Study On Important Toxic Factors Of Candida Albicans By Electron Microscopy

Candida albicans is an important threat to patients with diseases such as AIDS and cancer as well as immunocompromised people.Currently,morphological plasticity is considered as a key toxic factor in Candida albicans pathogenesis.In this thesis,the key proteins that play a role in different stages of the morphological transformation of Candida albicans were taken as the research objects.First,the urea amidolyase(UA)that plays a metabolic role in the first stage and produces the morphological transformation signal——CO2 was structurally elucidated to analyze its molecular mechanism;then the characteristics and structure of pH-regulated antigen(Pral),a key protein mediating immune escape in the second stage,were analyzed to explore the interaction between Pral and upstream proteins of the complement pathway.Through the analysis of key proteins at different stages of hyphal growth,this study attempts to provide a reference and basis for the mechanisms involved in the pathogenicity of Candida albicans.In the first part of this thesis,we focused on UA,which belongs to the biotin-dependent carboxylase family.Conjugation of the active site and hydrolysis of the substrate through the swinging arm have been recognized as the enzyme activity mechanism of this family,but the structures of multiple biotin-dependent carboxylase family proteins that have been solved show that relying solely on the swinging arm is not enough to achieve the transfer of biotin in the two active sites,the biotin carboxyl carrier protein(BCCP)must also be translocate during catalysis,this is referred to as the "swinging-domain" model.Although multiple evidence in the family of biotin--dependent carboxylase proteins and phosphoubiquitin-dependent enzymes support this theory,there is no definitive structural evidence that can prove the mechanism of the "swinging-domain".In this study,the unloaded state(KlUA-APO),ADP bound state(KlUA+ADP)and the ADP urea bound state(KlUA+ADP+Urea)of full-length UA(Kluyveromyces lactis Urea Amidolyase,KlUA)was observed by negative staining single-particle 3D reconstruction.By comparing the structural differences of KlUA in the three states,it was found that the BCCP position in the KlUA dimer was asymmetric,the BCCP in the KlUA dimer without substrate and ligand was more flexible and swung more significantly,and it was speculated that the BCCP of each monomer could swing spontaneously between biotin carboxylase(BC)and carboxyl transferase(CT)without relying on the binding of substrate or ligand,and the BCCP in each monomer moved independently of each other.This structural feature is captured by the computational "purification" of single-particle three-dimensional remodelling,providing a structural explanation for the efficient enzymatic activity of UA and,to some extent,a structural basis for the"swinging-domain" model of the biotin-dependent carboxylase family.In the second part of the thesis,we focused on pH-regulated antigen(Pral),which plays a key role when Candida albicans immune escape.As an endogenous complement inhibitor expressed by Candida albicans,Pral can inhibit the activation of alternative pathway(AP)by cleaving complement component C3.Studies have shown that Pral can also inhibit the exertion of classical pathway(CP)and lectin pathway(LP)functions,and has an inhibitory effect on C3b deposition and immunophagy in the CP/LP pathway comparable to that in the AP pathway.In order to investigate the interference of Pral on the upstream key proteins of the three complement pathways,we used prokaryotic and eukaryotic expression systems to construct and express and purify Pral and complement proteins C2 and C4,extracted and purified C3 from plasma,and analyzed the characteristics of each protein,provide experimental materials for the study.The interaction between Pral and each complement protein was analyzed by enzyme-linked immunosorbent assay.The results showed that Pral was able to interact with CP/LP pathway complement component C2 and C4,and this interaction was independent of glycosylation modification of the protein itself while required metal ions for support.And it was conserved among species.These results preliminarily verified our conjecture that Pral inhibits the CP/LP pathway,that is,Pral may interfere with the function of C3 convertase(C4b2a)by binding C2 and C4 proteins.Subsequently,we analyzed the biochemical properties of Pral protein and showed that Pral in the native state existed in the solution as multimers greater than 250 kDa,with reducing resistance,and the formation of multimers was independent of metal ion binding.When the protein concentration increased,a concentration-dependent precipitation was produced.On the basis of the analysis of the characteristics of Pral protein,we also tried to dissect the molecular mechanism of complement component C3 protein cleavage by Pral using electron microscopic 3D remodeling technique.Firstly,the structural characteristics of Pral monomer were predicted and analyzed by homology modeling.Subsequently,the structural model of C3 protein in the native state was obtained by negative staining single-particle 3D reconstruction.Finally,the formation conditions of C3-Pra1 complex were explored,and the concentration ratio,incubation temperature and incubation time were used as variables to finally determine the optimal conditions for electron microscopic observation of C3-Pral complex.The above experimental results provide an experimental basis and some initial models for the next step in the structural analysis of the C3-Pral complex,and build an experimental system for the structural biology study of the interaction between Pral protein and upstream complement component C2 and C4 of CP/LP during Candida albicans immune escape.