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 under...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.展开更多
Vibrio parahaemolyticus is associated with seafood,and its pathogenicity and antimicrobial resistance are closely related to lipid A.In V.parahaemolyticus,free lipid A species and complete lipopolysaccharide coexist b...Vibrio parahaemolyticus is associated with seafood,and its pathogenicity and antimicrobial resistance are closely related to lipid A.In V.parahaemolyticus,free lipid A species and complete lipopolysaccharide coexist but the reason is unknown.In Escherichia coli,this reaction to covert lipid A to the full-length lipopolysaccharide is catalyzed by a bifunctional 3-deoxy-d-manno-octulosonic acid(Kdo)transferase WaaA.In this study,a monofunctional Kdo transferase in V.parahaemolyticus encoded by VP_RS01035 was identified.When VP_RS01035 was deleted in V.parahaemolyticus ATCC33846,no full-length lipopolysaccharide was synthesized,but more free lipid A species were accumulated,and most of them were modified by phosphoethanolamine.When V.parahaemolyticus VP_RS01035 was overexpressed in a newly constructed E.coli waaA mutant WH001,and the full-length lipopolysaccharide was partially restored,suggesting that VP_RS01035 could replace waaA in E.coli.In addition,hexa-acylated lipid A and Kdo-lipid A species were directly extracted from WH001/pB2-RS01035,suggesting that V.parahaemolyticus VP_RS01035 is monofunctional and less efficient than E.coli WaaA.These results indicate that lipid A biosynthesis in V.parahaemolyticus can proceed without the addition of Kdo and V.parahaemolyticus VP_RS01035 plays an important role on lipid A diversity in V.parahaemolyticus.展开更多
基金funded by the Basic Research Program of Jiangsu and supported by the Jiangsu Basic Research Center for Synthetic Biology(Grant No.BK20233003)by National Natural Science Foundation of China(NSFC32270102).
文摘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.
基金supported by National Natural Science Foundation of China(NSFC32270102).
文摘Vibrio parahaemolyticus is associated with seafood,and its pathogenicity and antimicrobial resistance are closely related to lipid A.In V.parahaemolyticus,free lipid A species and complete lipopolysaccharide coexist but the reason is unknown.In Escherichia coli,this reaction to covert lipid A to the full-length lipopolysaccharide is catalyzed by a bifunctional 3-deoxy-d-manno-octulosonic acid(Kdo)transferase WaaA.In this study,a monofunctional Kdo transferase in V.parahaemolyticus encoded by VP_RS01035 was identified.When VP_RS01035 was deleted in V.parahaemolyticus ATCC33846,no full-length lipopolysaccharide was synthesized,but more free lipid A species were accumulated,and most of them were modified by phosphoethanolamine.When V.parahaemolyticus VP_RS01035 was overexpressed in a newly constructed E.coli waaA mutant WH001,and the full-length lipopolysaccharide was partially restored,suggesting that VP_RS01035 could replace waaA in E.coli.In addition,hexa-acylated lipid A and Kdo-lipid A species were directly extracted from WH001/pB2-RS01035,suggesting that V.parahaemolyticus VP_RS01035 is monofunctional and less efficient than E.coli WaaA.These results indicate that lipid A biosynthesis in V.parahaemolyticus can proceed without the addition of Kdo and V.parahaemolyticus VP_RS01035 plays an important role on lipid A diversity in V.parahaemolyticus.
