Identification and Biosynthesis of Pro-Inflammatory Sulfonolipids from an Opportunistic Pathogen Chryseobacterium gleum

Abstract

Sulfonolipids (SoLs) are a unique class of sphingolipids featuring a sulfonate group compared to other sphingolipids. However, the biological functions and biosynthesis of SoLs in human microbiota have been poorly understood. Here, we report the discovery and isolation of SoLs from a human opportunistic pathogen Chryseobacterium gleum DSM16776. We show for the first time the pro-inflammatory activity of SoLs with mice primary macrophages. Furthermore, we used both in vivo heterologous expression and in vitro biochemical reconstitution to characterize two enzymes, cysteate synthase and cysteate fatty acyltransferase, that are specifically involved in the biosynthesis of SoLs rather than other sphingolipids. Based on these two SoL-specific enzymes, our bioinformatics analysis showed a wider distribution of SoL biosynthetic genes in microbes that had not been reported as SoL producers. We selected four of these strains and verified their cysteate synthase and cysteate fatty acyltransferase activities in SoL biosynthesis. Considering this wider distribution of SoL-specific biosynthetic enzymes in the context of SoLs' activity in mediating inflammation, a common and fundamental biological process, it may suggest a more comprehensive function of SoLs at play.

Figure 1.

(A) Diagrammatic workflow for SoLs discovery from human opportunistic pathogen C. gleum DSM16776. (B) Negative ion mode feature-based molecular networking (FBMN) of C. gleum extracts. (C) Chemical structure of a major SoL compound, SoL A, isolated from C. gleum (capnine moiety in blue).

Figure 2.

SoL A induced inflammatory response in primary macrophages isolated from wild type male C57Bl/6 mice. (A). Increased mRNA levels of pro-inflammatory cytokines induced by 10 μM of SoL A. LPS (100 ng/mL) used as a control; (B) Increased mRNA levels of IL-6 and IL-1ß induced by SoL A (10 μM) and a mixture of SoL A (10 μM) + LPS (100 ng/mL). LPS (100 ng/mL) used as a control. For each treatment, n=3. *, p<0.05; **, p< 0.01; ***, p< 0.001, versus vehicle.

Figure 3.

SoL A induced IL-6 protein production and secretion in macrophages. LPS (100 ng/mL) used as a control. For each treatment, n=3. ****, p< 0.0001, versus vehicle.

Figure 4.

HPLC analysis of in vitro Cys assays followed by derivatization with L-FDAA. (A) Cys1, (B) Cys2, (C) Cys3. Reactions were performed as follows: (i) L-cysteate standard, (ii) L-phosphoserine standard, (iii) L-phosphoserine + Na₂SO₃ + boiled Cys, (iv) L-phosphoserine + Cys, and (v) L-phosphoserine + Na₂SO₃ + Cys. Cya: cysteate; Cya-FDAA: cysteate-FDAA derivative; Pi-Ser: phosphoserine; Pi-Ser-FDAA: phosphoserine-FDAA derivative.

Figure 5.. Comparative LC-ESI analysis of production of key enzymatic conversions.

The extracted ion chromatograms (EICs) were shown at m/z 334.20 [M - H]⁻ for 1, m/z 360.22 [M - H]⁻ for 2, m/z 362.23 [M - H]⁻ for 3, and m/z 388.25 [M - H]⁻ for 4. (A) Production of E. coli BL21(DE3)/pHis8::cfat1 fed with cysteate or not (i and ii) and in the in vitro CFAT1 reaction (iii and iv). (B) Production of E. coli BL21(DE3)/pACYC::cys1/pHis8::cfat1 (v) and E. coli BL21(DE3)/pACYC/pHis8 (vi). (C) One-pot assay of Cys1 and CFAT1(vii, viii, ix and x). (D) The biosynthetic pathway of 3.

Figure 6.

Distribution of Spt homologs in different organisms. SSN analysis of the Spt homologs in diverse organisms and visualized by Cytoscape. CFATs responsible for SoLs biosynthesis fell into a separate cluster as highlighted in the box, five of which (shown in red) were characterized in this study. Some reported prokaryote-derived Spt responsible for condensation of serine and fatty acyl-CoA are shown in green while some reported eukaryote-derived Spt responsible for condensation of serine and fatty acyl-CoA are shown in blue.