Study On Histone Acetylation In Regulation Of Development And Animal Pathogenecity In Aspergillus Flavus

Aspergillus flavus is a saprophytic filamentous fungus,and it is a pathogen of crops,animals and humans.This fungus can contaminate crops and cause severe economic losses,and it can produce one of the most toxic and carcinogenic secondary metabolite aflatoxin,which brings great threat to animal and human health.Furthermore,Aspergillus flavus is the second leading opportunistic animal and human pathogen causing invasive aspergillosis disease with a high teratogenicity and lethality.However,the diagnosis and treatments of invasive aspergillosis remain a difficult clinical problem.Therefore,clarifying the key regulators of Aspergillus flavus in development and pathogenesis can provide an important theory for prevention and control of aflatoxin and reduction of economic losses.Moreover,it also provides important theoretical basis for the clinical treatment of aspergillosis.In this study,the invasive aspergillosis animal model caused by A flavus was successfully established,indicating that Aspergillus flavus was indeed an important source pathogen of invasive aspergillosis.Further analysis showed that lung tissues appeared bleeding and necrosis lesions when infected by A.flavus.Blood index determination indicated that A flavus caused inflammation in mice,and the expression levels of mice pulmonary y-interferon were significantly increased when infected by A.flavus.Furthermore,the concentration of 1,3-?-D glucan was identified as an important reference index in diagnosis of apergillosis caused by A.flavus.Finally,invasive aspergillosis caused a significant decrease in the expression levels of fungal inflammatory factors genes IFN-?,TNF-?,TLR2 and Dectin-1 in lung tissue of mice.Furthermore,deletion mutant strains that previously displayed to be severely virulent or partially or fully virulent attenutaed in a crop infection showed comparable degrees of infection in the murine model.These results provided an important basis for clinical diagnosis and treatment of invasive fungal infection.Acetylation plays vital roles in regulating the development,secondary metabolic biosynthesis and pathogenesis in fungi,but acetylation in the pathogen of animals as well as plants,A.flavus,has not been reported.In this study,we identified five classical histone deacetylases(HDACs)of A.flavus through bioinformatics analysis,including class ? sub-family RpdA,Rpd3 and HosA,and class ?sub-family HdaA,HosB.By analysis of HDAC gene expression in different developmental stages,we found that the classical histone deacetylases played an important role in the conidiation,aflatoxin synthesis and sclerotia formation.For further investigation of the roles of classic HDAC in growth,aflatoxin synthesis,and pathogenesis of A.flavus,a classical HDACs inhibitor trichostatin A(TSA)was used to treat A.flavus.The results showed that it decreased growth rate and aflatoxin biosynthesis,indicating that HDACs was vital for development and aflatoxin synthesis in A.flavus.Then,the homologous recombination method was used to construct the single HDAC gene deletion strains,and we named it ?rpd3,?hosA,?hdaA and ?hosB strains.We found that a class ? HDAC HosA played vital roles in growth,conidation,sclerotia formation,oxidative stress response,aflatoxin synthesis,and a class ?HDAC,HdaA was involved in sclerotia production and aflatoxin biosynthesis.Further subcellular localization analysis revealed that HosA was mainly localized in the nucleus and barely in the cytoplasm,while HdaA was distributed in both the cytoplasm and the nucleus.To further elucidate the mechanism of HosA in the regulation of aflatoxin biosynthesis,HosA protein was purified and its HDAC activity was characterized.By co-culturing with HosA,we found that TSA could significantly inhibit HDAC activity,demonstrating that HosA harbored HDAC activity in vitro and was sensitive to TSA.Importantly,by addition of exogenous cAMP experiment,we then found that HosA functioned downstream of cAMP signalling pathway in regulation of aflatoxin biosynthesis.Further chromatin immunoprecipitation experiments indicated that HosA bound directly to AFB1 biosynthesis cluster genes to regulate their expression.These results reveal a novel mechanism by which classical HDACs mediate the activation of secondary metabolites genes in fungi.Furthermore,we found that ?hosA strains was defective in invasive aspergillosis.By bioinformatics analysis,a class ? histone deacetylases family,silent information regulator family(sirtuins family)was found in A.flavus,and this family included six members and named as Sir2,Hst2,Hst2p,Hst4,Sirt4 and Sirt5 respectively.To further study the function of class ? histone deacetylases family sirtuins in A.flavus,a sirtuins inhibitor nicotinamide(NAM)was used to treat A.flavus.As a result,we found that NAM could affect its growth and aflatoxin production.Next,the sirtuins gene deletion strains were constructed,and we named them?sir2,?hst2,?hst2p,?hst4,?sirt4 and ?sirt5 respectively.All sirtuins single gene deletion mutants showed no effect on the growth rate.The?hst4,?sirt4 and ?sirt5 strains were found to facilitate conidia germination,and both ?sir2 and ?hst4 significantly reduced conidiation.We also found that Sir2 inhibited sclerotia formation,while Hst4 and Sirt5 is vital for this progress.Addtionally,Hst4 was found to be involved in response to oxidative stresses and genotoxic stresses.Furthermore,the aspergillosis experiment results revealed that ?hst4 strains were less pathogenc than wild type when used to infect the mice.All these results revealed that Sirtuins family played an important role in growth,development and pathogenesis of A.flavus.In addition,a histone acetyltransferase GcnE was found in A.flavus.By fluorescence localization,GcnE was found to localize in both the nucleus and the cytoplasm on conidiation stage and germination stage,while mainly in the nucleus at hyphal stage.To study the function of GcnE,we constructed a gcnE deletion strain of A.flavus.Western blotting analysis revealed that the level of acetylation on H3 and H3K14 was significantly decreased in ?gcnE when compared to wild type.It also demonstrated that GcnE was involved in growth,conidiation,sclerotia formation,cell wall and genome intergrity response,and biosynthesis of aflatoxin.These results revealed that histone acetyltransferase was involved in the development and aflatoxin biosynthesis of A.flavus as well.In summary,the invasive aspergillosis animal model caused by Aspergillus flavus was successfully constructed in this study.We revealed that the epigenetic acetylation was involved in regulation of growth,development,aflatoxin synthesis and pathogenesis of Aspergillus flavus.All these results paved a basis for prevention and control of Aspergillus flavus pathogenicity at early stages and helped to understand the regulation of acetylation modification in other pathogenic fungi.These genes or proteins could be crucial targets for designing anti-fungus drugs.It also provided important theoretical basis for the clinical treatment of aspergillosis.