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
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.
李洁1 何智春2 郑芳林1 徐新平1. BMH2调控新型隐球菌致病感染能力的功能研究[J]. 中国医药导报, 2023, 20(21): 9-13,18.
LI Jie1 HE Zhichun2 ZHENG Fanglin1 XU Xinping1. Functional study of BMH2 in regulating the pathogenic infection ability of Cryptococcus neoformans. 中国医药导报, 2023, 20(21): 9-13,18.
[1] Littman ML，Borok R. Relation of the pigeon to cryptococcosis：natural carrier state，heat resistance and survival of Cryptococcus neoformans [J]. Mycopathol Mycol Appl，1968， 35（3）：329-345.
[2] Kamari A，Sepahvand A，Mohammadi R. Isolation and molecular characterization of Cryptococcus species isolated from pig eon nests and Eucalyptus trees [J]. Curr Med Mycol，2017，3（2）：20-25.
[3] Kronstad JW，Attarian R，Cadieux B，et al. Expanding fungal pathogenesis：Cryptococcus breaks out of the opportunistic box [J]. Nat Rev Microbiol，2011，9（3）：193-203.
[4] Byrnes EJ，Bartlett KH，Perfect JR，et al. Cryptococcus gattii：an emerging fungal pathogen infecting humans and animals [J]. Microbes Infect，2011，13（11）：895-907.
[5] Bratton EW，El Husseini N，Chastain CA，et al. Comparison and temporal trends of three groups with cryptococcosis：HIV-infected，solid organ transplant，and HIV-negative/non- transplant [J]. PLoS One，2012，7（8）：e43582.
[6] Beardsley J，Sorrell TC，Chen SC. Central Nervous System Cryptococcal Infections in Non-HIV Infected Patients [J]. J Fungi（Basel），2019，5（3）：71.
[7] Li Y，Zou M，Yin J，et al. Microbiological，Epidemiological，and Clinical Characteristics of Patients With Cryptococcal Meningitis at a Tertiary Hospital in China：A 6-Year Retrospective Analysis [J]. Front Microbiol，2020，11：1837.
[8] Rajasingham R，Smith RM，Park BJ，et al. Global burden of disease of HIV-associated cryptococcal meningitis：an updated analysis [J]. Lancet Infect Dis，2017，17（8）：873-881.
[9] Brown GD，Denning DW，Gow NA，et al. Hidden killers：human fungal infections [J]. Sci Transl Med，2012，4（165）：165rv113.
[10] Lin X. Cryptococcus neoformans：morphogenesis，infection，and evolution [J]. Infect Genet Evol，2009，9（4）：401-416.
[11] Huang SH，Wu CH，Chang YC，et al. Cryptococcus neoformans-derived microvesicles enhance the pathogenesis of fungal brain infection [J]. PLoS One，2012，7（11）：e48570.
[12] Li J，Chang YC，Wu CH，et al. The 14-3-3 Gene Function of Cryptococcus neoformans Is Required for its Growth and Virulence [J]. J Microbiol Biotechnol，2016，26（5）：918-927.
[13] van Hemert M，Steensma H，Van Heusden G. 14-3-3 proteins：key regulators of cell division，signalling and apoptosis [J]. Bioessays，2001，23（10）：936-946.
[14] van Heusden GP，Steensma HY. Yeast 14-3-3 proteins [J]. Yeast，2006，23（3）：159-171.
[15] Zhang TJ，Shi L，Chen DD，et al. 14-3-3 proteins are involvedin growth，hyphal branching，ganoderic acid biosynthesis，and response to abiotic stress in Ganoderma lucid- um [J]. Appl Microbiol Biotechnol，2018，102（4）：1769- 1782.
[16] Wang W，Shakes DC. Molecular evolution of the 14-3-3 protein family [J]. J Mol Evol，1996，43（4）：384-398.
[17] Rosenquist M，Sehnke P，Ferl RJ，et al. Evolution of the 14-3-3 protein family：does the large number of isoforms in multicellular organisms reflect functional specificity [J]. J Mol Evol，2000，51（5）：446-458.
[18] Aitken A. Functional specificity in 14-3-3 isoform interactionsthrough dimer formation and phosphorylation. Chromosome location of mammalian isoforms and variants [J]. Plant Mol Biol，2002，50（6）：993-1010.
[19] Aitken A. 14-3-3 proteins：a historic overview [J]. Semin Cancer Biol，2006，16（3）：162-172.
[20] Ramm G，Larance M，Guilhaus M，et al. A role for 14-3-3 in insulin-stimulated GLUT4 translocation through its interaction with the RabGAP AS160 [J]. J Biol Chem，2006， 281（39）：29174-29180.
[21] Kumar R，Srivastava S. Quantitative proteomic comparison of stationary/G0 phase cells and tetrads in budding yeast [J]. Sci Rep，2016，6：32031.
[22] Roberts RL，M?觟sch HU，Fink GR. 14-3-3 proteins are essentialfor RAS/MAPK cascade signaling during pseudohyphal development in S. cerevisiae [J]. Cell，1997，89（7）：1055-1065.
[23] van Heusden GP，Wenzel TJ，Lagendijk EL，et al. Characterization of the yeast BMH1 gene encoding a putative protein homologous to mammalian protein kinase Ⅱ activators and protein kinase C inhibitors [J]. FEBSLett，1992，302（2）：145-150.
[24] Gelperin D，Weigle J，Nelson K，et al. 14-3-3 proteins：potential roles in vesicular transport and Ras signaling in Saccharomyces cerevisiae [J]. Proc Natl Acad Sci U S A，1995，92（25）：11539-11543.
[25] Ford J，Al-Khodairy F，Fotou E，et al. 14-3-3 protein homologs required for the DNA damage checkpoint in fission yeast [J]. Science，1994，265（5171）：533-535.
[26] Koike A，Kato T，Sugiura R，et al. Genetic screening for regulators of Prz1，a transcriptional factor acting downstream of calcineurin in fission yeast [J]. J Biol Chem，2012， 287（23）：19294-19303.
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