rbk基因超表达对类植物乳杆菌LR-1生物被膜、黏附特性及代谢谱的调控作用

Overexpression of rbk affects the biofilm formation, adhesion, and metabolic profile of Lactiplantibacillus paraplantarum LR-1

摘要:

目的 生物被膜形成和黏附能力是评价益生菌功效的重要指标。然而乳杆菌中特定基因与这些能力的关系尚不清楚。rbk基因编码核糖激酶，参与核糖代谢，可能与被膜形成和黏附能力相关。本研究旨在分析rbk基因超表达对类植物乳杆菌LR-1的生物被膜形成和黏附能力的影响，探讨其对群感及相关基因的调控，并从代谢谱角度揭示rbk超表达对菌体的影响机制。方法 以类植物乳杆菌LR-1为目标菌株，通过穿梭载体pMG76e构建rbk-pMG76e-LR-1重组菌株。通过qRT-PCR及酶活试验证实rbk基因超表达。采用结晶紫染色、细胞黏附试验和qRT-PCR分析rbk超表达对生物被膜形成、黏附能力，以及tuf、luxS、rpoN基因表达的影响；进一步通过非靶向代谢组学分析rbk超表达对代谢谱的影响；最后通过外源添加代谢物验证其对LR-1生物被膜形成和黏附能力的影响。结果 rbk基因超表达显著提升了LR-1的生物被膜形成能力(在不同条件下提升1.55-2.34倍)和对HT-29细胞的黏附能力(约3.58倍)，同时显著提升了tuf、luxS、rpoN基因表达水平(分别提升70.30、96.94、45.61倍)。非靶向代谢组学分析显示，rbk超表达导致145种代谢物丰度变化。外源添加代谢物的结果显示，l-脯氨酸、鼠李糖及NADH显著提高了LR-1生物被膜形成(分别提升1.27、1.39和1.25倍)和黏附能力(分别提升1.40、1.41和1.52倍)。结论 本研究表明rbk基因可以作为提升乳杆菌生物被膜形成及黏附能力的关键靶点。

关键词:乳杆菌;rbk基因超表达;生物被膜;黏附;非靶向代谢组学

Abstract:

Objective Biofilm formation and adhesion are important indicators for evaluating the beneficial effects of probiotics. However, the relationship of specific genes with the biofilm formation and adhesion of Lactiplantibacillus remains unclear. The rbk gene encodes ribokinase, which is involved in ribose metabolism and may be related to biofilm formation and adhesion. This study aims to analyze the effects of rbk overexpression on the biofilm formation and adhesion of Lactiplantibacillus paraplantrum LR-1, explore the role of this gene in the regulation of quorum sensing (QS) and expression of related genes, and reveal the influencing mechanism of rbk overexpression in bacteria from a metabolic profile perspective.Methods L. paraplantarum LR-1 was selected as the target strain, and the shuttle vector pMG76e was used to construct the recombinant strain rbk-pMG76e-LR-1. The overexpression of rbk was confirmed by qRT-PCR and the enzyme activity assay. Crystal violet staining, cell adhesion assay, and qRT-PCR were employed to evaluate the effects of rbk overexpression on biofilm formation, adhesion, and expression of tuf, luxS, and rpoN. Furthermore, untargeted metabolomics analysis was conducted to assess the effect of rbk overexpression on the metabolic profile. Finally, the effect on the biofilm formation and adhesion of LR-1 was verified by exogenous addition of metabolites.Results The overexpression of rbk increased the biofilm formation of LR-1 and the adhesion to HT-29 cells by 1.55-2.34 folds and 3.58 folds, respectively. Moreover, the overexpression of rbk up-regulated the expression levels of tuf, luxS, and rpoN by 70.30, 96.94, and 45.61 folds, respectively. The untargeted metabolomics analysis revealed that rbk overexpression led to changes in the abundance of 145 metabolites. Finally, the exogenous addition of l-proline, rhamnose, and nicotinamide adenine dinucleotide (NADH) increased the biofilm formation of LR-1 by 1.27, 1.39, and 1.25 folds and the adhesion by 1.40, 1.41, and 1.52 folds, respectively.Conclusion This study demonstrates that rbk can serve as a key target for enhancing the biofilm formation and adhesion of Lactiplantibacillus.

Key words:Lactiplantibacillus;rbk overexpression;biofilm;adhesion;untargeted metabolomics