A novel antibiotic 3-isopropylhexahydro-4H-pyrido[1,2-α]pyrazine-1,4(6H)-dione isolated from the thermophilic bacterium Bacillus licheniformis QX928

doi:10.1016/j.jia.2024.04.021

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A novel antibiotic 3-isopropylhexahydro-4H-pyrido[1,2-α]pyrazine-1,4(6H)-dione isolated from the thermophilic bacterium Bacillus licheniformis QX928

Hulin Qiu¹*, Shaoxian Chen¹^,*, Aiguo Yin^{1, 2}, Tingting Miao¹, Fengfei Shen¹, Ying Li¹, Yunyi Xiao¹,Jinping Hai¹, Bo Xu^{1, 2}^#

¹College of Biological and Food Engineering, Guangdong University of Petrochemical Technology, Maoming 525000, China

²Maoming Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Maoming 525000, China

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摘要多重耐药菌对大多数天然抗生素和合成抗生素表现出抗药性，而细菌能够抵御抗生素杀伤作用的重要机制是形成生物膜，因此抑制或干扰生物膜的形成同时具有杀菌作用是一种有效应对细菌多重抗药性的策略。本研究从四川温泉中筛选到一株地衣芽孢杆菌命名为地衣芽孢杆菌QX928，该菌对金黄色葡萄球菌（Staphylococcus aureus）、肠炎沙门氏菌（Salmonellaenteritidis）、铜绿假单胞菌（Pseudomonas aeruginosa）、大肠杆菌（Escherichia coli）和梭状芽孢杆菌（Clostridium）等常见致病菌具有明显的抑制作用。从该菌发酵液中提取并纯化抑菌活性物质，采用核磁共振、气质联用、高效液相色谱、傅里叶红外结合薄层色谱检测QX928的抗菌活性组分后，鉴定该化合物为3-异丙基六氢-4H-吡哆[1,2-α]吡嗪-1,4(6H)-二酮（IPHPPD）。经SciFinder检索表明IPHPPD是一个由微生物合成同时具有抗菌和抗群体感应特性的抗生素化合物新分子。本研究以铜绿假单胞菌ATCC27853为实验菌，测定IPHPPD最低抑菌浓度为(13±0.17) mg L^-1，最低杀菌浓度为(22±0.72) mg L^-1。通过原子力显微镜、电子显微镜、荧光显微镜观察IPHPPD对铜绿假单胞菌生物膜形成过程和已生成生物膜的结构作用机制发现：在低浓度下，IPHPPD干扰了铜绿假单胞菌的信号因子和群体感应，从而改变了生物膜的形态和结构，而高浓度IPHPPD对铜绿假单胞菌的抑制作用更强烈，主要是通过降低其毒力因子、细胞膜通透性和能量代谢达到杀菌效果。经过1×MIC的IPHPPD处理后，转录组分析表明铜绿假单胞菌共有5,708个基因表达发生差异变化，2,516个基因表达被上调，3,192个基因表达被下调；其中与氨基酸代谢、ABC运输、丙酮酸代谢等相关基因表达被明显下调，而与细胞凋亡相关基因表达上调，表明IPHPPD能够与铜绿假单胞菌相互作用并干扰其细胞生理代谢进程，打破其稳定性，干扰细胞正常功能或者通过细胞膜与其胞内物质相互作用使其死亡。因此，本研究结果表明低浓度IPHPPD可以强烈抑制致病菌的运动、毒力因子的产生和生物膜的形成，为IPHPPD能够作为药物开发的潜在靶点提供了关键证据，证明IPHPPD在预防和治疗动物疾病药物开发方面具有巨大潜力。

Abstract A microbial strain designated Bacillus licheniformis QX928 was screened from hot springs in Sichuan Province, China, and a compound generated in the culture of this strain clearly inhibited Pseudomonas aeruginosa ATCC27853. The measured minimum inhibitory and lowest bactericidal concentrations were (13±0.17) and (22±0.72) mg L^-1, respectively. The compound was identified as 3-isopropylhexahydro-4H-pyrido[1,2-α]pyrazine-1,4(6H)-dione (IPHPPD). A SciFinder search revealed that IPHPPD could be the first compound synthesized by microorganisms that had both antibacterial and anti-quorum sensing properties. At low concentrations, IPHPPD interfered with the signaling factors and population effects of P. aeruginosa, thereby altering the biofilm morphology and structure. IPHPPD more strongly inhibited P. aeruginosa at high concentrations, primarily by reducing its virulence factors, cell membrane permeability and energy metabolism. A transcriptome analysis highlighted the role of IPHPPD in the transcriptional regulation of cellular metabolism and quorum sensing. Thus, the results of this study provide critical evidence that IPHPPD is a potential target for drug development to prevent and treat diseases in animals.

Keywords: thermophilic bacteria antibacterial mechanism Quorum sensing Pseudomonas aeruginosa

Online: 22 May 2024

Fund: This work was supported by the Guangdong Basic and Applied Basic Research Foundation (2022A1515012380, 2024A1515012511), the Guangdong Special Project on Key Fields of Colleges and Universities (Rural Revitalization, 2020ZDZX1020), Science and Technology Special Fund Project of Maoming Science and Technology Bureau (2021KJZXZJGSPDX003), and the Projects of Talents Recruitment of GDUPT (519033).

About author: Hulin Qiu, E-mail: 969152127@qq.com; Shaoxian Chen, E-mail: YUQChina@126.com;

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Hulin Qiu, Shaoxian Chen, Aiguo Yin, Tingting Miao, Fengfei Shen, Ying Li, Yunyi Xiao, Jinping Hai, Bo Xu. 2024. A novel antibiotic 3-isopropylhexahydro-4H-pyrido[1,2-α]pyrazine-1,4(6H)-dione isolated from the thermophilic bacterium Bacillus licheniformis QX928. Journal of Integrative Agriculture, Doi:10.1016/j.jia.2024.04.021

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