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Porphyromonas gingivalis Sphingolipid Synthesis Limits the Host Inflammatory Response.

ABSTRACT: Porphyromonas gingivalis, like other bacteria belonging to the phylum Bacteroidetes, synthesizes sphingolipids (SLs). However, their exact roles in microbial physiology and their potential role in mediating interactions with their eukaryotic host are unclear. Our working hypothesis for this study was that synthesis of SLs (host-like lipids) affords a mechanism that allows P. gingivalis to persist in homeostasis with its host. In a previous study, we deleted a gene (PG1780 in strain W83) predicted to encode a serine palmitoyl transferase (SPT)-the enzyme that catalyzes the first conserved step in the synthesis of SLs-and we determined that the mutant was unable to synthesize SLs. Here, we characterized the SPT enzyme encoded by PG1780, analyzed the impact of SPT deletion on P. gingivalis gene expression (RNA-Seq analysis), and began to define the impact of SL synthesis on its interactions with host cells. Enzymatic analysis verified that the protein encoded by PG1780 is indeed an SPT. RNA-Seq analysis determined that a lack of SL synthesis results in differential expression of extracytoplasmic function sigma factors, components of the type IX secretion system (T9SS), and CRISPR and cas genes. Our data demonstrate that when human THP1 macrophage-like cells were challenged with the wild type (W83) and the SL-null mutant (W83 ΔSPT), the SL-null strain elicited a robust inflammatory response (elevated IL-1β, IL-6, IL-10, IL-8, RANTES, and TNFα) while the response to the parent strain W83 was negligible. Interestingly, we also discovered that SLs produced by P. gingivalis can be delivered to host cells independent of cell-to-cell contact. Overall, our results support our working hypothesis that synthesis of SLs by P. gingivalis is central to its ability to manipulate the host inflammatory response, and they demonstrate the integral importance of SLs in the physiology of P. gingivalis.

SUBMITTER: Rocha FG

PROVIDER: S-EPMC7174802 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Periodontal disease is a chronic oral inflammatory disease that is triggered by bacteria such as Porphyromonas gingivalis. P. gingivalis strains exhibit great heterogeneity, with some strains being encapsulated while others are nonencapsulated. Although the encapsulated strains have been shown to be more virulent in a mouse abscess model, so far the role of the capsule in P. gingivalis interactions with host cells is not well understood and its role in virulence has not been defined. Here, we investigated the contribution of the capsule to triggering a host response following microbial infection, as well as its protective role following bacterial internalization by host phagocytic cells with subsequent killing, using the encapsulated P. gingivalis strain W50 and its isogenic nonencapsulated mutant, PgC. Our study shows significant time-dependent upregulation of the expression of various groups of genes in macrophages challenged with both the encapsulated and nonencapsulated P. gingivalis strains. However, cells infected with the nonencapsulated strain showed significantly higher upregulation of 9 and 29 genes at 1 h and 8 h postinfection, respectively, than cells infected with the encapsulated strain. Among the genes highly upregulated by the nonencapsulated PgC strain were ones coding for cytokines and chemokines. Maturation markers were induced at a 2-fold higher rate in dendritic cells challenged with the nonencapsulated strain for 4 h than in dendritic cells challenged with the encapsulated strain. The rates of phagocytosis of the nonencapsulated P. gingivalis strain by both macrophages and dendritic cells were 4.5-fold and 7-fold higher, respectively, than the rates of phagocytosis of the encapsulated strain. On the contrary, the survival of the nonencapsulated P. gingivalis strain was drastically reduced compared to the survival of the encapsulated strain. Finally, the encapsulated strain exhibited greater virulence in a mouse abscess model. Our results indicate that the P. gingivalis capsule plays an important role in aiding evasion of host immune system activation, promoting survival of the bacterium within host cells, and increasing virulence. As such, it is a major virulence determinant of P. gingivalis.

Project description:Periodontitis is a very common oral inflammatory disease that results in the destruction of supporting connective and osseous tissues of the teeth. Although the exact etiology is still unclear, Gram-negative bacteria, especially Porphyromonas gingivalis in subgingival pockets are thought to be one of the major etiologic agents of periodontitis. Endothelin (ET) is a family of three 21-amino acid peptides, ET-1, -2, and -3, that activate G protein-coupled receptors, ETA and ETB. Endothelin is involved in the occurrence and progression of various inflammatory diseases. Previous reports have shown that ET-1 and its receptors, ETA and ETB are expressed in the periodontal tissues and, that ET-1 levels in gingival crevicular fluid are increased in periodontitis patients. Moreover, P. gingivalis infection has been shown to induce the production of ET-1 along with other inflammatory cytokines. Despite these studies, however, the functional significance of endothelin in periodontitis is still largely unknown. In this study, we explored the cellular and molecular mechanisms of ET-1 action in periodontitis using human gingival epithelial cells (HGECs). ET-1 and ETA, but not ETB, were abundantly expressed in HGECs. Stimulation of HGECs with P. gingivalis or P. gingivalis lipopolysaccharide increased the expression of ET-1 and ETA suggesting the activation of the endothelin signaling pathway. Production of inflammatory cytokines, IL-1?, TNF?, and IL-6, was significantly enhanced by exogenous ET-1 treatment, and this effect depended on the mitogen-activated protein kinases via intracellular Ca2+ increase, which resulted from the activation of the phospholipase C/inositol 1,4,5-trisphosphate pathway. The inhibition of the endothelin receptor-mediated signaling pathway with the dual receptor inhibitor, bosentan, partially ameliorated alveolar bone loss and immune cell infiltration. These results suggest that endothelin plays an important role in P. gingivalis-mediated periodontitis. Thus, endothelin antagonism may be a potential therapeutic approach for periodontitis treatment.

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Porphyromonas gingivalis Sphingolipid Synthesis Limits the Host Inflammatory Response.

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