关键词: 白念珠菌, 氧化性应激, 巨噬细胞, ATP1基因

Abstract: 【Abstract】 Objective To investigate the physiological role of F1Fo-ATP synthase α-subunit encoding gene （ATP1） in promoting Candida albicans （C. albicans） to escape from macrophage killing through eliminating intracellular reactive oxygen species （ROS） by using a reverse genetics approach. Methods An ATP1 deletion strain and a parental strain of C. albicans were cultured on the YPD media, and the number of formed colonies on the plates was counted to evaluate in vitro viability of C. albicans. To evaluate their in vivo viability, the ATP1 deletion strain and parental strain of C. albicans were inoculated into mice through the caudal vein, kidney tissues were taken out from the mice 1-7 days after the infection, and inoculated onto the YPD medium followed by numeration of colonies after 48 hours of culture. After co-culture of overnight-cultured C. albicans suspensions with macrophages, some of the C. albicans suspensions were inoculated onto the YPD solid medium followed by numeration of colonies and determination of survival rate, and some culture supernatants were inoculated into the 96-well plate for detection of the level of lactate dehydrogenase （LDH） released by macrophages by LDH release assay. A model mimicking oxidative stress in macrophages was established by using hydrogen peroxide. After treatment with hydrogen peroxide, the number of colonies was counted to compare the viability of the C. albicans strains. DCFH-DA staining was conducted to detect the intracellular ROS level in C. albicans after co-culture with macrophages or treatment with hydrogen peroxide, and real-time fluorescence-based quantitative PCR to measure mRNA expression of catalase 1 （CAT1）, superoxide dismutase 4 （SOD4） and SOD5 genes in C. albicans after treatment with hydrogen peroxide. Statistical analysis was carried out by using two-way analysis of variance or Student t test. Results In vitro, the colony number in both the parental strain group and ATP1 deletion strain group gradually increased over time; after 24 hours, the colony number of the ATP1 deletion strain group was only 10% of that in the parental strain group （F = 481.84, P < 0.001）. The number of colony formed by the parental strain-infected mouse kidney tissues gradually increased over time, but that by the ATP1 deletion strain-infected mouse kidney tissues gradually decreased, and there was a significant difference between the two groups （F = 78.27, P = 0.001）. After in vitro co-culture of C. albicans with macrophages, the survival rate in the ATP1 deletion strain group （62.67% ± 3.51%） was significantly lower than that in the parental strain group （82.33% ± 2.52%, t = 7.88, P = 0.001）, and the percentage of LDH released by macrophages was also significantly lower in the ATP1 deletion strain group （27.80% ± 3.54%） than in the parental strain group （87.78% ± 0.17%, t = 33.89, P < 0.001）, which were consistent with the in vivo results. In the model mimicking oxidative stress, the viability of the ATP1 deletion strain group was significantly lower than that of the parental strain group （F = 3 440.65, P < 0.001）. Both in the co-culture model with macrophages and in the model mimicking oxidative stress in macrophages, the intracellular ROS levels were significantly higher in the ATP1 deletion strain group than in the parental strain group （both P < 0.001）. Furthermore, the mRNA expression of CAT1, SOD4 and SOD5 genes was significantly lower in the ATP1 deletion strain group than in the parental strain group after treatment with hydrogen peroxide （all P < 0.001）. Conclusion ATP1 deletion may reduce the capabilities of C. albicans to counteract oxidative stress and eliminate ROS, likely by down-regulating the expression of oxidative stress- and ROS clearance-related genes respectively, which may prevent C. albicans from escaping from the macrophage killing and lead it to be eliminated by the host ultimately.

Key words: Candida albicans, Oxidative stress, Macrophages, ATP1 gene