/invasion in endothelial cells.Outcome of innate immune responses is representative as a balance of functional, intact pathways (TLR-left panel) and dysregulated pathways (NLR-right panel). P. gingivalis represents a human pathogen that utilizes fimbriae for attachment and invasion of endothelial cells. Fimbriae are not only expressed on whole bacteria (A), but within outer membrane vesicles (OMV) that are released from the cell (B), as occurs with all Gram-negative bacteria identified to date. Fimbriae bind to TLR2 and MD2 to activate TLR2 and TLR4, resulting in the induction of NF-κB, leading to inflammation, and de novo protein synthesis (C) of cell adhesion molecules (CAM) and TLR (TLR) expressed at the cell surface (D). Many studies have demonstrated invasion of endothelial cells by P. gingivalis (E). However, recent studies have shown that OMV (containing fimbriae and active gingipain activity) gain entry into host cells rapidly in a gingipain-dependent manner (E), independent of whole organism. Upon entry, intracellular gingipain activity degrades RIPK1 and RIPK2 (abrogated by protease inhibitors) (F), resulting in a variety of possible consequences as a function of selective targeting of intracellular NOD1 or NOD2 (NOD1/2) signaling pathways and/or disruption of RIPK1 or RIPK2-mediated cell signaling. Alteration of immune signaling responses may result in decreased host cell death, decreased inflammatory mediator expression, and subsequent enhancement of intracellular bacterial cell survival, all of which contributes to an intracellular niche for P. gingivalis (G).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369954&req=5

ppat-1002723-g011: Model of P. gingivalis innate immune activation/invasion in endothelial cells.Outcome of innate immune responses is representative as a balance of functional, intact pathways (TLR-left panel) and dysregulated pathways (NLR-right panel). P. gingivalis represents a human pathogen that utilizes fimbriae for attachment and invasion of endothelial cells. Fimbriae are not only expressed on whole bacteria (A), but within outer membrane vesicles (OMV) that are released from the cell (B), as occurs with all Gram-negative bacteria identified to date. Fimbriae bind to TLR2 and MD2 to activate TLR2 and TLR4, resulting in the induction of NF-κB, leading to inflammation, and de novo protein synthesis (C) of cell adhesion molecules (CAM) and TLR (TLR) expressed at the cell surface (D). Many studies have demonstrated invasion of endothelial cells by P. gingivalis (E). However, recent studies have shown that OMV (containing fimbriae and active gingipain activity) gain entry into host cells rapidly in a gingipain-dependent manner (E), independent of whole organism. Upon entry, intracellular gingipain activity degrades RIPK1 and RIPK2 (abrogated by protease inhibitors) (F), resulting in a variety of possible consequences as a function of selective targeting of intracellular NOD1 or NOD2 (NOD1/2) signaling pathways and/or disruption of RIPK1 or RIPK2-mediated cell signaling. Alteration of immune signaling responses may result in decreased host cell death, decreased inflammatory mediator expression, and subsequent enhancement of intracellular bacterial cell survival, all of which contributes to an intracellular niche for P. gingivalis (G).
Mentions: The interactions between bacteria with host defense mechanisms are dynamic cellular processes involving pathogen-mediated factors and host signaling cascades, microenvironments and appropriate immune responses. We and others have demonstrated the role of membrane-bound TLR in the recognition and host defense against P. gingivalis. However, the roles of intracellular PRR in P. gingivalis infection and persistence are poorly understood. Our results demonstrate that P. gingivalis Kgp activity induces the proteolysis of key signaling proteins involved in TNF-mediated cell death and NOD-mediated host defense pathways. Further studies will be required to determine the functional consequence of these observations, in a manner that can distinguish between RIPK1 and RIPK2-mediated processes. In addition, studies will examine the internalization of OMV and the extent of gingipain colocalization with PRR, RIPK1, and RIPK2. We propose that the disruption of intracellular signaling facilitates the persistence of P. gingivalis in endothelial cells by evading host defense mechanisms and altering cell death pathways (Figure 11).

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