Danyang Huang , Lingyan Chen , Zhe Wang , Fenfang He , Xinrui Zhang , Xiaoyuan Wang
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Based on sequence alignment analysis, <em>V. parahaemolyticus VP_RS08405</em> has high homology to <em>E. coli lpxL</em>, <em>lpxM</em> and <em>lpxP</em> which encode the three secondary acyltransferases of lipid A. Therefore, <em>V. parahaemolyticus VP_RS08405</em> was cloned into pBAD33, and the resulting pB08405 was introduced in <em>E. coli</em> mutants WHL00 in which <em>lpxL</em> was deleted, WHM00 in which <em>lpxM</em> was deleted, WHP00 in which <em>lpxP</em> was deleted, and WH300 in which <em>lpxL</em>, <em>lpxM</em> and <em>lpxP</em> were deleted. The recombinant strains WHL00/pB08405, WHM00/pB08405, WHP00/pB08405, WH300/pB08405, as well as their vector controls, were grown at normal and low temperatures. Lipid A species were isolated from the above strains and analyzed by using high-performance liquid chromatography-tandem mass spectrometry and thin-layer chromatography. After comparing the secondary acyl alterations of lipid A from different recombinant strains, it is concluded that VP_RS08405 specifically catalyzed the addition of a palmitoleate to the 2′-position of lipid A and its activity is not temperature-sensitive. In addition, to determine the dependence of VP_RS08405 on Kdo, <em>VP_RS08405</em> was overexpressed in <em>E. coli</em> mutants WH001 in which <em>waaA</em> was deleted, and WH400 in which <em>waaA</em>, <em>lpxL</em>, <em>lpxM</em> and <em>lpxP</em> were deleted. Lipid A species were isolated from WH001/pB08405 and WH400/pB08405, and analyzed. The results show that the function of VP_RS08405 is Kdo-dependent. These findings provide a better understanding of the structural diversity of lipid A in <em>V. parahaemolyticus</em>.</p></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"180 ","pages":"Article 110504"},"PeriodicalIF":3.4000,"publicationDate":"2024-08-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Characterization of a secondary palmitoleoyltransferase of lipid A in Vibrio parahaemolyticus\",\"authors\":\"Danyang Huang , Lingyan Chen , Zhe Wang , Fenfang He , Xinrui Zhang , Xiaoyuan Wang\",\"doi\":\"10.1016/j.enzmictec.2024.110504\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>The detection of pathogenicity and immunogenicity in <em>Vibrio parahaemolyticus</em> poses a significant challenge due to its threat to human health and food safety, which is strongly correlated with lipid A. Lipid A, a critical component found in most Gram-negative bacteria, functions as a hydrophobic anchor for lipopolysaccharide. <em>V. parahaemolyticus</em> synthesizes multiple lipid A species with various secondary acyl chains. In this study, a secondary acyltransferase of lipid A encoded by <em>VP_RS08405</em> in <em>V. parahaemolyticus</em> was identified. Based on sequence alignment analysis, <em>V. parahaemolyticus VP_RS08405</em> has high homology to <em>E. coli lpxL</em>, <em>lpxM</em> and <em>lpxP</em> which encode the three secondary acyltransferases of lipid A. Therefore, <em>V. parahaemolyticus VP_RS08405</em> was cloned into pBAD33, and the resulting pB08405 was introduced in <em>E. coli</em> mutants WHL00 in which <em>lpxL</em> was deleted, WHM00 in which <em>lpxM</em> was deleted, WHP00 in which <em>lpxP</em> was deleted, and WH300 in which <em>lpxL</em>, <em>lpxM</em> and <em>lpxP</em> were deleted. The recombinant strains WHL00/pB08405, WHM00/pB08405, WHP00/pB08405, WH300/pB08405, as well as their vector controls, were grown at normal and low temperatures. Lipid A species were isolated from the above strains and analyzed by using high-performance liquid chromatography-tandem mass spectrometry and thin-layer chromatography. After comparing the secondary acyl alterations of lipid A from different recombinant strains, it is concluded that VP_RS08405 specifically catalyzed the addition of a palmitoleate to the 2′-position of lipid A and its activity is not temperature-sensitive. In addition, to determine the dependence of VP_RS08405 on Kdo, <em>VP_RS08405</em> was overexpressed in <em>E. coli</em> mutants WH001 in which <em>waaA</em> was deleted, and WH400 in which <em>waaA</em>, <em>lpxL</em>, <em>lpxM</em> and <em>lpxP</em> were deleted. Lipid A species were isolated from WH001/pB08405 and WH400/pB08405, and analyzed. The results show that the function of VP_RS08405 is Kdo-dependent. These findings provide a better understanding of the structural diversity of lipid A in <em>V. parahaemolyticus</em>.