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Porphyromonas gingivalis evasion of autophagy and intracellular killing by human myeloid dendritic cells involves DC-SIGN-TLR2 crosstalk.

ABSTRACT: Signaling via pattern recognition receptors (PRRs) expressed on professional antigen presenting cells, such as dendritic cells (DCs), is crucial to the fate of engulfed microbes. Among the many PRRs expressed by DCs are Toll-like receptors (TLRs) and C-type lectins such as DC-SIGN. DC-SIGN is targeted by several major human pathogens for immune-evasion, although its role in intracellular routing of pathogens to autophagosomes is poorly understood. Here we examined the role of DC-SIGN and TLRs in evasion of autophagy and survival of Porphyromonas gingivalis in human monocyte-derived DCs (MoDCs). We employed a panel of P. gingivalis isogenic fimbriae deficient strains with defined defects in Mfa-1 fimbriae, a DC-SIGN ligand, and FimA fimbriae, a TLR2 agonist. Our results show that DC-SIGN dependent uptake of Mfa1+P. gingivalis strains by MoDCs resulted in lower intracellular killing and higher intracellular content of P. gingivalis. Moreover, Mfa1+P. gingivalis was mostly contained within single membrane vesicles, where it survived intracellularly. Survival was decreased by activation of TLR2 and/or autophagy. Mfa1+P. gingivalis strain did not induce significant levels of Rab5, LC3-II, and LAMP1. In contrast, P. gingivalis uptake through a DC-SIGN independent manner was associated with early endosomal routing through Rab5, increased LC3-II and LAMP-1, as well as the formation of double membrane intracellular phagophores, a characteristic feature of autophagy. These results suggest that selective engagement of DC-SIGN by Mfa-1+P. gingivalis promotes evasion of antibacterial autophagy and lysosome fusion, resulting in intracellular persistence in myeloid DCs; however TLR2 activation can overcome autophagy evasion and pathogen persistence in DCs.

SUBMITTER: El-Awady AR

PROVIDER: S-EPMC4352937 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:ObjectivePeriodontitis is a microbe-induced chronic inflammatory disease. Previous exposure of the host to bacteria or their virulence factors leads to refractory responses to further stimuli, which is called tolerance. Porphyromonas gingivalis (P. gingivalis) is one of the most important pathogenic microorganisms associated with periodontitis, and is a potent inducer of pro- and anti-inflammatory cytokines. The aim of this study was to explore the roles and possible mechanisms of tolerance induced by P. gingivalis.MethodsTHP-1-derived macrophages were pretreated with 1x108 colony-forming units/ml P. gingivalis ATCC 33277 or 21 clinical isolates from moderate to severe chronic periodontitis patients (24 h), washed (2 h) and treated with P. gingivalis ATCC 33277 or the same clinical isolates again (24 h). Levels of pro-inflammatory cytokines TNF-? and IL-1? and anti-inflammatory cytokine IL-10 in supernatants were detected by ELISA. Moreover, to identify the possible mechanisms for the changes in cytokine secretion, Toll-like receptor 2 (TLR2) and TLR4 protein expressions were explored in these cells by flow cytometry.ResultsAfter repeated challenge with P. gingivalis ATCC 33277 or clinical isolates, production of TNF-? and IL-1? in macrophages was decreased significantly compared with that following a single stimulation (p<0.05), while only comparable levels of IL-10 were detected in P. gingivalis ATCC 33277 or clinical isolate-tolerized cells (p>0.05). In addition, there was interstrain variability in the ability to induce IL-1? and IL-10 production after repeated P. gingivalis stimulation. However, no significant changes in TLR2 or TLR4 were detected in macrophages that were repeatedly treated with P. gingivalis ATCC 33277 or clinical isolates compared with those stimulated with P. gingivalis only once (p>0.05).ConclusionsRepeated P. gingivalis stimulation triggered tolerance, which might contribute to limiting periodontal inflammation. However, tolerance induced by P. gingivalis might develop independently of TLR2 and TLR4 and be related to molecules in signaling pathways downstream of TLR2 and TLR4.

Project description:Porphyromonas gingivalis is a Gram-negative intracellular pathogen associated with periodontal disease. We have previously reported on whole-cell quantitative proteomic analyses to investigate the differential expression of virulence factors as the organism transitions from an extracellular to intracellular lifestyle. The original results with the invasive strain P. gingivalis ATCC 33277 were obtained using the genome sequence available at the time, strain W83 [GenBank: AE015924]. We present here a re-processed dataset using the recently published genome annotation specific for strain ATCC 33277 [GenBank: AP009380] and an analysis of differential abundance based on metabolic pathways rather than individual proteins.Qualitative detection was observed for 1266 proteins using the strain ATCC 33277 annotation for 18 hour internalized P. gingivalis within human gingival epithelial cells and controls exposed to gingival cell culture medium, an improvement of 7% over the W83 annotation. Internalized cells showed increased abundance of proteins in the energy pathway from asparagine/aspartate amino acids to ATP. The pathway producing one short chain fatty acid, propionate, showed increased abundance, while that of another, butyrate, trended towards decreased abundance. The translational machinery, including ribosomal proteins and tRNA synthetases, showed a significant increase in protein relative abundance, as did proteins responsible for transcription.Use of the ATCC 33277 specific genome annotation resulted in improved proteome coverage with respect to the number of proteins observed both qualitatively in terms of protein identifications and quantitatively in terms of the number of calculated abundance ratios. Pathway analysis showed a significant increase in overall protein synthetic and transcriptional machinery in the absence of significant growth. These results suggest that the interior of host cells provides a more energy rich environment compared to the extracellular milieu. Shifts in the production of cytotoxic fatty acids by intracellular P. gingivalis may play a role in virulence. Moreover, despite extensive genomic re-arrangements between strains W83 and 33277, there is sufficient sequence similarity at the peptide level for proteomic abundance trends to be largely accurate when using the heterologous strain annotated genome as the reference for database searching.

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Porphyromonas gingivalis evasion of autophagy and intracellular killing by human myeloid dendritic cells involves DC-SIGN-TLR2 crosstalk.

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