| Background and PurposeCryptococcus neoformans(C.neoformans)is an environmental pathogen found primarily in pigeon droppings and soil contaminated with avian guanos throughout the world.It is a heterothallic,basidiomycetous,pathogenic fungus that infects both immunocompetent and immunocompromised individuals.The prevalence of disease caused by this organism has increased dramatically as a result of human immunodeficiency virus(HIV)infection,organ transplantation,cytotoxic chemotherapy and corticosteroid use.And the infection caused by C.neoformans can result in chronic dormant infection with the potential for acute outbreaks.C.neoformans has several well established virulence factors:capsule,melanin,expression of urease and phospholipase,and the ability to grow at human body temperature.C.neoformans is strictly an obligate aerobic pathogenic yeast.In laboratory conditions,atmospheric levels of oxygen(21%)are required for optimal growth of C.neoformans and lower oxygen concentrations lead to a significant reduction in cell growth.The inhaled C.neoformans cells reach the central nervous system by haematogenous dissemination and cause life-threatening meningoencephalitis.It is well known that oxygen concentrations in the human brain are reported to be drastically lower than in the atmosphere and vary significantly among anatomical sites.Moreover,inflammation,thrombosis,and necrosis associated with infection are thought to lead to increased degrees of hypoxia.Thus,in order to establish infection in the host,C.neoformans cells must sense and adapt to rapidly changing oxygen level.Recent studies have demonstrated the obligate aerobic pathogenic yeast C.neoformans can adapt to and survive in hypoxic conditions,and indicated that the central strategy of C.neoformans lies in adjustment of its proliferation rate to the available oxygen levels in CNS,Sterol-response element binding protein(SREBP)and Tco pathways seem to be two sensing systems involved in hypoxia sensing in C.neoformans,while there is still a lack of information on the effect of hypoxia on virulence of C.neoformans.We found that Hypoxia may have some inhibiting effect on capsule formation,melanin production and the activities of phospholipase and urease of C.neoformans.Melanin is one of the most important virulence factors of Cryptococcus neoformans,which not only protects cryptococcus from external damage,but also is an important pathogenic factor,and also regulates immune function.L-dopa was converted into a dopone by laccase,and added the amino group to form the cyclodopa.The oxidized cyclic dopa is converted to the doped pigment,which forms the dihydroxyindole,which forms the melanin(negatively charged pigment)after its free polymerization.C.neoformans laccase has two gene codes:LAC1 and LAC2.The majority of C.neoformans melanin is produced by Lacl,although a second laccase enzyme Lac2.Therefore,the study of laccase is very important.However,due to the small amount of laccase produced by C.neoformans,it is not easy to purify.Therefore,it is very important to establish a large number of recombinant expression and purified and active laccase heterologous expression systems.In recent years,more and more laccase genes from different sources have been cloned and heterologously expressed in different hosts,but most of them belong to plant fungi.No related research has been found that heterologous expression of l.accase proteins of pathogenic fungi.Therefore,this experiment is intended to establish a heterologous expression system for C.neoformans laccase,which will lay the foundation for the further study of laccase properties.In summary,the purpose of this paper is 1)to explore the effect of hypoxia on the transcriptome of Cryptococcus neoformans.2)to express the recombinant protein of Cryptococcus neoformans in the prokaryotic,eukaryotic and insect expression systems;3)purification of the recombinant protein of Cryptococcus neoformans was performed to provide a basis for the study of the subsequent laccase crystal structure.Methods1)C.neoformans B3501A were cultured under hypoxic and normoxic conditions and subjected to RNA-Seq sequencing analysis to investigate the effect of hypoxia on the transcriptome of cryptococcosis strains.2)The laccase gene of C.neoformans was synthesized using the whole gene synthesis and optimized codons and constructed into Escherichia coli,Pichia pastoris expression strains,and insect cell sf9 cells.The newborn were explored by trying different temperatures,IPTG inducing concentrations,and culture time.Coccus laccase expression in prokaryotic,eukaryotic,and insect systems.3)Based on the above expressions,explore the different combinations of methods to purify C.neoformans laccase and detect its activity.Result1)There were 316 gene expression differences under hypoxic conditions,of which 212 genes were up-regulated and 104 genes were down-regulated.In the normoxic culture group,519 gene expressions changed compared with 0 h,of which 368 genes were up-regulated and 151 genes were down-regulated.The Cap59,Caap60,Cap64,Cap10,Lac1 and Ura5 genes showed no difference in expression at each time point under both culture conditions.Other virulence-related new gene expressions in C.neoformans have also undergone different changes.2)The recombinant laccase of C.neoformans can be successfully expressed in Escherichia coli.At 37℃ and 16℃,induced by 0.1 mM,0.5 mM,1 mM IPTG,most of them expressed as inclusion bodies.In the eukaryotic system,Pichia pastoris,it has not been successfully expressed.In the insect expression system,C.neoformans laccase is soluble in the form of secreted proteins.3)C.neoformans laccase recombinant protein inclusion bodies can be expressed by the renaturation method,Q column,Gel Filtration,Mono Q column method to obtain the purity of more than 90%of the protein.The recombinant protein expressed in the insect expression system can be purified by the NI-NTA method to obtain high purity active protein.ConclusionUnder anaerobic conditions,the transcriptome of C.neoformans was changed,and the expression of major virulence-related genes was changed,and the trends at different time points were different.After optimizing the synthesis of C.neoformans Lacl gene and cloning it into E.coli BL21,the prokaryotic system can successfully express a large number of C.neoformans laccase proteins,most of which exist as inclusion bodies.The recombinant laccase protein of C.neoformans can be purified by means of Q-column,and the purity can reach over 90%.In the insect expression system,C.neoformans laccase was secreted and expressed in a soluble form,and purified by NI-NTA to obtain high-purity active protein.In summary,the successful expression and purification of cryptozyme of C.neoformans can provide the basis for subsequent studies such as crystal structure analysis of laccase,which may provide new ideas for the study of pathogenic mechanism of C.neoformans. |
PDF Full Text Request