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<p>The human intestinal symbiotic microorganism <i>Bacteroides thetaiotaomicron</i> has a unique lipooligosaccharide structure, which promotes its beneficial symbiosis with the host. But its synthesis mechanism is not fully understood. In this study, protein sequence alignment revealed that the protein encoded by <i>B. thetaiotaomicron</i> VPI 5482 <i>BT_2747</i> gene shares 24% sequence identity with <i>Escherichia coli</i> WaaA. The expression vector was used to overexpress BT_2747 in <i>E. coli</i> lipopolysaccharide mutant strains constructed by knocking out the <i>waaC-waaF</i>, <i>lpxL</i> or <i>lpxM</i> genes, resulting in the recombinant strains WH001(Δ<i>waaA</i>)/pBT2747, WL003(Δ<i>waaAwaaC-F</i>)/pBT2747, WL004(Δ<i>waaAwaaC-FlpxL</i>)/pBT2747, WL005(Δ<i>waaAwaaC-FlpxM</i>)/pBT2747 and WL006(Δ<i>waaAwaaC-FlpxLlpxM</i>)/pBT2747. Lipid A/Kdo-lipid A were extracted from these recombinant strains and analyzed by liquid chromatography-mass spectrometry. The results showed that BT_2747 could convert a portion of the lipid IV<sub>A</sub> into Kdo-lipid IV<sub>A</sub>, but no Kdo<sub>2</sub>-lipid IV<sub>A</sub> structure was detected in <i>E. coli</i>. Small amounts of hexa-acylated Kdo-lipid A were also detected in the recombinant strains WH001/pBT2747 and WL003/pBT2747, and a small amount of penta-acylated Kdo-lipid A was found in WL004/pBT2747. The recombinant strain was further modified by introducing <i>lpxF</i> that enabled to synthesize Kdo-lipid IV<sub>A</sub>-1-phosphate. The results revealed that the <i>BT_2747</i> gene in <i>B. thetaiotaomicron</i> VPI 5482 encodes the Kdo transferase of lipid A which uses lipid IV<sub>A</sub> as a substrate and only transfers single Kdo residue to lipid IV<sub>A</sub>. This study extends our understanding of the Kdo-lipid A structure and synthesis mechanism of <i>B. thetaiotaomicron</i>, which might provide a new perspective on how intestinal commensal bacteria regulate host immune homeostasis through unique Kdo-lipid A structure.</p>

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Identification of lipid A Kdo transferase encoded by BT_2747 in Bacteroides thetaiotaomicron VPI 5482

  • 期刊论文

  • 发表日期：

    2025年9月2日

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  • Geer Liu,
  • Guihong Zhao,
  • He Liu,
  • Dezhi Zhang,
  • Yaqun Tang,
  • Zihan Li,
  • Xiaoyuan Wang

摘要

The human intestinal symbiotic microorganism Bacteroides thetaiotaomicron has a unique lipooligosaccharide structure, which promotes its beneficial symbiosis with the host. But its synthesis mechanism is not fully understood. In this study, protein sequence alignment revealed that the protein encoded by B. thetaiotaomicron VPI 5482 BT_2747 gene shares 24% sequence identity with Escherichia coli WaaA. The expression vector was used to overexpress BT_2747 in E. coli lipopolysaccharide mutant strains constructed by knocking out the waaC-waaF, lpxL or lpxM genes, resulting in the recombinant strains WH001(ΔwaaA)/pBT2747, WL003(ΔwaaAwaaC-F)/pBT2747, WL004(ΔwaaAwaaC-FlpxL)/pBT2747, WL005(ΔwaaAwaaC-FlpxM)/pBT2747 and WL006(ΔwaaAwaaC-FlpxLlpxM)/pBT2747. Lipid A/Kdo-lipid A were extracted from these recombinant strains and analyzed by liquid chromatography-mass spectrometry. The results showed that BT_2747 could convert a portion of the lipid IVA into Kdo-lipid IVA, but no Kdo2-lipid IVA structure was detected in E. coli. Small amounts of hexa-acylated Kdo-lipid A were also detected in the recombinant strains WH001/pBT2747 and WL003/pBT2747, and a small amount of penta-acylated Kdo-lipid A was found in WL004/pBT2747. The recombinant strain was further modified by introducing lpxF that enabled to synthesize Kdo-lipid IVA-1-phosphate. The results revealed that the BT_2747 gene in B. thetaiotaomicron VPI 5482 encodes the Kdo transferase of lipid A which uses lipid IVA as a substrate and only transfers single Kdo residue to lipid IVA. This study extends our understanding of the Kdo-lipid A structure and synthesis mechanism of B. thetaiotaomicron, which might provide a new perspective on how intestinal commensal bacteria regulate host immune homeostasis through unique Kdo-lipid A structure.