中华皮肤科杂志 ›› 2025, Vol. 58 ›› Issue (12): 1134-1140.doi: 10.35541/cjd.20240612

• 论著·真菌感染性皮肤病 • 上一篇    下一篇

白念珠菌转录因子RBF1基因缺失对巨噬细胞和小鼠毒力效应研究

周蒙1，2    杨航1    余宇1    梅嬛1    李冬梅3    刘维达1，2，4    佘晓东1，2   

  1. 1中国医学科学院、北京协和医学院皮肤病研究所真菌科，南京  210042；2江苏省皮肤医学重点实验室，南京  210042；3乔治敦大学医学中心微生物学和免疫学系，华盛顿特区  20007；4南京医科大学公共卫生学院全球卫生中心，南京  211166
  • 收稿日期:2024-11-11 修回日期:2025-10-21 发布日期:2025-12-04
  • 通讯作者: 佘晓东 E-mail:shexd1979@163.com
  • 基金资助:
    国家重点研发计划（2022YFC3601800）；国家自然科学基金（82304040，82473541）；中国医学科学院医学与健康科技创新工程/中国医学科学院临床与转化医学研究专项（2023-I2M-C&T-B-114）；江苏省科技能力提升工程江苏医学重点实验室（ZDXYS202204）

Effects of the transcription factor RBF1 gene deletion in Candida albicans on its virulence towards macrophages and mice

Zhou Meng1,2, Yang Hang1, Yu Yu1, Mei Huan1, Li Dongmei3, Liu Weida1,2,4, She Xiaodong1,2   

  1. 1Department of Medical Mycology, Hospital for Skin Diseases, Institute of Dermatology, Chinese Academy of Medical Sciences ＆ Peking Union Medical College, Nanjing 210042, China; 2Jiangsu Provincial Key Laboratory of Dermatology, Nanjing 210042, China; 3Department of Microbiology & Immunology, Georgetown University Medical Center, Washington, DC 20007, USA; 4Center for Global Health, School of Public Health, Nanjing Medical University, Nanjing 211166, China
  • Received:2024-11-11 Revised:2025-10-21 Published:2025-12-04
  • Contact: She Xiaodong E-mail:shexd1979@163.com
  • Supported by:
    National Key Research and Development Program of China （2022YFC3601800）; National Natural Science Foundation of China （82304040, 82473541）; CAMS Innovation Fund for Medical Sciences （2023-I2M-C&T-B-114）; Jiangsu Provincial Medical Key Laboratory, Jiangsu Province Capability Improvement Project through Science, Technology, and Education  （ZDXYS202204）

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摘要： 【摘要】 目的 探究白念珠菌敲除转录因子RBF1基因后对宿主毒力的影响。方法 通过构建RBF1敲除的白念珠菌菌株（rbf1Δ），分别感染巨噬细胞系RAW 264.7细胞和小鼠进行体外和体内实验。体外实验中，将RAW264.7细胞与野生型（WT）和rbf1Δ白念珠菌分别共培养6、12、24、48 h，采用菌计数法和乳酸脱氢酶（LDH）检测实验评估白念珠菌对巨噬细胞的毒力，实时荧光定量PCR检测毒力相关基因表达水平。体内实验中，通过尾静脉注射WT和rbf1Δ白念珠菌各感染20只小鼠并记录存活时间，在感染后14 d摘取肾脏，采用肾脏研磨法检测小鼠体内菌载量，采用HE染色检测肾脏中免疫细胞浸润程度和炎症水平。结果 rbf1Δ组白念珠菌在与巨噬细胞共培养6、12、24、48 h后，巨噬细胞释放的LDH水平（24.03% ± 3.69%、31.96% ± 7.65%、12.05% ± 2.82%、16.75% ± 6.03%）均显著低于WT组（41.11% ± 3.69%、67.42% ± 7.65%、25.12% ± 2.82%、44.67% ± 6.03%，均P < 0.05），其中共培养6、12 h时rbf1Δ组剩余菌落数［（26.33 ± 7.09） × 103菌落形成单位（CFU）和（17.33 ± 3.06） × 103 CFU］均低于WT组（234.67 ± 22.03） × 103 CFU和（462.00 ± 59.63） × 103 CFU，LSD-t值分别为9.29、19.83，均P < 0.001），而共培养24、48 h时两组剩余菌落数差异无统计学意义（P > 0.05）。实时荧光定量PCR显示，与WT白念珠菌相比，rbf1Δ白念珠菌中HWP1 mRNA表达量较高（P = 0.001），而PLD1、PLB1、SAP2和EGR11 mRNA表达量均较低（均P < 0.05）。体内实验中，20 d内rbf1Δ组小鼠仅2只死亡，WT组小鼠死亡14只；感染后14 d，rbf1Δ组小鼠肾脏菌载量［（31.33 ± 4.04） CFU］明显低于对照组［（165.78 ± 7.10） CFU，t = 28.5，P < 0.001］，且肾脏免疫细胞浸润程度低于对照组。结论 敲除RBF1的白念珠菌对巨噬细胞和小鼠的毒力均显著减弱。

