| Talaromycosis is an endemic and opportunistic deep fungal infection caused by Talaromyces marneffei（T.marneffei）.In recent years,the number of T.marneffei infection also increased year by year,and the epidemic scope showed an expanding trend.T.marneffei is temperature-dependent dimorphic fungus,it grows as hyphae at 25℃ and transforms into yeast at 37℃.The transformation of T marneffei from phase to yeast in human body is an important pathogenic mechanism but the mechanism has not been clarified.In this study,T.marneffei random mutant library was constructed by optimizing Agrobacterium tumefaciens-mediated transformation technology.Based on the random mutant library,mutants whose phase could not be transformed into yeast were screened,their T-DNA insertion sites were determined,unknown genes involved in regulating the transformation of T.marneffei phase into yeast were found,and the gene function was verified and annotated,so as to further analyze the pathogenic mechanism of T.marneffei.The research content is divided into the following three parts:Part I:Construction of T.marneffei insertion mutant library by ATMT and identification of phenotypic change mutants in dimorphic transformationObjectives:T.marneffei random insertional mutant library was constructed by ATMT.The library was used to screen T.marneffei mutants that could not be transformed from phase to single yeast under the condition of 25℃ to 37℃.The T-DNA insertion site of the mutant were identified by sequencing to identify the key regulatory gene involved in the transformation of T.marneffei.Methods:1.The transformation system of ATMT was optimized and co-transformed with T.marneffei.Then positive clones were screened using sabouraud dextrose agar plate containing hygromycin B.Positive transformants were randomly selected for PCR to determine whether T-DNA was inserted successfully.The positive clones successfully inserted with T-DNA were sequenced after amplified the flanking sequence to explicitly insertion sites and calculate insertion efficiency.Then expand transformation to construct random insertion mutant library.2.The mutants in T marneffei mutant library that could not be transformed into single yeast were further screened.The mutants were cultured with hygromycin plate at 25℃ for 21 days,and then the positive clones were transferred to the new culture plate one by one.They were cultured at 37℃ for 21 days.The morphology was observed under the microscope to screen the mutants that could not be transformed into single yeast cells.3.The target mutant was subcultured for many times to confirm the stability of its phenotype,.and then the whole genome sequencing was compared to determine whether the T-DNA was inserted at a single site,and the gene of the insertion site were identified.Then the amino acid sequence encoded by the t-DNA inserted gene was analyzed by systematic tree.Results:1.T.marneffei transfected by Agrobacterium tumefaciens could grow on solid medium containing hygromycin.40 mutants of 44 positive clones randomly selected amplified the gene fragment of hygromycin B with correct molecular weight.After flanking sequence amplification and sequencing verification of the mutants of the 40 gene fragments containing hygromycin B,the gene information of 43 insertion sites was obtained,and the probability of single site insertion was calculated to be≥92.5%.2.Through the monoclonal screening of T.marneffei mutant library,1270 mutants were obtained when cultured at 25℃,of which 7 strains had colony morphological changes.Phenotypic changes include slowing down of colony growth,albinism,deepening of pigment,wrinkle and so on.3.When cultured at 37℃,the mutant named TMM1119 could not be transformed into a single yeast cell and it appears as a long strip.By whole-genome exon sequencing,the insertion site of t-DNA was identified to be between the 664th and 665th bases of T.marneffei’s PMAA003940.Systematic tree analysis showed that PMAA003940 was a homologous gene of agn1.Conclusion（s）:1.T.marneffei mutant library was successfully constructed by ATMT and the unit point insert a probability of 92.5%or more.2.Seven mutants whose morphology changed when cultured at 25℃ were successfully screened by T.marneffei mutant library.3.The mutant TMM1119 could not be transformed into single yeast cell when cultured at 37℃ and the insertion site was located at agnl.Part Ⅱ:Study on gene function of T marneffei mutant TMM199Objectives:To explore the functions of agnl in T.marneffei biological process of dimorphic conversion,ultrastructure,stress response capacity,growth rate,escape from macrophage killing as well as the downstream regulatory network.Methods:1.The ultrastructure of agn1 inserted mutant and wild type T.marneffei was compared by scanning electron microscopy and transmission electron microscopy.The growth rate and morphology of agn1 inserted mutant and wild type T.marneffei were observed in solid medium containing different concentrations of hydrogen peroxide,sorbitol,potassium chloride,Calcium fluorescent white and Congo red.The growth curves were drawn to compare the growth rates of agnl inserted mutant and wild type T.marneffei.Macrophage killing experiment and dynamic observation of the killing of T.marneffei by macrophages were carried out to compare the difference in the killing ability of agn1 inserted mutant and wild type T.marneffei to escape from macrophages.2.The transcriptomic changes of T.marneffei after agn1 insertion mutation were studied under 37℃ and the same culture conditions.Results:1.When cultured at 37℃,most wild strains of T.marneffei were ovoid single yeast cells,and a few yeast