Physiological Mechanism Of Candida Glabrata Mediator Complex Subunit 3 In Response To Acid Stress

This dissertation chose the mutant strain C. glabrata ATCC 55 as a model system to elucidate the physiological role of mediator subunit 3(Cg MED3) in Candida glabrata to against acid stress. The regulation mechanism and the effect of gene Cg MED3 A and Cg MEDB deletion on cell growth, transcription level and cell membrane components, in response to acid stress were studied in detail with method of transcriptomics, mass spectrometry and fluorescence anisotropy was combined to determine the physiological properties of the cell membrane. The main results were described as follows.1. The double mutant strain Cgmed3ABΔ and the revertant strain Cgmed3ABΔ/Cg MED3 AB were constructed. Compared with control strains, the changes in Cgmed3ABΔ were exhibited as follows: at p H 6.0, Cgmed3ABΔ strains showed a similar growth ability, and from 0 to3 h was in the lag phase, 3 to15 h was in logarithmic phase, after 15 h was entering the platform phase, the average specific growth rate was 0.096 h-1. At p H 2.0, 0 to 21 h in the culture lag phase, logarithmic phase after 21 h, the average biomass and specific growth rate were 0.25 g L-1 and 0.052 h-1, respectively, reduced by 65% and 40%;2. Analysis transcriptomic data of mutant strain Cgmed3ABΔ, the changes of gene expression under the p H 2.0 condition, compared to control strains found that: RNA-seq revealed that the expression of genes in the mevalonate and sterol pathways markedly changed in the Cgmed3ABΔ strain in such a manner that should result in the accumulation of squalene. In addition, fatty acid biosynthesis gene expression level decrease and related fatty acid elongation and synthesis genes down- regulated,which may leads to a decrease in fatty acid synthesis. The key genes in the process of synthesis of phospholipids were down-regulated, may lead to PI, PC and PE synthesis inhibited;3. Combined the technique of gas and liquid chromatography mass spectrometry, the contents of fatty acids, phospholipids and sterol in the cell membranes of Cgmed3ABΔ strains were detected under p H 2.0, found that: squalene accumulation in the Cgmed3ABΔ strain was 30-fold greater than that in the control strains at p H 2.0. Furthermore, the sterol composition of the membrane was altered, as the levels of lanosterol, zymosterol, fecosterol, and ergosterol were 88%, 88%, 92% and 82% lower, respectively, in the Cgmed3ABΔ strain than in the wild-type strain at p H 2.0. The fatty acids contents of C16:0, C16:1, C18:0, C18:1 and C18:2 were only 55%, 54%, 29%, 39% and 41% of control strains respectively. The content of phosphatidylcholine(PC) and phosphatidylcholine(PE) were decreased by 60% and 73%, respectively, compared with wt.4. The physiological characteristics of the cell membrane was changed, including membrane rigidity, as well as H+-ATPase activity. In p H 2.0, the rigidity of cell membrane were decreased 12% in Cgmed3ABΔ strain compare with control strains, and the proton pump H+-ATPase activity of cell membrane decreased 75% than that of the control strains.