Study On The Effect And Mechanism Of Activated Retrotransposons For The Adaptive Ability In Candida Albicans

Candida albicans, the major human fungal pathogen, is capable of causing superficial to life-threatening infections. Recently, many predisposing factors, including immunosuppressive therapy, antibiotic therapy, human immunodeficiency virus infection and diabetes, cause the high rate of C.albicans infection; however the high adaptability to environment change for C. albicans cells leads to current critical infection situation. Retrotransposons are mobile genetic elements capable of autonomous transposition via RNA intermediates. They are widespread in multicellular eukaryotes, and have an important effect on the structure of eukaryotic genomic and genetic evolution. In recent years, researches about retrotansposons transposition activity mechanism and their impact on the host gene transcription are one of the hot topics on the life science in current international research. The aim for this study is to discover active retrotansposons involving in regulating host genes in C. albicans, preliminarily to investigate the possible mechanism for retrotansposons transcription and transposition activity, and finally to explain the relationship between activated retrotansposons and the high adaptive ability in C. albicans from the perspective of genetic evolution.In this study, real-time quantitative RT-PCR for C. albicans SC5314 was adopted to calculate the relative express quantity for genes involving in DNA damage repair parthway, meanwhile, according to the relative expression variation for retrotansposons protein encoded regions (represented by ORFs) in C. albicans genome in response to stresses, the transcriptionally active retrotansposons could be found. Based on the previous studies,it is considered that the activation of retrotansposons is associated with DNA damage. So carcinogenic agent MMS, which directly impacted on the DNA molecule, was first selected. Results showed that most of the genes participating in gene damage reparation, especially in homologous recombination (HR) progress, were transcriptionally activated, and most of the seleted retrotransposons ORFs were activated except TCA8 (ORF19.6078 and orf19.6079). Further studies were carried out to investigate the relative expression for retrotansposons protein encoded regions in response to kinds of environmental stresses such as (Hyperosmotic,H₂O₂,azole drug ). The concentration of all these stresses reached half of lethal dose (IC50). It is showed that genes involving in DNA damage reparation were widely upregulated in the response to miconzole (MCZ) and H₂O₂ stress, and TCA3 (ORF19.2219),TCA11 (ORF19.6469) and Zorro2 (ORF19.7274 and ORF19.7275) were transcriptionally activated under the treatment with MCZ for 6 h; And TCA8 was selectively activated under the treatment with H₂O₂ for 6 h. Moreover, the activated repair genes showed priority to retrotansposons activity in time. However, hyperosmotic stress, neither NaCl hyperosmotic nor sorbital hyperosmotic, couldn't increase the retrotansposons expression, and no up-regulated genes involving DNA-damage repair were found in response to hyperosmotic. It is suggested that the regulation function for retrotansposons activity is condition-selective, and closely related with cell gene damage repair pathway.To further explore the possible mechanism for retrotansposons transcriptionally activity induced by MCZ and H₂O₂ stress, anti-oxidant NAC was added before the two kinds of stresses. The combinated effects on the strains growth, the relayive expression changes of retrotansposons and damage repair related genes, and the cellular ROS level were acquired to evaluate the combination function between NAC and MCZ, H₂O₂ stresses. Results showed that NAC could inhibit the killing cells caused by MCZ and effectively protect the yeast state cells, while no function was found under H₂O₂ stress; The up- regulated retrotansposons induced by MCZ and H₂O₂ stress were decreased and almost all the activity of damage repair genes were significantly reduced with the function of NAC; Besides, the generated intracellular ROS caused by MCZ and H₂O₂ stress could be completely inhibited with the combination function of NAC according to fluorenscence intensity detection. We proposed that accumulation of ROS by MCZ and H₂O₂ stress could indirectly cause cell oxidative damage, start the DNA damage repair parthway, and finally regulate the expression of retrotansposons.In the last part, to evaluate the function of activated retrotransposon, southern blotting technique was adopted to in-depth analysis of those active retrotansposons element whether capable of performing all the steps of the replication cycle, up to and including the generation of full-length double-stranded DNA molecules. Several strains were selected under long time pressure in the liquid medium (YPD). The digested genome of parental and derivative strains, were hybridized to particular probe for retrotansposons elements. The results revealed that more copy numbers of Zorro2 were found in the MCZ induced strains, while decrease of TCA3 copy numbers in derivative strains. Besides, TCA8 copy number was also seemed to increase in H₂O₂ induced strains. Using broth microdilution method and spot assay, all the derivative strains represented increased tolerance to the matching stress respectively. It is indicated that stress could change the copy numbers of retrotansposons, and the genomic remodeling due to retrotransopsoms may change the host gene expression through a number of different ways, and be capable to improve the ability to adapt to environment.Therefore, we propsose that the potential regulation function for the genome in C.albicans would become internal power for genetic evolution, and the high adaptive ability of C. albicans may closely related to the regulation function of widespread retrotansposons in C. albicans genome.