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Mechanism of histidine kinases in responses to carvacrol stress in Neurospora crassa
CHEN Pengxu, LAN Ziyi, XI Juan, CHEN Yingying, ZHENG Weifa, ZHAO Yanxia
Mycosystema ›› 2025, Vol. 44 ›› Issue (4) : 240268.
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Mechanism of histidine kinases in responses to carvacrol stress in Neurospora crassa
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The role of histidine kinases (HKs) in response to carvacrol stimulation in Neurospora crassa was investigated by comparing the morphological changes, the formation of growth-inhibition zone, total superoxide dismutase (T-SOD) and catalase (CAT) activities, as well as carotenoid content between different hk-deficient mutants and the wild-type N. crassa under carvacrol stress. hk-deficient strains, with the exception of Δdcc1, exhibited a zone of growth-inhibition and increased malondialdehyde (MDA) synthesis under carvacrol stress. Analysis of T-SOD and CAT activities revealed that the deletion of hcp1 and sln1 resulted in inhibition of T-SOD activity, while the deletion of os1, phy2, and hk1 led to reduction of CAT activity. Carvacrol stress stimulated T-SOD activity in the wild-type strain and Δhcp1, Δsln1, Δdcc1, and Δhk16 mutants. However, it did not induce CAT activity in the strains Δhcp1, Δnik2, Δphy2, Δluxq, Δdcc1, and Δhk16. Carotenoids are the main components within N. crassa and have antioxidant properties. It was observed that the deletion of hk9, os1, sln1, phy1, phy2, luxq, hk1, and dcc1 resulted in inhibition of carotenoid synthesis, but the carvacrol stress promoted the synthesis of carotenoids in Δsln1, Δluxq, and Δhk16 mutants. In summary, N. crassa responded to carvacrol stress by regulating the activities of T-SOD and CAT and the synthesis of carotenoids through HKs which sense the signal from carvacrol.
histidine kinase / carvacrol / carotenoids / malondialdehyde / superoxide dismutase {{custom_keyword}} /
Table 1 Content of main active components in CME表1 CME中主要活性成分含量 |
| 成分 Components | 含量 Content (mg/g) |
|---|---|
| 多糖 Polysaccharide | 509.47±14.73 |
| 尿苷Uridine | 1.21±0.10 |
| 鸟苷Guanosine | 0.66±0.03 |
| 腺苷Adenosine | 1.47±0.11 |
| 虫草素 Cordycepin | 2.66±0.15 |
| N6-(2-羟乙基)腺苷 N6-(2-hydroxyethtl)-adenosine | 2.15±0.09 |
| 总多酚 Total polyphenols | 9.21±0.37 |
| 类胡萝卜素 Carotenoid | 0.95±0.04 |
Fig. 1 Determination of monosaccharide composition by HPLC. A: Mixed reference substance; B: Tested samples. 1: Man; 2: Rha; 3: Glc-UA; 4: Gal-UA; 5: Lactose (internal standard); 6: Glc; 7: Gal: 8: Xyl; 9: Ara; 10: Fuc.图1 HPLC检测单糖组成 A：混合对照品；B：供试品. 1：甘露糖；2：鼠李糖；3：葡萄糖醛酸；4：半乳糖醛酸；5：乳糖内标；6：葡萄糖；7：半乳糖；8：木糖；9：阿拉伯糖；10：岩藻糖 |
Table 2 Monosaccharide composition of Cordyceps militaris polysaccharide表2 蛹虫草多糖的单糖组成 |
| 样品 Sample | 甘露糖 Man | 半乳糖醛酸 Gal-UA | 葡萄糖 Glc | 半乳糖 Gal | 阿拉伯糖 Ara |
|---|---|---|---|---|---|
| 含量 Content (mol%) | 25.73 | 9.26 | 29.01 | 29.39 | 5.62 |
Fig. 2 Determination of nucleosides by HPLC. A: Mixed reference substance; B: Test sample. 1: Cytidine; 2: Uridine; 3: Guanosine; 4: Thymidine; 5: Adenosine; 6: Cordycepin; 7: N6-(2-hydroxyethtl)- adenosine.图2 HPLC检测核苷类成分 A：混合对照品；B：供试品. 1：胞苷；2：尿苷；3：鸟苷；4：胸苷；5：腺苷；6：虫草素；7：N6-(2-羟乙基)腺苷 |
