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| Functional study of BMH2 in regulating the pathogenic infection ability of Cryptococcus neoformans |
| LI Jie1 HE Zhichun2 ZHENG Fanglin1 XU Xinping1 |
1.Jiangxi Institute of Respiratory Disease, the First Affiliated Hospital of Nanchang University, Jiangxi Province, Nanchang 330006, China;
2.Department of Respiratory, Yichun People’s Hospital, Jiangxi Province, Yichun 336000, China
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Abstract Objective To study the functional role of BMH2 gene in Cryptococcus neoformans. Methods Using CRISPR-Cas9 combined electroporation, the original promoter of BMH2 gene was replaced with the inducible promoter CTR4 construction conditional knockout mutant strain. The original promoter of BMH2 gene was supplemented to construct a complement strain. The strains were cultured in basic YPD/copper-rich YPD solid medium to observe the growth of the strains; the growth curve was plotted in copper-rich YPD liquid medium; the cell morphology was observed in basic YPD/copper-rich YPD liquid medium; the hypha growth was observed in basic V8/copper-rich V8 solid medium; Basic YPD/copper-rich YPD solid medium was cultured at 25℃, 30℃, and 37℃, and the growth was observed at different temperatures; the growth was observed in copper-rich YPD and copper-rich YPD+stress solid medium. Results There was no difference in the growth of all strains in basic YPD solid medium； in copper-rich YPD solid medium, the growth of mutant train was weakened, but there was no difference between wild type and complement strain. Compared with the wild type, the survival curve of mutant train decreased, and the trend of complement strain was consistent with that of wild type. There was no difference in cell morphology of all strains in basic YPD liquid medium; in copium-rich YPD liquid medium, there was no difference in cell morphology between wild-type and complement strain, but the cells of mutant strain became larger and irregular. In copper-rich YPD liquid medium, the cell diameter of mutant strain was larger than that of wild type and complement strain (P＜0.05), but there was no significant difference in cell diameter between wild type and complement strain (P＞0.05). In base V8 solid medium, the mutant strain produced a small amount of hypha compared with the wild type. In copper-rich V8 solid medium, the mutant strains did not produce hypha at all compared with the wild type. In basic YPD solid medium, all strains showed no difference in growth at different temperatures; in copper-rich YPD solid medium, there was no difference in the growth of complement strain and wild type, but the growth of mutant strain showed high temperature sensitivity. Under different stress conditions, there was no difference in the growth of complement strain and the wild type. Compared with wild-type and normal growing strains without stress, mutant strain showed growth inhibition under the stress of 0.005% SDS, 0.250% CR, 1 mol/L KCL, 1 mol/L NaCl, and 1 mol/L Sorbitol; compared with the wild type, the mutant strain showed growth inhibition under the stress of 10, 15 mmol/L, and 20 mmol/L DTT; compared with the normal growing without stress, the growth of the mutant strain under the stress of 10, 15 mmol/L, and 20 mmol/L DTT was enhanced. Conclusion BMH2 gene plays an important role in normal growth, hyphae growth, high-temperature growth, and stress response of Cryptococcus neoformans.
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