Identification of a 3-deoxy-D-manno-octulosonic acid kinase of lipid A in Vibrio parahaemolyticus

IF 3.9 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology Pub Date : 2026-02-01 Epub Date: 2025-11-04 DOI:10.1016/j.enzmictec.2025.110775

Lingyan Chen , Danyang Huang , Xinrui Zhang , Hongchen Yin , Xiaoyuan Wang

{"title":"Identification of a 3-deoxy-D-manno-octulosonic acid kinase of lipid A in Vibrio parahaemolyticus","authors":"Lingyan Chen , Danyang Huang , Xinrui Zhang , Hongchen Yin , Xiaoyuan Wang","doi":"10.1016/j.enzmictec.2025.110775","DOIUrl":null,"url":null,"abstract":"<div><div>Vibrio parahaemolyticus is a major seafood-associated foodborne pathogen whose lipopolysaccharide (LPS) plays an important role in virulence and antimicrobial resistance. The LPS of V. parahaemolyticus contains a single 3-deoxy-D-manno-octulosonic acid (Kdo) sugar with phosphorylation. Previously, we have characterized the gene VP_RS01035 is responsible for the addition of Kdo; in this study, we characterized another gene VP_RS00960 which is responsible for the Kdo phosphorylation of V. parahaemolyticus LPS. To investigate its function, we first constructed an LPS-deficient Escherichia coli WH600 strain using CRISPR/Cas9. Heterologous expression of VP_RS01035 alone or in combination with VP_RS00960 yielded recombinant strains WH600/pB1–1 and WH600/pB2–12, respectively. Analysis of total lipids from the recombinant strains by thin-layer chromatography and high-performance liquid chromatography-tandem mass spectrometry demonstrated that the VP_RS00960 gene encodes a Kdo kinase responsible for phosphorylating the 4-OH site of the Kdo sugar in V. parahaemolyticus. To further validate its role, we deleted VP_RS00960 gene in V. parahaemolyticus, resulting in mutant ΔRS00960. Notably, ΔRS00960 failed to produce polysaccharide-linked lipid A, although free lipid A synthesis remained unaffected. Furthermore, defective long-chain LPS assembly compromised outer membrane integrity, increasing permeability and hydrophobicity while reducing biofilm formation. Consequently, ΔRS00960 exhibited heightened susceptibility to membrane-targeting antibiotics, such as erythromycin and novobiocin. Macrophage infection assays using RAW264.7 cells revealed that VP_RS00960 deletion attenuated bacterial pathogenicity. These findings enhance the understanding of the pathogenicity and drug resistance of V. parahaemolyticus, and provide novel insights and strategies for addressing antibiotic resistance and food safety challenges posed by V. parahaemolyticus.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"193 ","pages":"Article 110775"},"PeriodicalIF":3.9000,"publicationDate":"2026-02-01","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/S0141022925001954","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2025/11/4 0:00:00","PubModel":"Epub","JCR":"Q2","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}

引用次数: 0

Abstract

Vibrio parahaemolyticus is a major seafood-associated foodborne pathogen whose lipopolysaccharide (LPS) plays an important role in virulence and antimicrobial resistance. The LPS of V. parahaemolyticus contains a single 3-deoxy-D-manno-octulosonic acid (Kdo) sugar with phosphorylation. Previously, we have characterized the gene VP_RS01035 is responsible for the addition of Kdo; in this study, we characterized another gene VP_RS00960 which is responsible for the Kdo phosphorylation of V. parahaemolyticus LPS. To investigate its function, we first constructed an LPS-deficient Escherichia coli WH600 strain using CRISPR/Cas9. Heterologous expression of VP_RS01035 alone or in combination with VP_RS00960 yielded recombinant strains WH600/pB1–1 and WH600/pB2–12, respectively. Analysis of total lipids from the recombinant strains by thin-layer chromatography and high-performance liquid chromatography-tandem mass spectrometry demonstrated that the VP_RS00960 gene encodes a Kdo kinase responsible for phosphorylating the 4-OH site of the Kdo sugar in V. parahaemolyticus. To further validate its role, we deleted VP_RS00960 gene in V. parahaemolyticus, resulting in mutant ΔRS00960. Notably, ΔRS00960 failed to produce polysaccharide-linked lipid A, although free lipid A synthesis remained unaffected. Furthermore, defective long-chain LPS assembly compromised outer membrane integrity, increasing permeability and hydrophobicity while reducing biofilm formation. Consequently, ΔRS00960 exhibited heightened susceptibility to membrane-targeting antibiotics, such as erythromycin and novobiocin. Macrophage infection assays using RAW264.7 cells revealed that VP_RS00960 deletion attenuated bacterial pathogenicity. These findings enhance the understanding of the pathogenicity and drug resistance of V. parahaemolyticus, and provide novel insights and strategies for addressing antibiotic resistance and food safety challenges posed by V. parahaemolyticus.

查看原文本刊更多论文

副溶血性弧菌脂质a 3-脱氧-d -甘露糖醛酸激酶的鉴定

副溶血性弧菌是一种主要的海产食源性病原菌，其脂多糖（LPS）在毒力和耐药性中起着重要作用。副溶血性弧菌的LPS含有一个磷酸化的3-脱氧-d -甘露糖醛酸（Kdo）糖。之前，我们已经鉴定了VP_RS01035基因负责Kdo的添加；在这项研究中，我们鉴定了另一个基因VP_RS00960，该基因负责副溶血性弧菌LPS的Kdo磷酸化。为了研究其功能，我们首先利用CRISPR/Cas9构建了一株缺乏lps的大肠杆菌WH600菌株。VP_RS01035单独或与VP_RS00960联合异种表达分别产生重组菌株WH600/ pB1-1和WH600/ pB2-12。利用薄层色谱和高效液相色谱-串联质谱法对重组菌株的总脂质进行了分析，结果表明VP_RS00960基因编码Kdo激酶，负责磷酸化副溶血性弧菌Kdo糖的4-OH位点。为了进一步验证其作用，我们在副溶血性弧菌中删除VP_RS00960基因，导致突变ΔRS00960。值得注意的是，ΔRS00960不能产生多糖连接的脂质A，尽管游离脂质A的合成不受影响。此外，有缺陷的长链LPS组装破坏了外膜的完整性，增加了渗透性和疏水性，同时减少了生物膜的形成。因此，ΔRS00960对膜靶向抗生素（如红霉素和新生物素）表现出更高的敏感性。RAW264.7细胞的巨噬细胞感染实验显示，VP_RS00960缺失降低了细菌的致病性。这些发现增强了对副溶血性弧菌致病性和耐药性的认识，并为解决副溶血性弧菌的抗生素耐药性和食品安全挑战提供了新的见解和策略。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

求助全文

约1分钟内获得全文求助全文

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.