关键词: 白色念珠菌, 基因敲除技术, RBF1, 毒力, 巨噬细胞, 小鼠

Abstract: 【Abstract】 Objective To investigate effects of the transcription factor RPG-box binding factor （RBF1） gene deletion in Candida albicans on its virulence towards the host. Methods RBF1-knockout（rbf1Δ） Candida albicans strains were constructed and used to infect macrophages RAW 264.7 and mice for in vitro and in vivo experiments. In vitro, RAW 264.7 cells were co-cultured with wild-type （WT） or rbf1Δ Candida albicans for 6, 12, 24, and 48 hours. The virulence of Candida albicans towards macrophages was assessed by the colony-counting method and lactate dehydrogenase （LDH） release assay, and the expression levels of virulence-related genes were detected by real-time fluorescence-based quantitative PCR （qRT-PCR）. In vivo, mice were infected with WT or rbf1Δ strains via tail vein injection （20 mice in each group）, and survival time was recorded. Fourteen days after infection, mouse kidneys were harvested, and the fungal load in mouse kidneys was determined by tissue homogenization. The degree of immune cell infiltration and inflammation in the kidneys was assessed by hematoxylin-eosin （HE） staining. Results After 6, 12, 24, and 48 hours of co-culture with macrophages, the levels of LDH released by macrophages were significantly lower in the rbf1Δ group （24.03% ± 3.69%, 31.96% ± 7.65%, 12.05% ± 2.82%, and 16.75% ± 6.03%, respectively） than in the WT group （41.11% ± 3.69%, 67.42% ± 7.65%, 25.12% ± 2.82%, and 44.67% ± 6.03%, respectively; all P < 0.05）. The remaining fungal colony counts at 6 and 12 hours were significantly lower in the rbf1Δ group （［26.33 ± 7.09］ × 103 colony forming unit ［CFU］ and ［17.33 ± 3.06］ × 103 CFU, respectively） than in the WT group （［234.67 ± 22.03］ × 103 CFU and ［462.00 ± 59.63］ × 103 CFU, respectively; least significant difference-t values were 9.29 and 19.83, respectively, both P < 0.001）, while there were no significant differences in the remaining fungal colony counts at 24 and 48 hours between the two groups （both P > 0.05）. As qRT-PCR showed, compared with the WT strains, the rbf1Δ strains exhibited significantly increased HWP1 mRNA expression （P = 0.001）, but significantly decreased mRNA expression of PLD1, PLB1, SAP2, and EGR11 （all P < 0.05）. In the in vivo experiment, only 2 mice died in the rbf1Δ group, whereas 14 mice died in the WT group within 20 days; 14 days after infection, the kidney fungal load was significantly lower in the rbf1Δ group （31.33 ± 4.04 CFU） than in the control group （165.78 ± 7.10 CFU; t = 28.5, P < 0.001）, so was the degree of immune cell infiltration in the kidneys. Conclusion The virulence of RBF1-knockout Candida albicans towards macrophages and mice was significantly attenuated.

Key words: Candida albicans, Gene knockout techniques, RBF1, Virulence, Macrophages, Mice

引用本文

周蒙, 杨航 余宇 梅嬛 李冬梅 刘维达, 佘晓东, . 白念珠菌转录因子RBF1基因缺失对巨噬细胞和小鼠毒力效应研究[J]. 中华皮肤科杂志, 2025,58(12):1134-1140. doi:10.35541/cjd.20240612

Zhou Meng, Yang Hang, Yu Yu, Mei Huan, Li Dongmei, Liu Weida, She Xiaodong, . Effects of the transcription factor RBF1 gene deletion in Candida albicans on its virulence towards macrophages and mice[J]. Chinese Journal of Dermatology, 2025, 58(12): 1134-1140.doi:10.35541/cjd.20240612