| Backgrounds and Objectives:In recent years, with the increasing of organ transplant patients, the widespread use ofimmunosuppressive agents, as well as the extensive use of broad-spectrum antibiotics, thepopularity and development of the interventional medical treatment, worldwide incidenceof invasive fungal infections is rising sharply. The threat of invasive fungal infections tohuman health and life has become a worldwide medical problem. In clinical fungalinfections, Candida is the most common pathogenic fungi, with the detection rate rankedfirst for all pathogenic fungi in hospital blood samples. Of the Candida infections, Candidaalbicans is the most common, which has the highest infection rate. Therefore, the study ofthe pathogenesis of C. albicans is important for the prevention and treatment of fungalinfections.The growth of C. albicans is characterized by the remarkable state of yeast, pseudohyphaeand hypha. And they intertwined to be biofilm. Recently more and more studies showedthat biofilm and hypha are important for the C. albicans virulence. Research of genesrelated to the C. albicans biofilm formation and hypha formation of is important for theclarification of the pathogenesis of C. albicans. This study focuses on the screening anddiscovery of C. albicans biofilm, hypha and virulence related genes, and exploring theunderlying mechanism.Animal models are important for new drug discovery. Notably, insect infection modelshave significant advantages and provide a rapid evaluation of the efficacy and toxicity ofagents in vivo. Among the insects available, Galleria mellonella emerged at the forefront.Methods:Bioinformatics analysis found that ECM17might be involved in the biosynthesis of sulfuramino acids and play an important role in the formation of hyphae and biofilm in C.albicans. In this study, we used HIS1-LEU2-ARG4strategy to knockout and reintegrateECM17gene in C. albicans strain SN152. BIGGY agar assay was performed to detect theH2S generation in theecm17/mutant. XTT experiment was performed to detect thebiofilm formation of theecm17/mutant. SLD solid culture experiment was performedto investigate the hyphae formation and Real-Time RT PCR was performed to detect theexpression of many different hyphae and adhesion related genes. Finally we detected the intracellular cAMP levels of the mutant and analyzed the possible signal pathway.In addition, before this study, we screened a library of C. albicans mutants using theheterologous host Caenorhabditis elegans infection model and got six genes which mightbe important to the hyphae formation and virulence of C. albicans. In this study, we usedthe caSAT1knockout strategy to construct six C. albicans mutants with the deletion ofthese genes. Then growth curves assay, biofilm formation assay, hyphae formation assay,drug susceptibility test and animal experiments were performed to detect the difference ofthe gene knockout strains and the parental strain. Based on these results, we found thatNGG1is important for the hyphae formation and virulence of C. albicans. After that,microarray experiment was performed to detect the gene expression changes of thengg1/mutant.G. mellonella-C. albicans infection model has been widely used in studies of fungalpathogenesis. However, the established model was not suitable to evaluate antifungalagents. In this study, we optimized the G. mellonella-C. albicans infection model and usedthis model to evaluate different antifungals successfully.Results：1. The result showed that C. albicansecm17/mutant was unable to catalyze thebiochemical reaction from sulfite to H2S, and hardly grew in medium lacking Met and Cys.With the supply of H2S donor, the growth ofecm17/was improved in theMet/Cys-lacking medium. These results confirmed the important role of ECM17in Metand Cys biosynthesis. Biofilm formation was attenuated byecm17/mutant comparedwith the wild-type or reintegrated strains. In addition, adhesion and filamentous growth ofecm17/declined. Further results indicated that the defective biofilm formation ofecm17/was possibly related to the down-regulation of ALS3, CSH1, HWP1and ECE1,four important genes in adherence and filamentous growth. It is known that HWP1, ALS3and ECE1are regulated by cAMP-PKA pathway, and our further results indicated thatdisruption of ECM17decreased cAMP level under all conditions in this study. In addition,the filamentous growth ofecm17/on SLD lacking Met or Cys was improved markedlywith the addition of exogenous cAMP, indicating that cAMP-PKA pathway is associatedwith ECM17and Met/Cys biosynthesis pathway, especially for filamentous growth.2. In this study, we successfully knocked out six C. albicans genes which mightimpact the hyphae formation and virulence of C. albicans. Then we found that the ngg1/ mutant grew slowly in YPD medium, and showed decreased hyphae formation, decreasedbiofilm formation ability, reduced sensitivity to azoles, oxidative stress and cell wallrelated compounds. Also, the results showed that ngg1/is avirulent to mice at theconcentration of1×106CFU/mice. Microarray results showed that under hyphae inducingcondition, the expression of several membrane, vacuole, peroxisomes and microsomesrelated genes were up regulated in ngg1/mutant. These genes are involved in theintracellular oxidation-reduction process of C. albicans, as well as the biosynthesis,transport and metabolism of oxygen acids, organic acids, carboxylic acids and fatty etc.Meanwhile, some genes relate to C. albicans cellular components organization regulation,chromosome separation and organization, cell cycle regulation, mitosis regulation, and cellgrowth regulation were down regulated. Changes in expression of these genes might beassociated with the decreased growth rate, reduced hyphae formation ability, attenuatedvirulence of the ngg1/mutant.3. To evaluate antifungals against C. albicans, we modified the G. mellonella-C.albicans infection model. We tested a range of inocula and chose5×105CFU/larva as thesuitable inoculum for the infection model to evaluate antifungals. Using this G.mellonella-C. albicans infection model, the antifungal activity of FLC, AMB and5-FC,and the synergy between amphotericin B and flucytosine were verified.Conclusion:1. It is the first study showed that in C. albicans, ECM17regulated the biosynthesisof Met and Cys by controlling the generation of H2S, and ECM17-dependent Met/Cysbiosynthesis played an important role in biofilm formation through affecting adhesion andfilamentous growth. This work provides new insights into the role of Met/Cys as signalmolecules and is helpful to better understand the associations between Met/Cysbiosynthesis pathway, cAMP-PKA pathway and biofilm formation.2. As an invertebrate model, C. elegans has obvious advantages in screening C.albicans hyphae formation and virulence related genes. Genes obtained based on thescreening results have important potential research value. NGG1is involved in the growthand hyphae formation of C. albicans. The disruption of NGG1can disturb the biosynthesis,transport and metabolism of some nutrients in C. albicans. Also, NGG1can impact the cellcycle and mitosis of C. albicans. All these effects can lead to the slowly growth of ngg1/,as well as the decreased hyphae formation ability and weakened virulence. The mechanism might be related to the possible biological function of the gene: encode histoneacetyltransferase.3. G. mellonella-C. albicans infection model is reliable to evaluate antifungals invivo and promising in drug screening work. |
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