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