had a membrane with strong fluorescence staining in the middle while most of agn1 mutants showed long-rod shape or V shape and more than two transverse strong fluorescent diaphragms were distributed in most yeast.The proportion of single yeast cells,diaphragm yeast and V shape yeast in wild strains was 82.9%,17.1%and 3.9%,respectively.The proportion of single yeast cells,diaphragm yeast and V type yeast in agnl mutant strains was 30.4%,69.6%and 47%,respectively.Transmission electron microscopy（TEM）results showed that the intermediate septum of agn1 mutant had degraded,and the secondary septum of yeast cells separated,while the cell wall at the edge of septum remained connected and the yeast cell wall became thinner.2.After 21 days of continuous culture from 37℃ to 25℃,the mycelia of agnl mutant showed short and thick,increased bifurcation,and some mycelia were malformed,with more septa between mycelia and a bead-like contraction at the diaphragm,and some mycelia broke,and the sporogenic structure atrophy and variation.3.The growth ability of agn1 mutant was significantly lower than that of wild strain.With the increase of the concentration of KCl,sorbitol,hydrogen peroxide,calcium fluorescent white and Congo red,the growth was inhibited more seriously,which showed that the colony diameter was smaller,and even the colony morphology changed.4.After coculture with macrophages for 48 hours,the survival number of T.marneffei yeast in macrophages decreased significantly of agn1 mutant.Under the fluorescence microscope,it can be seen that with the extension of coculture time,the wild strain T.marneffei yeast gradually increased in macrophages,while the agn1 mutant T.marneffei yeast gradually decreased in macrophages and was finally killed.5.Transcriptome results showed that the angl mutant was significantly different from the wild strain,and 2744 genes were differentially expressed,among which 774 genes were up-regulated and 1970 genes were down-regulated.GO enrichment analysis of down-regulated genes with significant differences showed that down-regulated genes were mainly involved in "transmembrane transporter activity","catalytic activity","zinc ion binding" and other biological processes,and were mainly located in "cell membrane".The pathways with the highest concentration of differential genes were "biosynthesis pathway of secondary metabolites","microbial metabolism pathway in different environments" and"amino sugar and nucleotide sugar metabolism".Conclusions:1.The transformation of T.marneffei yeast phase into mycelium phase was not inhibited after agn1 insertion mutation,but the transformation into single yeast cells was inhibited after agn1 mutation.after Agn1 mutation of T.marneffei contribute to suppressing the formation of single yeast cells by block the edge diaphragm cell wall degradation.2.Agnl plays an important role in the regulation of the structure of T marneffei cell wall,mycelial growth,the production of broom branches and conidia formation.3.Agn1 participates in the ability of T.marneffei to resist environmental stress.4.Agnl is involved in the viability of T.marneffei yeast in escape macrophage killing.5.Transcriptome analysis suggested that agn1 might be involved in transmembrane transporter activity,catalytic activity,zinc ion binding and other biological processes in T.marneffei yeast cells.Part Ⅲ:Construction of T.marneffei infection animal model and study on the pathogenicity of agn1 mutantObjectives:Construct an animal model that infected by T.mareffei and use the animal model to study the pathogenicity of agn1 mutant.Methods:1.The invasive infection model of T.marneffei was established by intraperitoneal injection of nude mice.The accidental death of nude mice was observed,the survival curve and body weight curve were drawn,and the nude mice were killed regularly in batches.The gross morphology,HE and PASM staining of important organs were observed;2.Using the above model,the same dose of wild strain and agn1 mutant yeast were injected,respectively,to draw the survival curve and body weight curve,and the nude mice were killed in batches at regular time,then the gross morphology was observed,HE and PASM staining of important organs,and the pathogenicity of T.marneffei agn1 mutant was studied in vivo;3.The wild type strains and agn1 mutant strains of T.marneffei injected into the intraperitoneal nodules of nude mice.Fungal staining was performed for living tissue,morphological differences were observed and the maximum diameter was measured by fluorescence inverted microscope.Results:1.T.marnefei intraperitoneal injection infected nude mice showed weight loss,shortened survival time,rash,hepatosplenomegaly and other symptoms consistent with clinical patients.Fungal infiltration was found in the lung,skin,rectum,small intestine,mesenteric nodules,spleen,kidney,liver and bone marrow of infected nude mice.2.Virulence of the mutant was studied in vivo by intraperitoneal injection infection,and the survival time of the agn1 mutant was 3 times that of the wild strain compared with the wild strain,and the agl mutant had lighter visceral lesions,slower disease progression and fewer fungal infiltrating lesions in nude mice.3.Agn1 mutant strains also showed v-shaped and long rods in nude mice,with the maximum diameter of some cells exceeding 10μm.Conclusions:1.Intraperitoneal injection of T.marneffei successfully cause disseminated infection in nude mice.2.Agn1 insertion mutation significantly reduced the pathogenicity of T.marneffei.3.Agn1 also promote the formation of T.marneffei single yeast cells in vivo. |
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