TpcA In Aspergillus Fumigatus Participates In The Functional Study Of Thiamine Pyrophosphate Transport And Iron Absorption

Owing to its capacity to be an indispensable cofactor for a variety of cellular processes including amino acid metabolism,respiration and biosynthesis of sterols and DNA,iron is an essential nutrient for virtually every organism.The availability of free iron is very limited by pathogens due to it exists in the form of ferritin,lactoferrin or transferrin in host.Previous results strongly suggest that most fungal pathogens to obtain iron from their hosts are critical for fungi to cause disease in humans.Aspergillus fumigatus,the most common airborne fungal pathogen of humans,which causes life-threatening invasive disease especially in immunocompromised patients.In Aspergillus fumigatus,regulation of iron homeostasis is mediated by two central transcription factors,HapX and SreA,which are interconnected in a negative feedback loop:HapX represses expression of sreA during iron starvation,while SreA represses hapX during iron sufficiency.During iron starvation,HapX functions include the repression of iron-consuming pathways to spare iron and activation of iron uptake by siderophores.Instead,under iron-replete conditions,expression of the GATA factor SreA is increased,which results in repression of the hapX and induces the expression of iron assimilation genes.On the other hand,SreA binds to the ATCWGATAA sequences and also inhibits the expression of siderophore genes.In order to finding new genes which are potentially involved in iron uptake in A.fumigatus,a large T-DNA insertional mutagenesis library(～2000 transformants)was constructed.All of the transformants were first screened for colonies impairment in the presence of the iron chelator bathophenanthroline disulfonate(BPS)on solid media.The mutant,named H0421,which obtained from the first round screening was sensitive in low iron.Here,we report the identification of one novel gene,tpcA,which has been cloned in an insertional mutagenesis screen for pathogenic mutants of A.fumigatus by TAIL-PCR technique.Moreover,we confirmed that deletion of tpcA resulted in the colonies phenotype of AtpcA mutant was similar to T-DNA insertional mutagenesis(H0421)in low iron.Our results showed that we had successfully screened a gene,tpcA,which plays important roles in low iron.The homologous gene of tpcA in saccharomyces cerevisiae is tpcl which transports ThPP into the mitochondria in exchange for thiamine monophosphate(ThMP).However,tpcA,involved in the iron uptake under iron limitation which is not studied in A.fumigatus.The main results were as follows:(1)In this study,we found that TpcA is an integral protein of the inner mitochondrial membrane by the construction of strains with green fluorescent protein(GFP)fused to TpcA.And this result is consistent with the cellular location of S.cerevisiae.(2)The AtpcA strains,but not wild-type strains,which were not able to grow on minimal medium(MM)in the absence of thiamine.In addition,the growth phenotype of ΔtpcA strains on thiamine-less MM was fully restored by complementing the knock-out strain with the TPC1-pAN7-1 plasmid.(3)We used site-directed mutagenesis to introduce individual mutations in the predicted conserved function site Arg53,Asp60,Gly153,Gly205,Lys255 and Lys315 and examined the effect of each mutation on function of the TpcA in Thpp transport.Our results showed that mutating Gly 153,Asp60,Lys255 and Lys315 to lead to a significant retardant hyphal growth and sharp reduction in conidia production,while mutating Arg53 and Gly205 was without an effect.Our findings that with adding extracellular Thpp was able to rescue the growth defects on MM.In addition,the colony phenotype of two sites mutation strains in the K255 and K315,with similar the ΔtpcA strains,which were not able to grow and exhibited an auxotrophy for thiamine on minimal medium(MM).Thus,among the six residues predicted,only Gly 153,Asp60,Lys 255 and Lys315 were found to be play a role in transport function.(4)In the tpcA disruption mutant;the transcript levels of selected genes involved in iron metabolism no dramatically changes.However,the extracellular siderophore production of the ΔtpcA mutant was decreased by at least 30%compared with the wild-type and ΔtpcA::tpcA-complemented strains under iron starvation conditions.Collectively,our finding indicates that TpcA not only involved in as a mitochondrial transporter for thiamine pyrophosphate,but also in iron acquisition in the first time.TpcA inactivation decreases production of TAFC,which was not well able to use the way of siderophore-mediated iron uptake and ΔtpcA exhibited severely growth defect under iron starvation.In conclusion,our data suggest that a new role for TpcA in iron acquisition in A.fumigatus,via influencing the production of siderophore.Howere,future studies will continue to seek to elucidate the siderophore production mechanisms mediated by TpcA in A.fumigatus and its relationship to fungal virulence.