| Aspergillus fumigatus is a clinically important opportunistic pathogenic filament fungus and the major pathogen for human aspergillosis.A.fumigatus mainly infects the immunocompromised patients and invasive aspergillosis(IA)caused by A.fumigatus has higher mortality rate,up to 80-95%.In recent decades immunocompromised patients increased along with the increase of interventional therapies and usage of immunosuppressants;meanwhile,the resistance of A.fumigatus to the first-line clinical antifungal drug,triazoles including itroconazole,voriconazole and posaconazole is increasingly reported in many contries and regions.Thus,searching for a new target of antifungal therapy and analysis on the new mechanism of drug resistance become the reseach hotspot in anti-infection of A.fumigatus.The virulence of A.fumigatus refers to multi-factors including cell wall components,toxins,allergens and proteases et al;on the other hand,the host could resist or fight against A.fumigatus through internalization,phagocytosis and release of inflammatory factors by the innate immune system which is the first-line defence against pathogens.Among them,the ability to be resistant to extracellular stresses and to escape host immune attack are the key factors of A.fumigatus virulence.Actin cytoskeleton has an important role in cell plorized growth,spore production,stress response and maintenance of cell morphology of fungi.The network regulating actin cytoskeleton rearrangement is quite complicated and the function of many actin-related factors need to be investigated.As an actin depolymerizing factor,Cofilin is a core protein for regulating actin cytoskeleton rearrangements.Cofilin depolymerizes F-actin and this function would be lost when the serine at the N-terminal of Cofilin is phosphorylated.In mammalian cells,Cofilin is involved in many physiological processes,e.g.cell locomotion,cytokinesis,endocytosis,and exocytosis.In saccharomyces cerevisiae,Cofilin is indispensable and its site-mutation has great influence on growth phenotype and multi-drug resistance.However,the function of Cofilin in A.fumigatus is still unknown.So the purposes of our research here are to clarify the regularity of expression and distribution of Cofilin in different growth stage of A.fumigatus,and to investigate the role of Cofilin in germination,hyphal growth,response to extracellular stresses and virulence of A.fumigatus through site-mutation,alteration of expression and phosphorylated status of Cofilin.This work would contribute to some theoretical basis or drug target for a potential antifungal drug development.Firstly,we found that the expression of cofilin goes up along with conidial germination of A.fumigatus with a peak at around 8 h.Under flourecent microscope,we oberseved the hyphae of A.fumigatus expressing the GFP-fused Cofilin and found the dispersed distribution of Cofilin in the cytoplasm.We constructed the cofilinteton mutant expressing cofilin in the presence of doxycycline conditionally,cofilin OE overexpressing cofilin,cofilinS5 A mutant using CEA17ku80Δ as a parental strain and cofilinteton/cofilinS5 E mutant strain using cofilinteton as a parental strain based on homologous recombination and protoplasts transformation.The expression level of cofilin in cofilinteton in the presence of 3 ?g/ml doxycyline was significantly lower and the expression of Cofilin in cofilin OE was always higher than its parental strain CEA17ku80Δ.The site-mutation from serine to alanine in CofilinS5 A resulted in a nonphosphorylated and constitutively active Cofilin;whereas the S5 E mutation make Cofilin mimick the constitutive phosphorylation status in cofilinteton/cofilinS5 E mutant.It was found that Cofilin is essential for the viability and growth of A.fumigatus.Downregulation of Cofilin reduced the growth rate of A.fumigatus greatly and impaired the hyphal polarity and radial growth of colony on solid aspergillus minimum medium(AMM).When cultured in liquid AMM,cofilinteton mutant appeared octopus-like structure at the early stage of growth,hyperbranched and even cytoplasmic leakage was also present.The presence of sorbitol in the medium was able to rescue the growth inhibition of cofilinteton.The overexpression of Cofilin had hardly any effect on A.fumigatus morphology,but the growth rate of cofilin OE on AMM was higher than its parental strain at 28°C.With the growth test of A.fumigatus under different extracellular stresses,it was found that the sensitivity of cofilinteton to calfolour white,fanesol and congo red was similar to its parental strain,but more sensitive to SDS,H2O2 and pH 9.0.Except the sensitivity to H2O2 being reduced,the sensitivity of cofilin OE to other extracellular stresses had no difference with its parental strain.The genes catA,cat1,skn7 and yap1,which are involved in regulation in oxidative stress and pacC which regulated the response to alkaline pH stress were downregulated in cofilinteton and upregulated in cofilin OE.Using western-blot,we also found overexpression of Cofilin could activate the high osmolarity glycerol pathway with the increased phosphorylation of SakA and mRNA accumulation of three genes including cat A,dprA and dprB which were closely associated with activation of SakA.The virulence assay in vivo showed that the survival rate of Galleria mellonella infected by cofilinteton was significantly higher than its parental strain;however,the survival rates of hydrocortisone acetate and cyclophosphamide immunosuppressed mice or Galleria mellonella infected by cofilin OE had no significant difference from its parental strain.These data indicated Cofilin might regulate virulence of A.fumigatus.Next,the effect of Cofilin expression on the interaction between A.fumigatus and host cells were studied in vitro.Compared to their parental strain,the adherence of cofilinteton and cofilin