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

Abstract

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 IV_A into Kdo-lipid IV_A, but no Kdo₂-lipid IV_A 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 IV_A-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 IV_A as a substrate and only transfers single Kdo residue to lipid IV_A. 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.

Keywords

Bacteroides thetaiotaomicron / Escherichia coli / Lipopolysaccharide / Lipid a kdo transferase / WaaA

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Funding

National Natural Science Foundation of China(NSFC32270102)

Jiangsu Basic Research Center for Synthetic Biology(BK20233003)

RIGHTS & PERMISSIONS

Jiangnan University