Vacuolar Protein Sorting-associated Protein Vps17 Regulates Sexual Reproduction And Virulence In Cryptococcus Neoformans

Cryptococcus neoformans is a capsular opportunistic fungal pathogen that can infect individuals with immune deficiency,such as AIDS patients or organ transplant recipients,through the respiratory pathway.C.neoformans usually causes primary pulmonary cryptococcosis first and then disseminates to the brain,causing fatal cryptococcal meningitis,resulting in about 200000 deaths worldwide every year.Despite a large number of deaths,the treatment options for cryptococcosis are still limited.Currently,only the three major classes of drugs are approved for clinical use and there are still problems,such as the continuous evolution of drug-resistant strains.The virulence of C.neoformans depends on many factors,including polysaccharide capsules,melanin production,cell wall integrity,heat resistance and secreted extracellular enzymes,and basidiospores of infected propagules produced by sexual reproduction.Basic research on the growth and virulence mechanism of Cryptococcus will help us formulate more efficient treatment strategies.Vesicle transportation ensures the normal operation of various life activities in the organism,and the closely related Vps protein can regulate the morphological development,sexual reproduction,and pathogenicity of fungi by affecting the sorting of many important proteins.The Vps17 protein studied in this subject is one of the main components of Retromer,the reverse vesicle transport complex.By analyzing the spatiotemporal expression pattern of VPS17 and its functions in the growth,sexual reproduction,and virulence of C.neoformans,we obtained the following results:1.Identification of VPS17 and analysis of temporal and spatial expression patternsThrough sequence analysis,we found that the coding region of the VPS17 gene is 2967bp in length and contains six introns.It can encode a Vps17 protein containing 818 amino acids,and the protein contains a PX domain and a BAR domain,which is a kind of progressive conservative SNx protein that can bind to inositol phosphate.To study the spatiotemporal expression pattern of VPS17,we constructed two fluorescent strains,P_VPS17-mCherry and GFP-Vps17.After microscopic examination of the GFP-Vps17strain,we found that Vps17 is located on the vesicle and is not affected by external stress conditions;at the same time,the results of the mating experiment of GFP-Vps17 fluorescent strain also showed that Vps17 is located on the vesicle of the yeast,mycelium,basidiospore or basidiospore morphology after mating of C.neoformans.According to the analysis of the fluorescence signal of the P_VPS17-mCherry fluorescent strain after mating,we found that the VPS17 is expressed in all stages of C.neoformans development.RT-qPCR analysis showed that VPS17 was expressed in the early stages of Cryptococcus mating（0～7 d）.Based on the above results,we speculate that VPS17 may play an important role in the sexual reproduction of C.neoformans.2.Construction and phenotypic analysis of VPS17 gene knock out,complementation and overexpression strainsIn this study,we explored the function of VPS17（CNAG＿00508）in C.neoformans through methods such as gene knockout,complementation,and overexpression.Therefore,we constructed the vps17Δmutants,vps17Δ::VPS17 complementation strains,and VPS17^OEoverexpression strain for subsequent phenotypic experiments and pathogenicity studies.Polysaccharide capsule,melanin and growth at 37℃ are the three typical virulence factors of C.neoformans.The virulence factor test results show that the VPS17 gene knock out only affects the formation of C.neoformans polysaccharide capsules.The results of the stress tolerance assay showed that vps17Δmutants are sensitive to SDS,NaCl,and KCl,indicating that VPS17 may regulate the integrity of the cryptococcal cell membrane.The results of mating experiments showed that the Vps17 protein plays an important role in the sporulation process of C.neoformans,and the vps17Δmutants can not produce basidiospores after mating.In order to explore the role of Vps17 in the mating process of Cryptococcus,we constructed the Nop1-mCherry strain in the background of vps17Δmutant and detected the nuclear dynamics of C.neoformans during the mating process based on the red fluorescent signal of each developmental stage after examination of the strain.Fluorescence observation results showed that the vps17Δmutant was unable to undergo subsequent meiosis after the nuclei fused during the mating process,which resulted in the blocking of the sporulation process of C.neoformans.3.The role of Vps17 on the pathogenicity of C.neoformansThe virulence of the vps17Δmutant strain was tested using a mouse nasal infection model.The mouse survival curve showed that the virulence of vps17Δmutant was significantly reduced;CFU statistical results and pathological sections showed that the vps17Δmutant had minor damage to the lungs and spleen of the mice but caused severe damage to the brain tissue.No cryptococcal cells and lesions were found in the brain and spleen of mice infected with vps17Δmutant on the 7th and 14th day;cryptococcal cells were detected in the brain,lung and spleen tissues were taken on the 21st day,but the CFU was still significantly lower than that in the wild-type strain,and the range of lesions caused was also smaller.Therefore,we speculate that the reduced virulence of the vps17Δmutant may be due to its slower proliferation rate in the host than the wild-type strain..In summary,our results indicated that the Vps17 protein plays an important role in the sexual reproduction and pathogenicity of C.neoformans.