</p></div>\",\"PeriodicalId\":11770,\"journal\":{\"name\":\"Enzyme and Microbial Technology\",\"volume\":\"180 \",\"pages\":\"Article 110504\"},\"PeriodicalIF\":3.4000,\"publicationDate\":\"2024-08-22\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Enzyme and Microbial Technology\",\"FirstCategoryId\":\"5\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S014102292400111X\",\"RegionNum\":3,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"BIOTECHNOLOGY & APPLIED MICROBIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Enzyme and Microbial Technology","FirstCategoryId":"5","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S014102292400111X","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
引用次数: 0
摘要
由于副溶血性弧菌对人类健康和食品安全的威胁与脂质 A 密切相关,因此对其致病性和免疫原性的检测是一项重大挑战。脂质 A 是大多数革兰氏阴性细菌中的重要成分,具有脂多糖疏水锚的功能。副溶血性弧菌可合成多种具有不同仲酰基链的脂质 A。本研究鉴定了副溶血弧菌中由 VP_RS08405 编码的脂质 A 二级酰基转移酶。根据序列比对分析,副溶血性弧菌 VP_RS08405 与大肠杆菌 lpxL、lpxM 和 lpxP 编码的三种脂质 A 二级酰基转移酶具有高度同源性。因此,将副溶血性弧菌 VP_RS08405 克隆到 pBAD33 中,并将得到的 pB08405 导入大肠杆菌突变株 WHL00(其中删除了 lpxL)、WHM00(其中删除了 lpxM)、WHP00(其中删除了 lpxP)和 WH300(其中删除了 lpxL、lpxM 和 lpxP)。重组菌株 WHL00/pB08405、WHM00/pB08405、WHP00/pB08405、WH300/pB08405 及其载体对照均在常温和低温条件下生长。利用高效液相色谱-串联质谱法和薄层色谱法从上述菌株中分离并分析了脂质 A 的种类。通过比较不同重组菌株的脂质 A 的仲酰基变化,得出结论:VP_RS08405 能特异性地催化脂质 A 的 2′位上棕榈油酸酯的添加,且其活性对温度不敏感。此外,为了确定 VP_RS08405 对 Kdo 的依赖性,在删除了 waaA 的大肠杆菌突变体 WH001 和删除了 waaA、lpxL、lpxM 和 lpxP 的 WH400 中过表达了 VP_RS08405。从 WH001/pB08405 和 WH400/pB08405 中分离并分析了脂质 A 的种类。结果表明,VP_RS08405 的功能依赖于 Kdo。这些发现有助于更好地了解副溶血性弧菌中脂质 A 的结构多样性。
Characterization of a secondary palmitoleoyltransferase of lipid A in Vibrio parahaemolyticus
The detection of pathogenicity and immunogenicity in Vibrio parahaemolyticus poses a significant challenge due to its threat to human health and food safety, which is strongly correlated with lipid A. Lipid A, a critical component found in most Gram-negative bacteria, functions as a hydrophobic anchor for lipopolysaccharide. V. parahaemolyticus synthesizes multiple lipid A species with various secondary acyl chains. In this study, a secondary acyltransferase of lipid A encoded by VP_RS08405 in V. parahaemolyticus was identified. Based on sequence alignment analysis, V. parahaemolyticus VP_RS08405 has high homology to E. coli lpxL, lpxM and lpxP which encode the three secondary acyltransferases of lipid A. Therefore, V. parahaemolyticus VP_RS08405 was cloned into pBAD33, and the resulting pB08405 was introduced in E. coli mutants WHL00 in which lpxL was deleted, WHM00 in which lpxM was deleted, WHP00 in which lpxP was deleted, and WH300 in which lpxL, lpxM and lpxP were deleted. The recombinant strains WHL00/pB08405, WHM00/pB08405, WHP00/pB08405, WH300/pB08405, as well as their vector controls, were grown at normal and low temperatures. Lipid A species were isolated from the above strains and analyzed by using high-performance liquid chromatography-tandem mass spectrometry and thin-layer chromatography. After comparing the secondary acyl alterations of lipid A from different recombinant strains, it is concluded that VP_RS08405 specifically catalyzed the addition of a palmitoleate to the 2′-position of lipid A and its activity is not temperature-sensitive. In addition, to determine the dependence of VP_RS08405 on Kdo, VP_RS08405 was overexpressed in E. coli mutants WH001 in which waaA was deleted, and WH400 in which waaA, lpxL, lpxM and lpxP were deleted. Lipid A species were isolated from WH001/pB08405 and WH400/pB08405, and analyzed. The results show that the function of VP_RS08405 is Kdo-dependent. These findings provide a better understanding of the structural diversity of lipid A in V. parahaemolyticus.
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