OE to A549 cells decreased obviously and also the adherence-related factors medA,stuA and uge3 were down-regulated in cofilinteton whereas only stuA was downregulated in cofilin OE.The ability of A.fumigatus strains to invade into pulmonary epithelial cell,A549 cells was detected by nystatin protection assay,which also reflected equally the intracellular survival of A.fumigatus in host cells.The conidial internalization of into A549 cells was reduced by decrease of Cofilin expression and increased contrastly by overexpression of Cofilin.The mRNA level of inflammatory factors MCP-1,IL-8 and TNF-α in A549 cells when stimulated by cofilinteton,cofilin OE,their parental strain and their culture supernatants,was detected by RT-qPCR.It was demonstrated that less MCP-1 was expressed in the A549 cell challenged by cofilinteton compared to its parental strain;On the contrary,expression of MCP-1 in A549 cells challenged by cofilin OE was upregulated.But the expression of MCP-1 in A549 cells challenged by culture supernatants of these strains were similar.These data indicated that A.fumigatus Cofilin somehow is involved in affecting the inflammatory factor release of host cells and this influence might be attributed to some components on the surface of A.fumigatus instead of the secreted components in the culture.To test this hypothesis,we first used anti-dectin-1 antibody to pretreat the A549 cells and found that this block can neutralize the difference of MCP-1 expression in A549 cells challenged by cofilin OE and its parental cells.Further we demonstrated that the β-1,3-glucan and glycol-conjugates were reduced in cofilinteton and increased in cofilin OE by immunofluorescence assay.These data indicated that the increasing expression of MCP-1 in A549 cells challenged by cofilin OE might be dependent on the increased β-1,3-glucan on the surface of A.fumigatus.For further evidence,we found by RTPCR that the gene for β-1,3-glucan synthesis and genes for chitin synthesis were also downregulated in cofilinteton and upregulated in cofilin OE.Interestingly,it was shown by western blot assay that the MpkA got activated significantly and the phosphorylation of MpkA significantly increased by the down-regulation of Cofilin in A.fumigatus;however,no fluence on MpkA phosphorylation was found by overexpression of Cofilin.These results indicated that the effect of Cofilin on polarized growth,oxidative and alkaline pH stresses and virulence of A.fumigatus is related to the synthesis of cell wall polysaccharides which might be independent on MpkA-mediated cell wall integrity signaling pathway.Sequentially,we studied the effect of phosphorylation changes of Cofilin on growth,stress response and virulence of A.fumigatus.Firstly,no change was found on morphology and growth rate of colony,the sensitivity of A.fumigatus to cell wall perturbing agents,H2O2 and alkaline pH when Ser5 of Cofilin was substituted to Ala5.Besides,the internalization of cofilinS5 A strain to A549 cells and the virulence of cofilinS5 A strain in hydrocortisone acetate murine model were similar to its parental strain.The CofilinS5 E mutation inhibited growth of A.fumigatus.If Cofilin was phosphorylated on this position completely,the strain was not able to keep alive.The cofilinS5 E mutation could not aggravate the stress-response phenotype of cofilinteton.This indicated the phosphorylation of Cofilin was vital to survival and growth of A.fumigatus and excess of unphosphorylated Cofilin had little effect on physiological function of A.fumigatus,of which the mechanism need to be further investigated.Finally,to further study the function of Cofilin of A.fumigatus,we constructed several strains with site-mutations at multiple potential residues of Cofilin;meanwhile,put forward a new strategy of gene mutagenesis by construction of parental gene mutation cassette.This strategy was quite simple and save time without requirement on plasmid construction which includes restriction-ligation,transformation and screen of Escherichia coli.This strategy was best applied to construct multiple mutants on the same gene which have different mutations at native loci all at once.Based on this strategy,we succeeded to construct three cofilin mutants,cofilinD21 A,R21A,cofilinK36 A and cofilinE48 A,respectively.All these site-mutated amino acids are charged.Compared with their parental strain,cofilinD21 A,R21A and cofilinK36 A produced less conidia,but produced more pigment.The hyphae of cofilinK36 A was wider with calcoflour white stain.By clonogenic assay,cofilinD21 A,R21A and cofilinK36 A strains were more resistant to voriconazole and itraonazole than their parental strain,but cofilinE48 A strain was only resistant to voriconazole.The internalization of these cofilin mutant strains into A549 cells was reduced greatly compared to their parental strain by nystatin protection assay;however,the virulence of the cofilin mutants was similar to their parental strain in hydrocortisone acetate murine model.The detailed mechanism of three mutants regulating drug resistance and virulence of A.fumigatus would be elucidated in the future.In brief,our research systematically illustrated the function and regulation of Cofilin on polarized growth,stress response and virulence in A.fumigatus;discovered a new link between actin cytoskeleton-regulatory protein and stress response mechanism of cell wall and virulence in A.fumigatus.In the light of rather distant phylogenetic relationships of Cofilin between A.fumigatus and Homo sapiens,our research provided a new theoretical basis and potential drug target for antifungal therapy.Besides,we set up a novel strategy of gene mutagenesis,which facilitated greatly to screen key residues and study the relation between structure and function of protein in A.fumigatus. |
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