Calmodulin In Aspergillus Fumigatus Depends On The Molecular Characteristics Of Its Calcium Ion Binding Site And Its Important Physiological Functions

Calmodulin(CaM)is a highly conserved and ubiquitous Ca2+-sensing protein in eukaryotes,which includes four nonidentical EF-hand motifs to bind Ca2+.The primary function of CaM is to sense the changes in intracellular calcium concentrations and transduce the Ca2+ signal by regulating various target proteins.Previous studies have revealed that calmodulin is an essential protein and it peforms the essential functions relying on its ability of binding calcium in most organisms.However,in the opportunistic pathogen A.fumigatus,the reports of CaM remain limited yet.In this study,we explored the basic role of calmodulin in A.fumigatus and its dependence on calcium.Frist,we constructed a cam conditional mutant,which displayed severe defects in hyphal growth and conidiation.And we found that Aspergillus fumigatus CaM(AfCaM)was located to the active growing sites,which is similar to other species.Through homologous protein alignment and SMART analysis,AfCaM contains four EF-hand loops directly combined with Ca2+.A truncated mutant lacking these four loops showed similar defective phenotype to the conditional strain,indicating that these Ca2+-binding loops are important for fully function of AfCaM.Futher point mutation experiments in each Ca2+-binding site of CaM indicate that different sites are not identical for colony growth and the second one is the most important.Moreover,mutations to site2 lead to hypersensitivity to the thermal stress while high osmotic pressure can partly restore the abnormal phenotype(especially conidiation defects)in most mutants.In addition,upon calcium stimuli,Crzl from the CaM(124).CaM(134)mutants can't be translocated to the nucleus,indicating nuclear accumulation of Crz induced by calcium depends on CaM's Ca2+ binding ability.Mutations in Ca2+ binding sites can also lead to the disorder of cytoplasmic calcium.Taken together,our data provide direct evidence that calcium binding is required for various calmodulin function in Aspergillus fumigatus,which is significant for the reseach about the regulation of important cell process.