Study On Roles Of H2B Monoubiquitination In Regulating Dimorphic Transition And Biosynthesis Of Virulence Factors In Cryptococcus Neoformans

Cryptococcus neoformans is an important zoonotic pathogenic fungus,which mainly exists in saprophytic soil and pigeon manure.Spores or dried yeast cells enter host lungs through the respiratory tract,establishing asymptomatic infection in host with competent immunity.Once the host’s immune system gets compromised,the pathogen of latent infection can be reactivated and rapidly proliferated,causing systemic infection and fatal meningitis.The mortality rate of meningitis caused by C.neoformans is extremely high.Even with systematic antifungal drug treatment,the mortality rate can still be as high as 70 %.C.neoformans is the most common pathogen causing meningitis in HIV carriers in sub-Saharan Africa and causes more than 1 million human infections worldwide each year resulting in more than 600,000 deaths.C.neoformans is a dimorphic fungus that generates different morphotypes,including yeast,mycelium and spores in its life cycle.Studies have shown that yeast-to-hypha transition is closely related to its virulence and pathogenicity in C.neoformans.The yeast cells can effectively evade the host immune attack and is the virulent form;the hyphae can trigger host immune protection response,which is the avirulent form.The genetic regulatory mechanism of dimorphic transition in C.neoformans has been widely studied.However,its epigenetic regulation mechanism is largely unknown.Therefore,investigation of epigenetic regulation mechanism of the yeast-to-hypha transition is helpful to systematically and comprehensively understand the morphogenesis in C.neoformans,and provide a theoretical basis for the development of new control strategies against cryptococcal infection.On the basis of previous studies,we identified and characterized the homologous protein of ubiquitin-conjugating enzyme Rad6 involved in H2B monoubiquitination(H2Bub1)in C.neoformans by amino acid sequence conservation analysis.We also identified the core subunits Paf1,Ctr9,Rtf1,and Cdc73 of the Paf1 complex(PAF1C),which is necessary for H2Bub1 in eukaryotes.To investigate the effects of H2Bub1 on the dimorphic transition and virulence,we constructed mutants with deletion and complementation of the above-mentioned genes in C.neoformans.The mycelial growth and virulence of the mutants were tested,and the results are as follows:1.Phylogenetic and conservation analyses of the ubiquitin-conjugating enzyme Rad6 and PAF1C core subunits in C.neoformans.According to the protein sequence ubiquitin-conjugating enzyme Rad6 and PAF1C subunit in Saccharomyces cerevisiae,we obtained the corresponding protein candidates in C.neoformans by BLAST function of Fungi DB(https://fungidb.org/).The protein sequence was subjected to phylogenetic analysis and protein domain composition analyses.The results showed that ubiquitin-conjugating enzyme Rad6 and PAF1C core subunits,including Paf1,Ctr9,Rtf1,and Cdc73 were highly conserved across eukaryotes.2.Deletion and complementation of genes encoding Rad6 and PAF1C core subunits in C.neoformans,and evaluation of their effects on H2Bub1.The coding genes of Rad6 and PAF1C subunit candidates were knocked out and complemented by CRISPR/Cas9-based genetic manipulation technique.Then,the level of H2Bub1 in each mutant was analyzed by Western blotting.The results showed that deletion of Rad6 and PAF1C core subunits Paf1,Ctr9,Rtf1 and Cdc73 caused a significant decrease in H2Bub1 level.In addition,the complementary strains with either green fluorescent-or FLAG-tagged fusion proteins restored the H2Bub1 level in corresponding deletion mutants.Fluorescence observation showed that the fluorescence signals of were enriched in the nucleus,indicating the nuclear localization of Rad6 and PAF1C subunits in C.neoformans.3.Effects of Rad6 and PAF1C on dimorphic transition and virulence factors of Cryptococcus neoformans.In order to explore whether Rad6 and PAF1C regulate the dimorphic transition and virulence factors of C.neoformans,we cultured rad6Δ,paf1Δ,rtf1Δ,ctr9Δ and cdc73Δ on V8 mycelium inducing medium.The results showed that the mycelium of rad6Δ,paf1Δ,rtf1Δ,ctr9Δ and cdc73Δ was significantly reduced,and the complementary strains recovered the mycelium growth.In addition,cell growth and melanin formation of each mutant strain were significantly inhibited at 37 ℃,while the capsular formation induced on LIM medium was not significantly affected by gene deletion and overexpression relative to that of the wild type strain.In summary,ubiquitin-conjugating enzyme Rad6 and PAF1C-mediated H2Bub1 regulate the dimorphic transition and virulence factors of C neoformans.4.Effects of RAD6 and PAF1C core subunit RTF1 deletion on the transcriptional profile of C.neoformans.In order to explore the effect of Rad6 and PAF1C-mediated H2Bub1 on the gene transcription of C.neoformans,we performed a comparative transcriptomic analysis of the coding gene deletion mutants of Rad6 and PAF1C core subunit Rtf1 between yeast-to-hypha transition inducing and inhibiting conditions,with the wild-type strain as control.The results showed that RTF1 and RAD6 co-regulated the transcription levels of genes known to be involved in dimorphic transition,such as ZNF2,CFL1,MF2,3 and STE3α,indicating that Rad6 and PAF1C-mediated H2Bub1 can specifically regulate the transcription of dimorphic transition-related genes,thereby regulating dimorphic transition.In summary,we identified the ubiquitin conjugating enzyme Rad6 and the core subunits of PAF1C including Paf1,Ctr9,Rtf1 and Cdc73 in C.neoformans,and verified their functional conservation in mediating H2Bub1.Next,we determined the effects of Rad6 and PAF1C on the dimorphic transition and virulence in C.neoformans by analyzing the mycelial development and production of virulence factors in deletion and complementation mutants.Finally,we conducted the comparative transcriptomic analysis with RAD6 and RTF1 gene deletion mutants under mycelial inducing and inhibiting conditions,and demonstrated that Rtf1 and Rad6 regulate transcripts level of known genes that are involved in dimorphic transition in C.neoformans.This study preliminarily investigated the roles of Rad6 and PAF1C-mediated H2Bub1 in regulating the dimorphic transition and virulence factor production,and provided knowledge for systematic and comprehensive understanding of the epigenetic regulation of dimorphic transition and virulence factors in C.neoformans.It is of great significance for development of new prevention and control strategies based on epigenetic regulatory pathways.