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.

Sphingolipid-Containing Outer Membrane Vesicles Serve as a Delivery Vehicle To Limit Macrophage Immune Response to Porphyromonas gingivalis

“…As previously demonstrated, the direct challenge with the SLs null mutant elicited a hyperinflammatory profile from the host cells (17). Here, a highly similar cytokine profile was detected from macrophages cultured with wild type and SL-deficient P. gingivalis when bacteria to the host cell contact was prevented, by using a transwell system (Fig.…”

“…Purified OMVs suppress the host cell inflammatory response. To evaluate the possibility that purified P. gingivalis OMVs are able to recapitulate the immune stimulating activity previously observed by direct bacterial challenge (17), and nocontact / transwell challenge, purified OMVs from WT and SPT-mutant were added directly to THP-1 cells. We detected a hyperinflammatory response from THP-1 cells cultured directly with OMVs from the SL-null mutant in comparison to the elicited response by the OMVs from WT (Fig.…”

Role for Lipids Secreted by Irradiated Peripheral Blood Mononuclear Cells in Inflammatory Resolution in Vitro

“…If oral pathogens control the production of lipids other than prostanoids by cells of the oral cavity has not been identified but for example, macrophages can release sphingolipids [51]. However, it is the oral pathogens including P. gingivalis producing lipids such as sphingolipids [52], serine-glycine dipeptide lipid [53] and short-chain fatty acids [54] that can in turn manipulate the host inflammatory response in periodontitis and other inflammatory diseases.…”

Virulence of the Pathogen Porphyromonas gingivalis Is Controlled by the CRISPR-Cas Protein Cas3

“…As described previously ( 83 ), human monocyte/macrophage cell line THP-1 (ATCC, TIB-202) was cultured at 37°C in a 5% CO 2 incubator in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, l -glutamine (2 mM), penicillin/streptomycin (100 U/100 μg/ml), HEPES (10 mM), sodium pyruvate (1 mM), glucose (4.5 mg/ml), sodium bicarbonate (1.5 mg/ml), and 2-mercaptoethanol (Sigma-Aldrich) (0.05 mM). Cells were adjusted to 5 × 10 5 viable cells/ml, and, to induce differentiation into a macrophage-like state, THP-1 cells were placed into fresh medium containing 100 ng/ml phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich) and 1 ml of THP-1 cells was added to each well of 24-well cell culture plates.…”