粪肠球菌HHT-1拮抗镉和苯胺单一及复合胁迫的转录调控

Transcriptional regulation of Enterococcus faecalis HHT-1 against single and combined stress of cadmium and aniline

摘要:

目的 土壤微生物耐受单一重金属或有机污染物的生物调控机制已被广泛研究，但微生物应对复合污染的生物学机制仍不清楚。本研究旨在通过转录组学揭示粪肠球菌HHT-1耐受镉(Cd)和苯胺(aniline, AN)单一及复合污染胁迫的生物学应对机制。方法 采用转录组学方法，探究在半数抑制浓度(IC₅₀)下HHT-1在Cd (150 mg/L)和苯胺(2 g/L)单一及复合胁迫(Cd浓度150 mg/L+苯胺浓度2 g/L)的转录调控机制。结果 在Cd单一胁迫下，HHT-1通过增强金属结合蛋白和转运蛋白基因的表达，促进Cd²⁺的固定或排出。高浓度Cd引发细胞内的氧化应激反应，HHT-1上调核糖体和核苷酸相关基因的表达，以修复由氧化应激引起的蛋白质和DNA损伤，并产生过氧化氢酶清除细胞内的活性氧(reactive oxygen species, ROS)。此外，Cd胁迫还诱导HHT-1中致病基因和抗生素抗性相关基因的上调。在苯胺单一胁迫下，HHT-1激活苯胺降解酶基因的表达以降低其毒性，并上调药物外排蛋白基因将苯胺排出细胞外。苯胺同样引起细胞内的氧化应激，HHT-1通过上调谷胱甘肽合成酶基因来清除ROS。在Cd和苯胺复合胁迫下，HHT-1展现出复杂的应对机制。首先，HHT-1通过上调金属转运蛋白和药物外排蛋白基因，有效地将Cd和苯胺这2种有害物质排出细胞外，减轻了它们对细胞的毒性影响。同时，HHT-1通过过氧化氢酶和谷胱甘肽双重途径清除细胞内的ROS。在复合胁迫下，HHT-1上调致病基因和抗生素抗性相关基因，可能增强其致病性和耐药性。值得注意的是，在复合胁迫下，HHT-1并未显著表达苯胺降解酶相关基因。结论 粪肠球菌HHT-1对Cd和苯胺复合胁迫的转录调控机制结合了单一Cd胁迫和单一苯胺胁迫下的转录调控机制，但与单一Cd胁迫下的机制更为接近。在复合胁迫下，HHT-1主要通过增强细胞壁合成、Cd²⁺外排、DNA修复、清除ROS等机制进行调控，同时结合了苯胺胁迫下的药物外排蛋白、谷胱甘肽等相关基因的表达。Cd单一及Cd和苯胺复合胁迫都可能增加HHT-1的致病潜力和耐药性。

关键词:粪肠球菌;转录组学;镉;苯胺;复合污染

Abstract:

Objective The biological regulatory mechanisms of soil microorganisms in response to single heavy metals or organic pollutants have been extensively studied, while the biological mechanisms of microbial responses to combined pollution remain unclear. This study aims to reveal the biological mechanisms of Enterococcus faecalis HHT-1 in response to single and combined stress of cadmium (Cd) and aniline (AN) by transcriptomics.Methods Transcriptomics was employed to explore the transcriptional regulation of HHT-1 under single (Cd: 150 mg/L; AN: 2 g/L) and combined stress (150 mg/L Cd and 2 g/L AN) at half inhibitory concentrations (IC₅₀).Results Under single Cd stress, HHT-1 upregulated the expression of genes encoding metal-binding proteins and transporters to promote the sequestration or efflux of Cd²⁺. High concentrations of Cd induced oxidative stress in cells, and HHT-1 upregulated the expression of ribosome-related genes and nucleotide-related genes to repair the protein damage and DNA damage caused by oxidative stress, and cleared intracellular reactive oxygen species (ROS) by producing catalase. Moreover, Cd stress upregulated the expression of genes related to virulence and antibiotic resistance in HHT-1. Under the single stress of AN, HHT-1 activated the expression of AN-degrading enzyme genes to reduce its toxicity and upregulated the expression of genes encoding efflux pumps to excrete AN from the cell. AN also caused intracellular oxidative stress, and HHT-1 cleared the ROS by upregulating the expression of glutathione synthase genes. Under the combined stress of Cd and AN, HHT-1 exhibited a complex array of adaptive mechanisms. Initially, HHT-1 upregulated the expression of genes encoding metal transporters and efflux pumps to transport Cd and AN out of the cell, thereby mitigating their toxic effects on the cell. At the same time, HHT-1 cleared intracellular ROS via the dual pathway of catalase and glutathione. In addition, HHT-1 upregulates the expression of virulence and antibiotic resistance genes under combined stress, which may enhance its pathogenicity and resistance. It is worth noting that HHT-1 did not significantly upregulate the expression of AN-degrading enzyme-related genes under the combined stress.Conclusion The transcriptional regulation mechanism of E. faecalis HHT-1 in response to the combined stress of Cd and AN is a combination of the transcriptional regulation mechanisms observed under single Cd stress and single AN stress, and it is more similar to the mechanism observed under single Cd stress. HHT-1 mainly responds to the combined stress of Cd and AN by enhancing cell wall synthesis, Cd²⁺ efflux, DNA repair, and ROS scavenging. Genes associated with efflux pumps and glutathione under AN stress continue to be expressed under the combined stress. Both single Cd stress and combined stress of Cd and AN may increase the potential pathogenicity and drug resistance of HHT-1.

Key words:Enterococcus faecalis;transcriptomics;cadmium;aniline;combined pollution