| Background and purpose:With global aging and more cases of organ transplantation,tumor radiotherapy and chemotherapy,as well as the explosive growth of immune deficiency caused by HIV infection,fungal infection has been gradually become a clinically serious disease.Among many clinically common pathogenic fungi,Cryptococcus neoformans is currently one of the most pathogenic and lethal pathogenic fungi worldwide.Cryptococcus neoformans is an opportunistic fungal pathogen that widely exists in environments around the world,causing approximately 220000 fatal fungal infections worldwide each year.The risk of cryptococcosis and the use of biological immunotherapy in elderly individuals is further increasing.The most recent warning that Cryptococcus neoformans ranks among the four most harmful fungi,indicating the increasingly serious threat it poses to human public health human pathogenic fungi was released by the World Health Organization last year.However,the current treatments anti cryptococcal infections are very limited.Currently,the main anti cryptococcal drugs rely on three drugs,namely,azoles,polyenes,and5-fluorocytosine.And,the period of treatment to invasive cryptococcal infections is long,which highly causes drug resistance,leading to unusual difficulties and difficulties in clinical treatment.Therefore,it is extremely urgent to explore new strategies.According to current issues mentiond above,our group had been keeping on studying of Cryptococcus.And in this thesis,we found that the expression of PMP3 was up-regulated under antifungal drugs and certain stresses by analyzing the online transcriptomics data of Cryptococcus neoformans.Pmp3 is a highly conserved plasma membrane protein and has important functional roles in the model fungus Saccharomyces cerevisiae.Therefore,we put effort to dissect the roles of Pmp3 in Cryptococcus neoformans.We firstly constructed PMP3 deletion mutant strains of Cryptococcus neoformans,and observed the functional impact of PMP3 deletion in Cryptococcus neoformans according to the phenotype experiments of the mutant strains.Secondly,the complementation and overexpression strains were constructed with the mutant strain as the background,and the effect of the mutant strain on the function was further confirmed.Finally,the relevant mechanisms of the affected functions were explored.The purpose is that the research on PMP3 can provide new ideas and directions for the development of antifungal drugs and other problem.Methods:1.Construction of strains through CRISPR-Cas9 combined with electroporation technologyWith Cryptococcus H99 as the background,CRISPR-Cas9 combined with electroporation technology was used to knock out the target gene.The complementation and overexpression plasmids were constructed through plasmid digestion,seamless cloning technology,PCR and CRISPR-Cas9 combined with electroporation technology with mutant strain as the background.The plasmids were introduced into the strains to obtain overexpression strains and complementation strains.2.Bioinformatics analysis of PMP3Analysis of the transcriptomic data of Cryptococcus neoformans revealed changes in the transcript level of PMP3.The PMP3 gene sequence and amino acid sequence were obtained from the FungiDB website.Using PMP3 gene sequence as a reference sequence,similar sequences were found by NCBI-BLAST comparison,and a phylogenetic tree was constructed for all sequences.3.Temperature and chemical stress stress experiments for strains(1)In order to analyze the growth of the strains at different temperatures,monitor the growth of the strains at different temperatures(30°C and 39°C)on YPD solid medium,and take pictures after 48 hours.(2)In order to analyze the growth of the strains under different stress pressures,the strains were cultured on YPD solid medium added with specified compounds(sorbitol,Congo red,hydrogen peroxide,SDS and other chemical stress stress),and YPD common solid medium was used as a control.Photograph and record after incubation at 30°C for 48 hours.4.Drug susceptibility test for strainsIn order to analyze the drug susceptibility of the strains,the strains were cultured on the YPD solid medium added with the specified antifungal drugs(amphotericin B,fluconazole,5-fluorocytosine),and the YPD solid medium was used as the control.After incubation at 30°C for 48 h,photographs were taken and recorded.5.RNA extraction and RT-QPCRThe strains were collected and cultured overnight in YPD liquid medium for 20 hours to get bacterial precipitation,ground with liquid nitrogen,and after the wall was broken by a wall breaking machine,RNA samples were extracted according to the RNA extraction reagent.After the measured concentration and A260/280 value reach the standard,select a certain amount(no more than 1 μg)of RNA to react with the reverse transcription reagent to obtain cDNA.Perform QPCR reaction under the instructions of QPCR reaction reagents and analyze the relative expression levels of ERG1,ERG2,ERG3,ERG4,ERG5,ERG6,ERG11,and PMP3 in the strain samples.6.Fluorescence LocalizationThe cellular localization of PMP3 was observed under an inverted fluorescence microscope by constructing PMP3 overexpression strain with green fluorescent protein.7.Ergosterol Extraction and DetectionErgosterol is extracted by alcohol-alkali saponification method,quantitative measurements were then performed in HPLC and LC-MS systems.After Filipin staining of strains,the sterol-lipid distribution of the strains was observed under an inverted fluorescence microscope.8.Plasma membrane proteomicsThe overnight cultured strains were lysed and centrifuged in an ultra-high speed centrifuge to obtain plasma membrane proteins,and the plasma membrane protein samples were analyzed by proteomics.Result:1.The sequence of PMP3 gene is conserved in eukaryotes and PMP3 also exits in Cryptococcus neoformans2.Transcriptomics analysis shows that external pressure can up-regulate PMP3 transcription level3.PMP3 deletion affects the expression of Cryptococcus neoformans under external pressure(AmB drug)growth4.The mechanism of PMP3 function has nothing to do with ergosterol synthesis pathway-related gene expression,ergosterol content and distribution,and sphingolipid synthesis pathway.5.PMP3 mutants and wild-type plasma membrane proteomic analysis shows significant differences,and the mechanism of PMP3 function may be related to this difference Conclusions:1.Deletion of PMP3 affects the growth of Cryptococcus neoformans under AmB pressure2.The mechanism of PMP3 function has nothing to do with ergosterol and sphingolipid3.The mechanism of PMP3 function may be related to the significant difference between the plasma membrane protein and wild type after PMP3loss... |
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