Porphyromonas gingivalis induce apoptosis in human gingival epithelial cells through a gingipain-dependent mechanism

Center for Oral Health and Systemic Disease, School of Dentistry, University of Louisville, Louisville, KY, USA.
BMC Microbiology (Impact Factor: 2.73). 02/2009; 9(1):107. DOI: 10.1186/1471-2180-9-107
Source: PubMed


The oral pathogen Porphyromonas gingivalis has been shown to modulate apoptosis in different cell types, but its effect on epithelial cells remains unclear.
We demonstrate that primary human gingival epithelial cells (HGECs) challenged with live P. gingivalis for 24 hours exhibit apoptosis, and we characterize this by M30 epitope detection, caspase-3 activity, DNA fragmentation and Annexin-V staining. Live bacteria strongly upregulated intrinsic and extrinsic apoptotic pathways. Pro-apoptotic molecules such as caspase-3, -8, -9, Bid and Bax were upregulated after 24 hours. The anti-apoptotic Bcl-2 was also upregulated, but this was not sufficient to ensure cell survival. The main P. gingivalis proteases arginine and lysine gingipains are necessary and sufficient to induce host cell apoptosis. Thus, live P. gingivalis can invoke gingival epithelial cell apoptosis in a time and dose dependent manner with significant apoptosis occurring between 12 and 24 hours of challenge via a gingipain-dependent mechanism.
The present study provides evidence that live, but not heat-killed, P. gingivalis can induce apoptosis after 24 hours of challenge in primary human gingival epithelial cells. Either arginine or lysine gingipains are necessary and sufficient factors in P. gingivalis elicited apoptosis.

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    • "We also examined mRNA levels of BID and TRADD (Figure 8), which are pro-apoptotic genes that have previously been shown to be induced by P. gingivalis [24]. Basal levels of BID mRNA were reduced approximately 50% by FOXO1/FOXO3 knockdown (P<0.05). "
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    ABSTRACT: P. gingivalis is a prominent periodontal pathogen that has potent effects on host cells. In this study we challenged gingival epithelial cells with P. gingivalis with the aim of assessing how mRNA levels of key target genes were modulated by P. gingivalis via the transcription factors FOXO1 and FOXO3. Primary mono- and multi-layer cultures of gingival epithelial cells were challenged and barrier function was examined by fluorescent dextran and apoptosis was measured by cytoplasmic histone associated DNA. Gene expression levels were measured by real-time PCR with and without FOXO1 and FOXO3 siRNA compared to scrambled siRNA. P. gingivalis induced a loss of barrier function and stimulated gingival epithelial cell apoptosis in multilayer cultures that was in part gingipain dependent. P. gingivalis stimulated an increase in FOXO1 and FOXO3 mRNA, enhanced mRNA levels of genes associated with differentiated keratinocyte function (keratin-1, -10, -14, and involucrin), increased mRNA levels of apoptotic genes (BID and TRADD), reduced mRNA levels of genes that regulate inflammation (TLR-2 and -4) and reduced those associated with barrier function (integrin beta-1, -3 and -6). The ability of P. gingivalis to modulate these genes was predominantly FOXO1 and FOXO3 dependent. The results indicate that P. gingivalis has pronounced effects on gingival keratinocytes and modulates mRNA levels of genes that affect host response, differentiation, apoptosis and barrier function. Moreover, this modulation is dependent upon the transcription factors FOXO1 or FOXO3. In addition, a new function for FOXO1 was identified, that of suppressing TLR-2 and TLR-4 and maintaining integrin beta -1, beta -3 and beta -6 basal mRNA levels in keratinocytes.
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    • "The present findings corroborate another study [26] that found a higher number of Bcl-2 positive cells in the inflammatory infiltrate of periodontitis patients, suggesting that the Bcl-2 protein may play a role in the control of apoptosis in inflammatory cells. The up-regulation of Bcl-2 was observed in epithelial cells in response to Porphyromonas gingivalis gingipains, [27,28] which indicates that this bacteria can survive in the cellular environment by evading the host immune response. "
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    • "C. histolyticum α-clostripain has been shown to induce cell death and cytopathic effects on tissue culture cells, even at low dilutions [32]. Furthermore, purified HrgpA and RgpB, live P. gingivalis cells, and filtered culture supernatants were capable of inducing apoptosis in primary human gingivial epithelial cells unlike heat-killed P. gingivalis cells [33]. Since previous work suggested that mutation of the α-clostripain gene had the potential to alter the levels of secreted C. perfringens proteins [13], [34], and therefore to potentially affect their interaction with host cells, we decided to determine the effect of isogenic culture supernatants on host cell survival. "
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