Functional Characterization Of Phosphomannose Isomerase In Pathogenic Fungi Fusarium Oxysporum F. Sp. Cubense And Aspergillus Flavus

Fungal infections,posing an increasing threat to global public health,involve over six million fungal species worldwide,with less than 1%known to infect humans.Simultaneously,fungal phytopathogens significantly impact global crop production,causing an estimated 15%annual average loss.Within the Fusarium genus,comprising over 300 species,Fusarium oxysporum stands out for causing vascular wilt in plants.The Fusarium oxysporum f.sp.cubense(Foc)variant,responsible for banana wilt disease,results in substantial economic losses in the banana industry.Similarly,Aspergillus flavus,an opportunistic fungal pathogen,is capable of causing invasive aspergillosis and contaminates seed crops such as maize and peanuts with the carcinogenic aflatoxin.Unfortunately,the strategies available to combat fungal infections are limited,and the increasing incidence of fungicide resistance compounds the challenge.The cell wall,a crucial component enveloping the fungal cell,plays a vital role in maintaining cellular integrity and shielding the cell from external aggressors,such as environmental fluxes and host infections.Notably absent in animal cells,it differs from plant cell walls,making it a potential target for developing antifungal drugs.Comprising glucan,chitin,mannan,and glycoproteins,the fungal cell wall relies on three precursors for biosynthesis:UDP-glucose(UDP-Glc),UDP-GlcNAc,and GDP-Man.The biosynthesis of GDP-Man involves the consecutive catalysis by phosphomannose isomerase(PMI),phosphomannose mutase(PMM),and GDP-mannose pyrophosphorylase(GMPP).PMI,the initiating enzyme in the GDP-Man biosynthesis pathway,converts fructose-6-phosphate(Fru-6-P)into mannose-6-phosphate(Man-6-P),effectively linking the glycolytic pathway with cell wall biosynthesis.The pivotal role of PMI in growth and virulence is documented in various organisms,including Tobacco mosaic virus,Cryptococcus neoformans,Leishmania mexicana,and Aspergillus fumigatus.However,the function of PMI in the pathogenic fungi Fusarium oxysporum f.sp.cubense(Foc TR4)and A.flavus remains unclear.This study aims to elucidate the functional role of PMI in Foc TR4 and A.flavus,shedding light on its significance in these specific fungal species.The blast search using Saccharomyces cerevisiae PMI revealed two PMIencoding orthologues,FocPMIl and FocOMI2,within the Foc TR4 genome.Quantitative real-time PCR gene expression analysis demonstrated consistent expression of Focpmil across all developmental stages of Foc TR4.Mutant strains lacking Focpmil(ΔFocpmi1)exhibited a dependency on exogenous mannose for growth,while the ΔFocpmi2 mutant could grow without mannose supplementation.Consequently,FocPMIl was identified as the key enzyme responsible for mannose isomerase activity.Deletion of Focpmil resulted in severe growth abnormalities,with the mutant displaying optimal growth when the medium was supplemented with 5 mM mannose.Neither low nor high mannose concentrations supported the growth of the ΔFocpmil mutant.Analysis of cell wall contents,in comparison to the wild type(WT),indicated reduced chitin content in the mutant cell wall,rendering it susceptible to cell wall stressors.Additionally,the mutant exhibited heightened sensitivity to the oxidative stressor hydrogen peroxide(H2O2).Transcriptomic comparison between the mutant and the WT revealed down-regulation of several genes involved in host cell wall degradation.This down-regulation impaired the mutant’s ability to traverse the plant cell wall,resulting in a loss of pathogenicity.Consequently,FocPMI1 is a potential antifungal target against Foc TR4,given its crucial role in fungal growth,cell wall integrity,and pathogenicity.Likewise,the blast search using A.fumigatus PMI identified only one putative homolog of PMI in A.flavus.The pmi-deficient strain in A.flavus(ΔpmiA)displayed mannose auxotrophy,necessitating exogenous mannose for growth.The mutant exhibited optimum growth at 3 mM exogenous mannose,with partial growth observed at 0.5 and 5 mM,while concentrations exceeding 10 mM completely inhibited the mutant’s growth.Despite the presence of optimal mannose concentration,the mutant exhibited pronounced growth abnormalities,including impaired conidiation,early germination,and disruptions in stress responses.The mutant displayed hypersensitivity to cell wall stressors,demonstrating the crucial role of pmiA in cell wall biosynthesis and maintaining its integrity.Additionally,the ΔpmiA mutant strain exhibited increased sensitivity to cell membrane,osmotic,and oxidative stressors.Pathogenicity assays unveiled the mutant’s incapacity to colonize peanut and corn hosts,with no aflatoxins detected in infected corn and peanut seeds.Furthermore,the mutant demonstrated attenuated virulence in animal infection models,utilizing Caenorhabditis elegans and Galleria mellonella,thereby emphasizing the significant contribution of pmiA to the pathogenicity of A.flavus.In conclusion,this study underscores the crucial significance of PMI in Foc TR4 and A.flavus,shedding light on its vital roles in development,cell wall biogenesis,stress responses,and pathogenicity for both organisms.The findings suggest that PMI holds potential as a promising drug target for combating infections and addressing threats posed by Foc TR4 and A.flavus.This insight opens avenues for further research and the development of targeted strategies to mitigate the impact of these fungal pathogens.