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The monoterpene carvacrol, the major component of oregano and thyme oils, is known to exert potent antifungal activity against the pathogenic yeast Candida albicans. This monoterpene has been the subject of a considerable number of investigations that uncovered extensive pharmacological properties, including antifungal and antibacterial effects. However, its mechanism of action remains elusive. Here, we used integrative chemogenomic approaches, including genome-scale chemical-genetic and transcriptional profiling, to uncover the mechanism of action of carvacrol associated with its antifungal property. Our results clearly demonstrated that fungal cells require the unfolded protein response (UPR) signaling pathway to resist carvacrol. The mutants most sensitive to carvacrol in our genome-wide competitive fitness assay in the yeast Saccharomyces cerevisiae expressed mutations of the transcription factor Hac1 and the endonuclease Ire1, which is required for Hac1 activation by removing a nonconventional intron from the 3' region of HAC1 mRNA. Confocal fluorescence live-cell imaging revealed that carvacrol affects the morphology and the integrity of the endoplasmic reticulum (ER). Transcriptional profiling of pathogenic yeast C. albicans cells treated with carvacrol demonstrated a bona fide UPR transcriptional signature. Ire1 activity detected by the splicing of HAC1 mRNA in C. albicans was activated by carvacrol. Furthermore, carvacrol was found to potentiate antifungal activity of the echinocandin antifungal caspofungin and UPR inducers dithiothreitol and tunicamycin against C. albicans. This comprehensive chemogenomic investigation demonstrated that carvacrol exerts its antifungal activity by altering ER integrity, leading to ER stress and the activation of the UPR to restore protein-folding homeostasis. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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Studies on bacterial physiology are incomplete without knowledge of the signalling and regulatory systems that a bacterium uses to sense and respond to its environment. Two-component systems (TCSs) are among the most prevalent bacterial signalling systems, and they control essential and secondary physiological processes; however, even in model organisms, we lack a complete understanding of the signals sensed, the phosphotransfer partners and the functions regulated by these systems. In this review, we discuss several tools to map the genes targeted by transcriptionally acting TCSs. Many of these tools have been used for studying individual TCSs across diverse species, but systematic approaches to delineate entire signalling networks have been very few. Since genome sequences and high-throughput technologies are now readily available, the methods presented here can be applied to characterize the entire DNA-binding TCS signalling network in any bacterial species and are especially useful for non-model environmental bacteria.© 2020 The Authors. Environmental Microbiology Reports published by Society for Applied Microbiology and John Wiley & Sons Ltd.
