Inactivation of Candida albicans CaMIT1 gene with CRISPR/Cas9 approach and the phenotype analysis of its homozygous mutant

Abstract

Abstract:

Objective To construct mutants for C.albicans CaMIT1 and investigate its functions in C. albicans.Methods We constructed the inactivated mutant for CaMIT1 with the novel approach CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)/Cas9. Serial dilution assay was used to analyze the function of CaMIT1. Results We successfully constructed the homozygous inactivation mutant for CaMIT1, and phenotypic analysis revealed that the homozygous mutant was sensitive to calcium and lithium ions, sodium dodecyl sulfate, clotrimazole, ketoconazole and anidualafungin, but tolerant to Congo Red. Conclusions CaMIT1 is involved in the integrity of plasma membrane and cell wall as well as the calcium homeostasis.

Key words: Candida albicans, CaMIT1, CRISPR, Phenotypic analysis, Plasma membrane and cell wall stress

CLC Number:

R379.4

XU Hui-hui, JIANG Ling-huo. Inactivation of Candida albicans CaMIT1 gene with CRISPR/Cas9 approach and the phenotype analysis of its homozygous mutant[J]. Chinese Journal of Mycology, 2018, 13(4): 201-207.

References

[1] Sardi JC, Scorzoni L, Bernardi T, et al. Candida species:current epidemiology, pathogenicity, biofilm formation, natural antifungal products and new therapeutic options[J]. J Med Microbiol, 2013, 62(1):10-24.
[2] Wisplinghoff H, Ebbers J, Geurtz L, et al. Nosocomial bloodstream infections due to Candida, spp. in the USA:species distribution, clinical features and antifungal susceptibilities[J]. Int J Antimicrob Agents, 2014, 43(1):78-81.
[3] Klis FM, Groot PD, Hellingwerf K. Molecular organization of the cell wall of Candida albicans[J]. Med Mycol, 2001, 39(1):1-8.
[4] Fabre E, Sfihi-Loualia G, Pourcelot M, et al. Characterization of the recombinant Candida albicans β-1,2-mannosyltransferase that initiates the β-mannosylation of cell wall phosphopeptidomannan[J]. Biochem J, 2014, 457(2):347-360.
[5] Netea MG, Joosten LA, van der Meer JW, et al. Immune defence against Candida fungal infections[J]. Nat Rev Immunol, 2015, 15(10):630-642.
[6] Jouault T, El Abed-El Behi M, Martínez-Esparza M, et al. Specific recognition of Candida albicans by macrophages requires galectin-3 to discriminate Saccharomyces cerevisiae and needs association with TLR2 for signaling[J]. J Immunol, 2006, 177(7):4679-4687.
[7] Navarroarias MJ, Defosse TA, Dementhon K, et al. Disruption of protein mannosylation affects Candida guilliermondii cell wall, immune sensing, and virulence[J]. Front Microbiol, 2016, 7(7):1951.
[8] Dutton LC, Nobbs AH, Jepson K, et al. O-mannosylation in Candida albicans enables development of interkingdom biofilm communities[J]. MBio, 2014, 5(2):e00911.
[9] Gonzálezhernández RJ, Jin K, Hernándezchávez MJ, et al. Phosphomannosylation and the functional analysis of the extended Candida albicans MNN4-like gene family[J]. Front Microbiol, 2017, 8:2156.
[10] Bates S, Hall RA, Cheetham J, et al. Role of the Candida albicans MNN1 gene family in cell wall structure and virulence[J]. BMC Res Notes, 2013, 6(1):294.
[11] Jouault T, Ibataombetta S, Takeuchi O, et al. Candida albicans phospholipomannan is sensed through Toll-Like receptors[J]. J Infect Dis, 2003, 188(1):165-172.
[12] Mille C, Bobrowicz P, Trinel PA, et al. Identification of a new family of genes involved in β-1,2-mannosylation of glycans in Pichia pastoris and Candida albicans[J]. J Biol Chem, 2008, 283(15):9724-9736.
[13] Mille C, Janbon G, Delplace F, et al. Inactivation of CaMIT1 inhibits Candida albicans phospholipomannan β-mannosylation, reduces virulence, and alters cell wall protein β-mannosylation[J]. J Biol Chem, 2004, 279(46):47952-47960.
[14] Doudna JA, Charpentier E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9[J]. Science, 2014, 346(6213):1258096.
[15] Terns RM, Terns MP. CRISPR-based technologies:prokaryotic defense weapons repurposed[J]. Trends Genet, 2014, 30(3):111-118.
[16] Vyas VK, Barrasa MI, Fink GR. A Candida albicans CRISPR system permits genetic engineering of essential genes and gene families[J]. Sci Adv, 2015, 1(3):e1500248.
[17] Noble SM, Johnson AD. Strains and strategies for large-scale gene deletion studies of the diploid human fungal pathogen Candida albicans[J]. Eukaryot Cell, 2005, 4(2):298-309.
[18] Zhao Y, Du J, Xiong B, et al. ESCRT components regulate the expression of the ER/Golgi calcium pump gene PMR1 through the Rim101/Nrg1 pathway in budding yeast[J]. J Mol Cell Biol, 2013, 5(5):336-344.
[19] Jiang L, Alber J, Wang J, et al. The Candida albicans plasma membrane protein Rch1p, a member of the vertebrate SLC10 carrier family, is a novel regulator of cytosolic Ca2+ homoeostasis[J]. Biochem J, 2012, 444(3):497-502.
[20] Min K, Ichikawa Y, Woolford CA, et al. Candida albicans gene deletion with a transient CRISPR-Cas9 System[J]. mSphere, 2016, 1(3):e00130-16.
[21] Beeler TJ, Fu D, Rivera J, et al. SUR1 (CSG1/BCL21), a gene necessary for growth of Saccharomyces cerevisiae in the presence of high Ca2+ concentrations at 37 degrees C, is required for mannosylation of inositolphosphorylceramide[J]. Mol Gen Genet Mgg, 1997, 255(6):570-579.
[22] Roncero C, Durán A. Effect of calcofluor white and Congo red on fungal cell wall morphogenesis:in vivo activation of chitin polymerization[J]. J Bacteriol, 1985, 163(3):1180-1185.
[23] Ko A, Wheeler L J, Mathews CK, et al. Hydroxyurea arrests DNA replication by a mechanism that preserves basal dNTP pools[J]. J Biol Chem, 2004, 279(1):223-230.
[24] Shirahige K, Hori Y, Shiraishi K, et al. Regulation of DNA-replication origins during cell-cycle progression[J]. Nature, 1998, 395(6702):618-621.