State Key Laboratory of Food Science and Resources, Jiangnan University, Wuxi 214122, China; School of Food Science and Technology, Jiangnan University, Wuxi 214122, China; Ningbo Institute of Marine Medicine Peking University, Ningbo 315832, China.
School of Biotechnology, Jiangnan University, Wuxi 214122, China.
Enzyme Microb Technol. 2024 Oct;180:110504. doi: 10.1016/j.enzmictec.2024.110504. Epub 2024 Aug 22.
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.
副溶血弧菌的致病性和免疫原性检测具有挑战性，因为它对人类健康和食品安全构成威胁，而这与脂多糖的脂质 A 密切相关。脂质 A 是大多数革兰氏阴性菌的关键组成部分，作为脂多糖的疏水性锚。副溶血弧菌合成多种具有不同次级酰基链的脂质 A 种类。在这项研究中，鉴定了副溶血弧菌 VP_RS08405 编码的脂质 A 次级酰基转移酶。基于序列比对分析，副溶血弧菌 VP_RS08405 与编码脂质 A 三个次级酰基转移酶的大肠杆菌 lpxL、lpxM 和 lpxP 具有高度同源性。因此，将副溶血弧菌 VP_RS08405 克隆到 pBAD33 中，得到的 pB08405 被引入 lpxL 缺失的大肠杆菌突变体 WHL00、lpxM 缺失的 WHM00、lpxP 缺失的 WHP00 和 lpxL、lpxM 和 lpxP 缺失的 WH300 中。重组菌株 WHL00/pB08405、WHM00/pB08405、WHP00/pB08405、WH300/pB08405 及其载体对照在正常和低温下生长。从上述菌株中分离脂质 A 种类，并通过高效液相色谱-串联质谱法和薄层色谱法进行分析。比较不同重组菌株中脂质 A 的次级酰基变化后，得出结论，VP_RS08405 特异性催化棕榈油酸在脂质 A 的 2'位的添加，其活性不受温度影响。此外，为了确定 VP_RS08405 对 Kdo 的依赖性，在 waaA 缺失的大肠杆菌突变体 WH001 和 waaA、lpxL、lpxM 和 lpxP 缺失的 WH400 中过表达 VP_RS08405。从 WH001/pB08405 和 WH400/pB08405 中分离脂质 A 种类，并进行分析。结果表明，VP_RS08405 的功能依赖于 Kdo。这些发现为更好地了解副溶血弧菌中脂质 A 的结构多样性提供了依据。
