SPT20 Regulates Hyphal Formation Through Ras1/cAMP/PKA Pathway In Candida Albicans

Background:Candida albicans can be isolated from oral-pharyngeal,gastrointestinal,and urogenital tracts of healthy humans and cause infectious diseases in immunocompromised patients.Hyphal formation,the major virulence factor of C.albicans,is critical for biofilm formation,drug resistant and pathogenicity.Thus,exploring the regulation of hyphal formation and its related mechanisms is important to elucidate the pathogenic mechanism and develope new antifungal drugs.Hyphal formation is regulated by a range of environmental signals and transcriptional factors.Our previous study showed that SPT20 was involved in hyphal formation in C.albicans.However,the role of SPT20 in hyphal formation and its mechanism were obscure.Aim:To explore the relationship between SPT20 and hyphal formation in C.albicans,and further investigate how SPT20 regulated hyphal formation.Method:We tested the morphology of C.albicans spt20Δ/Δ mutant grown in various hypha-inducing media,and used quantitative reverse transcription-PCR studies to measure the expression levels of hyphal formation-association genes.Also,SPT20 expression was test in hyphal growth form and yeast gowth form of wild type,respectively.Furthermore,to explore the relationsip between SPT20 and Ras1/cAMP/PKA signaling,we tested transcript levels of genes acted in this pathway as well.Meanwhile,the concentration of cAMP and the activity of PKA were determined using an enzyme immunoassay and a non-radioactive protein kinase assay kit,respectively.And the morphology of C.albicans spt20Δ/Δ mutant strain was observed after the exogenous addition of cAMP.The release of LDH was measured to evaluate the effect SPT20 has on C.albicans cytotoxicity on cells,and the survival rate of fungal cells was calculated to evaluate C.albicans ability to escape from killing by macrophages.Result:We found that compared to wild type,C.albicans spt20Δ/Δ mutant strain was defective in all tested hypha-inducing media,and the expression level of genes associated with hyphal formation was significantly decreased.The SPT20 expression was upregulated when C.albicans cells were grown in hyphal form.Furthermore,genes acted in the Ras1/cAMP/PKA pathway were downregulated,and the cAMP level and the PKA activity were decreased in spt20Δ/Δ mutant strain.Notably,the exogenous addition of cAMP rescued the hyphal formation defect.Both the release of LDH and the survival rate of fungal cells were decreased when SPT20 mutant cells were co-cultrued with macrophages.Conclusion:SPT20 plays an important role in hyphal formation.It affacts hyphal formation,biofilm formation,and cytotoxicity of C.albicans,and influences C.albicans ability to excape from the killing by immune cells.SPT20 involved in the regulation of hyphal formation through Ras1/cAMP/PKA pathway,which make it could be a target of novol antifungal drugs.