The Molecular Characteristics And Functions Of Aspergillus Nidulans Calcium Signaling System Related Proteins AnPcpA And AnPefA

As we all know,the calcium signal is very conservative second messenger in eukaryotes,and it is also very critical for cell survival and death.Calcium signaling system involves in almost all physiological activities including growth,development,proliferation,secretion,material transport and stress responses.Although it has been confirmed that there are many important members of calcium signaling systems in the fungus,such as calcium ion channels,calmodulin and calcineurin phosphatase,which play very important roles in fungal growth,stress responses and so on.However,it is still unknown about what are calcium signaling system interaction proteins and their target proteins and how their networks are regulated.Moreover,it is also poorly understood whether the calcium signal system is involved in drug-stress responses or drug resistance in fungi.Aspergillus is a large class of fungi and distributed widely in nature.It contains the model fungus---Aspergillus nidulans(A.nidulans),Aspergillus oryzae(A.oryzae)and Aspergillus niger(A.niger)mostly used in industrial production,and also there are other classes associated with invasive aspergillosis such as Aspergillus fumigatus(A.fumigatus),Aspergillus flavus(A.flavus),Aspergillus terreus(A.terreus)and so on.In this study,A.nidulans,which is a model filamentous fungus for the basic biology research and a opportunistic pathogen for medical research,was used as our research material.Two candidate protein associated with calcium signaling systems,AnPcpA and AnPefA,were found in A.nidulans using bioinformatics methods.Through the application of gene fusion PCR for gene knockout,fluorescent protein labeling techniques and technology to replace the promoter,we found and characterized firstly their critical biological function in the polar growth,cytokinesis,stress response and other important life events in A.nidulans.Bioinformatics analysis indicated AnPcpA protein has calmodulin binding domain,which is the core protein of calcium signaling system,so we speculated that AnPcpA might be related with calcium signaling system.In this study,we found that it is essential for conidial germination and hyphal development.Compared to the hyphal apex localization pattern of calmodulin(CaM),which has been identified as an interactive partner of the pericentrin homolog,GFP-AnPcpA fluorescence dots are associated mainly with nuclei,while the accumulation of CaM at the hyphal apex depends on the function of AnPcpA.In addition,the depletion of AnPcpA by an inducible alcA promoter repression results in severe growth defects and abnormal nuclear segregation.Most interestingly,in mature hyphal cells,knockdown of AnPcpA was able to significantly induce changes in cell shape and cytoskeletal remodeling;it resulted in some enlarged compartments with condensed nuclei and anucleate small compartments as well.Moreover,defects in AnPcpA significantly disrupted the microtubule organization and nucleation,suggesting that AnPcpA may affect nucleus positioning by influencing microtubule organization.This is the first report of AnPcpA biological function in live cells in filamentous fungi.AnPefA is a predicted calcium signaling-related protein based on bioinformatic analysis,which contains multiple calcium binding sites and EF-hand structure.Since AnPefA homolog in mammalian affects the efficacy of anti-cancer drugs,we speculated that AnPefA may be involved in calcium signaling which is related to stress-response in A.nidulans.In this study,GFP-AnPefA fluorescence are distributed in cytoplasm in live cells.In addition,loss of it enhance drug resistance in A.nidulans,which is dependent on the calcineurin.Compared to the wild-type strain,AnPefA knock-out strain has higher survival rate.Most interestingly,loss of AnPefA by knock out reduce the cell apoptosis rate and overexpression of AnPefA by an inducible alcA promote increase cell apoptosis rate under ITZ condition.Moreover,knockdown of AnPefA results in ergosterol content increase not drug pump activity up.Our data suggeste that loss of AnPefA may enhance the drug resistance by influencing cell survival rate and ergosterol content in A.nidulans.This is the first report of AnPefA biological function in drug resistance in filamentous fungi.In conclusion,we have firstly investigated the critical biological function of AnPcpA and AnPefA in the polar growth and drg resistance in the model filamentous fungus and the opportunistic pathogens A.nidulans.This study will provide an important theoretic foundation about what are calcium signaling system interaction proteins and their target proteins and how their networks are regulated,and it is good for us to understand the functional genes in human.Moreover,it has a great significance in providing a new idea for the control of polar growth and fungal drug resistance and the current fungal disease treatment strategies.