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<p>Sphingolipids are essential membrane constituents composed of aliphatic amino alcohols. They are widely distributed in eukaryotes and certain prokaryotes, acting as key signaling molecules. Bacteroidota are the only known group of gut bacteria capable of synthesizing sphingolipids, which dominate their membrane lipid composition and have been shown to modulate host immune responses and metabolic homeostasis. However, it remains unclear whether Bacteroidota synthesize sphingolipids via a eukaryote-like sphinganine-dependent pathway and/or a&#xa0;<i>Caulobacter crescentus</i>-like oxidized dihydroceramide pathway. In this study, two genes <i>bt3032</i> and <i>bt3075</i> in <i>Bacteroides thetaiotaomicron</i> were predicted to be related to sphingolipid biosynthesis by homologic alignments with the enzymes sphinganine acyltransferase and ceramide reductase characterized in <i>C. crescentus</i>. The genes <i>Spt (bt0870)</i><i>, </i><i>Kdsr (bt0972)</i><i>, </i><i>bt3032</i> and <i>bt3075</i> were deleted individually in <i>B. thetaiotaomicron </i><i>VPI 5482</i>, resulting the mutant strains ΔSPT, ΔKDSR, ΔBT3032 and ΔBT3075, respectively. The lipids were isolated from these mutants and analyzed, using thin-layer chromatography and liquid chromatography–mass spectrometry. Deletion of <i>Spt</i> resulted in complete loss of sphinganine, dihydroceramide, and downstream sphingolipids, confirming its role as the serine palmitoyltransferase catalyzing the first step of dihydroceramide biosynthesis. Deletion of <i>Kdsr</i> caused accumulation of 3-ketodihydrosphingosine while sphingolipid synthesis was partially retained, indicating an alternative dihydroceramide biosynthetic route. In contrast, deletion of <i>bt3032</i> abolished dihydroceramide and derived sphingolipids, demonstrating its essential role as a dihydroceramide synthase. Deletion of <i>bt3075</i> reduced sphingolipid levels. Collectively, these results reveal multiple ceramide biosynthetic pathways in <i>B. thetaiotaomicron</i>.</p>

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Characterization of sphinganine acyltransferase and ceramide reductase in Bacteroides thetaiotaomicron

  • 期刊论文

  • 发表日期：

    2026年4月13日

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  • He Liu,
  • Geer Liu,
  • Jiaxin Wu,
  • Yongfa Liu,
  • Xuebing Cao,
  • Xiaoyuan Wang

摘要

Sphingolipids are essential membrane constituents composed of aliphatic amino alcohols. They are widely distributed in eukaryotes and certain prokaryotes, acting as key signaling molecules. Bacteroidota are the only known group of gut bacteria capable of synthesizing sphingolipids, which dominate their membrane lipid composition and have been shown to modulate host immune responses and metabolic homeostasis. However, it remains unclear whether Bacteroidota synthesize sphingolipids via a eukaryote-like sphinganine-dependent pathway and/or a Caulobacter crescentus-like oxidized dihydroceramide pathway. In this study, two genes bt3032 and bt3075 in Bacteroides thetaiotaomicron were predicted to be related to sphingolipid biosynthesis by homologic alignments with the enzymes sphinganine acyltransferase and ceramide reductase characterized in C. crescentus. The genes Spt (bt0870), Kdsr (bt0972), bt3032 and bt3075 were deleted individually in B. thetaiotaomicron VPI 5482, resulting the mutant strains ΔSPT, ΔKDSR, ΔBT3032 and ΔBT3075, respectively. The lipids were isolated from these mutants and analyzed, using thin-layer chromatography and liquid chromatography–mass spectrometry. Deletion of Spt resulted in complete loss of sphinganine, dihydroceramide, and downstream sphingolipids, confirming its role as the serine palmitoyltransferase catalyzing the first step of dihydroceramide biosynthesis. Deletion of Kdsr caused accumulation of 3-ketodihydrosphingosine while sphingolipid synthesis was partially retained, indicating an alternative dihydroceramide biosynthetic route. In contrast, deletion of bt3032 abolished dihydroceramide and derived sphingolipids, demonstrating its essential role as a dihydroceramide synthase. Deletion of bt3075 reduced sphingolipid levels. Collectively, these results reveal multiple ceramide biosynthetic pathways in B. thetaiotaomicron.