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香芹酚是一种存在于精油中的天然化合物,对多种微生物具有抗菌作用。该研究旨在研究香芹酚对蜡样芽孢杆菌的抑菌作用及其在白腐乳后发酵过程中的应用。研究中测定了香芹酚对2种蜡样芽孢杆菌的最小抑菌浓度(minimum inhibitory concentration, MIC)和最小杀菌浓度(minimum bactericidal concentration, MBC),分别为0.312 5 mg/mL和0.625 0 mg/mL,第24 h的抑菌率分别为94.06%和94.07%。同时考察了香芹酚对菌落形成、孢子萌发、分子泄露和细胞形态的影响,并通过在白腐乳后发酵阶段添加香芹酚来评估其应用效果。结果显示,在最小抑菌浓度下,香芹酚显著降低了孢子的萌发率,并导致蜡样芽孢杆菌细胞形态的改变和细胞膜的破坏,引起核酸、蛋白质和酶的泄露。此外,香芹酚处理组的白腐乳菌落总数、蜡样芽孢杆菌数和孢子数分别减少了84.62%、47.37%和93.15%。因此,香芹酚有望作为一种有效的抑菌剂,用于控制腐乳中的蜡样芽孢杆菌。
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γ-氨基丁酸(γ-aminobutyric acid，GABA)是一种功能性非蛋白氨基酸，在维持人体健康方面发挥着重要作用。为探明食用菌子实体的GABA含量水平和影响食用菌GABA产量的因素，本研究通过对食用菌GABA提取和衍生条件优化，建立了食用菌GABA检测的9-芴甲氧羰酰氯(FMOC-Cl)柱前衍生HPLC法;利用该方法测定了20种中国主要食用菌子实体的GABA含量，并进一步探究了培养料氮源含量和种类、栽培时间对GABA含量的影响。食用菌GABA提取的最佳料液比、时间和温度分别为1:100、1 h和65 ℃。20种食用菌子实体的GABA含量在0.20-3.02 mg/g DW，存在显著种间差异，其中黄白银耳GABA含量最高(3.02 mg/g DW)，香菇、银耳、金针菇、双孢蘑菇的GABA含量也较高，为1-2 mg/g DW。增加培养料含氮量可显著促进GABA积累，且添加尿素的促进效果明显优于添加麦麸。栽培时间也可影响子实体GABA产量，第二茬和三茬子实体的GABA含量显著高于第一茬子实体。综上可知，食用菌GABA含量不仅与品种密切相关，而且受到培养料中氮源含量和种类、栽培时间的影响。
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纽芬兰鸡油菌是北美东部一种深受欢迎的美味食用菌。火烧山，位于加拿大格罗莫恩国家公园中心、三面环水，该地纽芬兰鸡油菌的持续出菇时间至少可追溯至20世纪60年代且未见于周边地区。研究该持续存在的孤立种群，对于资源保护和开发具有重要意义。本研究在火烧山共采集纽芬兰鸡油菌子实体109个，包括间距100 m以上的6个局域群体共81个个体以及5 d后随机采集的28个个体。基于微卫星标记的基因分型结果显示，3个位点上分别有3、5和2个等位基因，而每个局域群体的各位点均被1到2个等位基因主导；共有多位点基因型29个。每个局域群体和总样本中的基因型频率总体上符合哈迪-温伯格平衡。虽然总样本中存在显著的遗传分化，但克隆校正后未检测到差异。比较间隔5 d的两次取样，等位基因和基因型频率存在微小差异。此外，与纽芬兰岛另外两地(分别相距约200 km和600 km)和安大略省哈密尔顿市一地(约2 000 km外)共3个地区群体样本进行了比较。结果表明，突变、有性生殖、基因流、选择和遗传漂变都对纽芬兰鸡油菌种群的遗传多样性产生了影响。
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Table 1 Content of main active components in CME
Fig. 1 Determination of monosaccharide composition by HPLC. A: Mixed reference substance; B: Tested samples. 1: Man; 2: Rha; 3: Glc-UA; 4: Gal-UA; 5: Lactose (internal standard); 6: Glc; 7: Gal: 8: Xyl; 9: Ara; 10: Fuc.Table 2 Monosaccharide composition of Cordyceps militaris polysaccharideFig. 2 Determination of nucleosides by HPLC. A: Mixed reference substance; B: Test sample. 1: Cytidine; 2: Uridine; 3: Guanosine; 4: Thymidine; 5: Adenosine; 6: Cordycepin; 7: N6-(2-hydroxyethtl)- adenosine.Fig. 3 Scavenging effects of CME on radicals. A: DPPH radical scavenging activities; B: ABTS radical scavenging activities; C: ·OH radical scavenging activities.Fig. 4 Effects of different concentrations of CME on the viabilities of HaCaT cells.Fig. 5 Effects of different concentrations of H2O2 on the viabilities of HaCaT cells.Fig. 6 Effects of different concentrations of CME on the viabilities of HaCaT cells induced by H2O2. Compared with control group, ##P<0.01; Compared with model group, *P<0.05, **P<0.01. The same below.Fig. 7 Effects of different concentrations of CME on ROS levels in HaCaT cells.Fig. 8 Effects of different concentrations of CME on MDA contents (A) and the activities of SOD (B), GSH-Px (C), CAT (D) in HaCaT cells.
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