Characterization of a secondary palmitoleoyltransferase of lipid A in Vibrio parahaemolyticus

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Enzyme and Microbial Technology Pub Date : 2024-08-22 DOI:10.1016/j.enzmictec.2024.110504

Danyang Huang , Lingyan Chen , Zhe Wang , Fenfang He , Xinrui Zhang , Xiaoyuan Wang

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Abstract

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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副溶血性弧菌中脂质 A 的次级棕榈酰基转移酶的特征

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。

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来源期刊

Enzyme and Microbial Technology 生物-生物工程与应用微生物

CiteScore

7.60

自引率

5.90%

发文量

142

审稿时长

38 days

期刊介绍： Enzyme and Microbial Technology is an international, peer-reviewed journal publishing original research and reviews, of biotechnological significance and novelty, on basic and applied aspects of the science and technology of processes involving the use of enzymes, micro-organisms, animal cells and plant cells. We especially encourage submissions on: Biocatalysis and the use of Directed Evolution in Synthetic Biology and Biotechnology Biotechnological Production of New Bioactive Molecules, Biomaterials, Biopharmaceuticals, and Biofuels New Imaging Techniques and Biosensors, especially as applicable to Healthcare and Systems Biology New Biotechnological Approaches in Genomics, Proteomics and Metabolomics Metabolic Engineering, Biomolecular Engineering and Nanobiotechnology Manuscripts which report isolation, purification, immobilization or utilization of organisms or enzymes which are already well-described in the literature are not suitable for publication in EMT, unless their primary purpose is to report significant new findings or approaches which are of broad biotechnological importance. Similarly, manuscripts which report optimization studies on well-established processes are inappropriate. EMT does not accept papers dealing with mathematical modeling unless they report significant, new experimental data.