Functional Characterization Of The Stimulatory Activity Of Cell Wall Component N-acetylglucosamine And A Proposed M Rna Binding Protein Spo5 In Cryptococcus Neoformans During Unisexual Reproduction

Cryptococcus neoformans is the model organism among human environmental fungal pathogens,whose infections of hosts are not obligated for their survival.This pathogen can cause severe cryptococcal meningitis,mainly in immunodeficient populations,and claims ~ 600,000 death annually.Treatment options for this infectious disease are limited and the mortality rate is unacceptably high.Thus it is essential to understand the mechanisms underling Cryptococcus infection and virulence evolution to develop better preventative or therapeutic options and manage the disease.Sexual reproduction(sex)is a fundamental feature in genetically divergent eukaryotes including fungi.In human fungal pathogens,sex can serve as a key biological strategy to promote virulence evolution due to its exceptional capability to create genetic diversity through genetic exchanging(meiotic recombination).In C.neoformans,sexual reproduction is highly associated with its infection and virulence evolution.The expression of the genes involved in cryptococcal sexs requires the induction by a combination of diverse stimuli,including environmental factors and metabolite signals.However,little is known regarding the extracellular and intracellular signalings that coordinate the infection-related sexual reproduction in C.neoformans.N-acetylglucosamine(GlcNAc)is the monomer of the chitin,which is an important component of fungal cell wall,and is of a variety of biological functions due to its key acitivities in signaling trusdution.This study attempts to understand the biological functions of GlcNAc in C.neoformans.We found that GlcNAc displays a strong stimulatory effect on the progression of Cryptococcus neoformans unisexual reproduction.This induction is specific for unisexual program since its impact on bisexual mating is not evident.By means of a combination of in silico and experimental approches,we systemically identified the enzymes involved in the catabolism of GlcNAc,including GlcNAc transporter Ngt1,GlcNAc kinase Hxk1,6-PO4-GlcNAc deacetylase Dac1,6-PO4-GLN Deaminase Nag1,metabolic regulator protein GlcR1 and oxidative dehydrogenase Str I.Of note,the kinase Hxk1 and regulator protein GlcR1,which are previously unassigned,are widespread exclusively among Basidiomycota species,where the catabolism of GlcNAc remains poorly understood.Their deletions abolish the utilization of GlcNAc by Cryptococcus cells,suggesting their essential role in GlcNAc metabolism.The molecular investigations of these proteins are still ongoing.In addition,we found that the genes engaged in GlcNAc utilization is not required for it-mediated induction of unisex.This indicates that C.neoformans may involve independent pathways specifying the metabolism and signal activity by GlcNAc.For identification of intraceullar signaling component responsible for unisexual reproduction,we reveals a proposed mRNA binding protein Spo5 in C.neoformans and demonstrates its core role in guiding the formation of basidia,which is a morphological hull-mark for sexual reproduction in Basidiomycota species.As far as we know,SPO5 represents the first determiant for basidia formation.Collectively,we evaluate the functional roles of GlcNAc in Cryptococcus biology,for the first time unveil the genetic basis underlying GlcNAc catabolism in Basidiomycota,demonstrate its regulatory function on inducing unisexual reproduction and identify a key gene SPO5 required for an important stage of unisexual reproduction-basidia formation.Our findings provide important insight into how extracellular and intracellular cues orchestrate sexual reproduction in Cryptocococus neoformans.