In the standard genetic code, the CUG triplet is translated as leucine. The pathogenic yeast Candida albicans and other CTG-clade yeasts contain tRNACAG, which is recognized by both leucine- and serine-tRNA synthetases. The CUG codon in these yeasts is translated most often as serine, and only in 3-5% of cases as leucine. Therefore, CTG Candida species have unstable proteomes. The effect of serine-leucine exchange on the structure and function of proteins has only been experimentally examined for a few cases. In C. albicans, CUG codons occur even in genes deemed to be essential. This means that serine-leucine ambiguity either does not affect the structure and function of the respective proteins, or that the presence of these amino acids at specific positions is associated with meaningful alteration of the proteins' function. This study employed AlphaFold2 to evaluate the potential effects of serine-to-leucine exchange in 12 proteins encoded by essential genes lacking orthologs in other yeasts and human genomes. The low homology with known proteins allowed us to make only low-confidence predictions. The analyzed proteins could be grouped into subsets based on the structural outcomes. Structural changes were observed only in four proteins. The remaining eight proteins showed no significant differences between serine and leucine variants.

• Keywords
• AlphaFold2, CUG codon, Candida albicans, codon usage, essential gene, orphan gene,
• Publication type
• Journal Article MeSH

Pathogenic Candida albicans yeasts frequently cause infections in hospitals. Antifungal drugs lose effectiveness due to other Candida species and resistance. New medications are thus required. Secreted aspartic protease of C. parapsilosis (Sapp1p) is a promising target. We have thus solved the crystal structures of Sapp1p complexed to four peptidomimetic inhibitors. Three potent inhibitors (Ki: 0.1, 0.4, 6.6 nM) resembled pepstatin A (Ki: 0.3 nM), a general aspartic protease inhibitor, in terms of their interactions with Sapp1p. However, the weaker inhibitor (Ki: 14.6 nM) formed fewer nonpolar contacts with Sapp1p, similarly to the smaller HIV protease inhibitor ritonavir (Ki: 1.9 µM), which, moreover, formed fewer H-bonds. The analyses have revealed the structural determinants of the subnanomolar inhibition of C. parapsilosis aspartic protease. Because of the high similarity between Saps from different Candida species, these results can further be used for the design of potent and specific Sap inhibitor-based antimycotic drugs.

• Keywords
• Inhibitor, crystal structure, hydrogen bonds, noncovalent interactions, peptidomimetics,
• MeSH
• Aspartic Acid Endopeptidases antagonists & inhibitors   metabolism   MeSH
• Candida parapsilosis enzymology   MeSH
• Fungal Proteins antagonists & inhibitors   metabolism   MeSH
• Protease Inhibitors chemical synthesis   chemistry   pharmacology   MeSH
• Models, Molecular MeSH
• Molecular Structure MeSH
• Peptidomimetics chemical synthesis   chemistry   pharmacology   MeSH
• Dose-Response Relationship, Drug MeSH
• Structure-Activity Relationship MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Aspartic Acid Endopeptidases MeSH
• Fungal Proteins MeSH
• Protease Inhibitors MeSH
• Peptidomimetics MeSH
• SAPP1 protein, Candida parapsilosis MeSH Browser

Candida parapsilosis is an emerging non-albicans Candida species that largely affects low-birth-weight infants and immunocompromised patients. Fungal pathogenesis is promoted by the dynamic expression of diverse virulence factors, with secreted proteolytic enzymes being linked to the establishment and progression of disease. Although secreted aspartyl proteases (Sap) are critical for Candida albicans pathogenicity, their role in C. parapsilosis is poorly elucidated. In the present study, we aimed to examine the contribution of C. parapsilosisSAPP genes SAPP1, SAPP2, and SAPP3 to the virulence of the species. Our results indicate that SAPP1 and SAPP2, but not SAPP3, influence adhesion, host cell damage, phagosome-lysosome maturation, phagocytosis, killing capacity, and cytokine secretion by human peripheral blood-derived macrophages. Purified Sapp1p and Sapp2p were also shown to efficiently cleave host complement component 3b (C3b) and C4b proteins and complement regulator factor H. Additionally, Sapp2p was able to cleave factor H-related protein 5 (FHR-5). Altogether, these data demonstrate the diverse, significant contributions that SAPP1 and SAPP2 make to the establishment and progression of disease by C. parapsilosis through enabling the attachment of the yeast cells to mammalian cells and modulating macrophage biology and disruption of the complement cascade.IMPORTANCE Aspartyl proteases are present in various organisms and, among virulent species, are considered major virulence factors. Host tissue and cell damage, hijacking of immune responses, and hiding from innate immune cells are the most common behaviors of fungal secreted proteases enabling pathogen survival and invasion. C. parapsilosis, an opportunistic human-pathogenic fungus mainly threatening low-birth weight neonates and children, possesses three SAPP protein-encoding genes that could contribute to the invasiveness of the species. Our results suggest that SAPP1 and SAPP2, but not SAPP3, influence host evasion by regulating cell damage, phagocytosis, phagosome-lysosome maturation, killing, and cytokine secretion. Furthermore, SAPP1 and SAPP2 also effectively contribute to complement evasion.

• Keywords
• Candida parapsilosis, complement, host-pathogen interactions, proteases, virulence,
• MeSH
• Aspartic Acid Endopeptidases genetics   metabolism   MeSH
• Cell Line MeSH
• Candida parapsilosis enzymology   pathogenicity   MeSH
• Virulence Factors genetics   metabolism   MeSH
• Fungal Proteins genetics   metabolism   MeSH
• Immune Evasion MeSH
• Complement System Proteins immunology   MeSH
• Humans MeSH
• Macrophages microbiology   MeSH
• Virulence MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Aspartic Acid Endopeptidases MeSH
• Virulence Factors MeSH
• Fungal Proteins MeSH
• Complement System Proteins MeSH
• SAPP1 protein, Candida parapsilosis MeSH Browser

Candida parapsilosis produces secreted aspartic proteinases (Saps), which contribute to the virulence of this opportunistic pathogen. Gene family containing as many as 14 sequences potentially encoding secreted aspartic proteinases was identified in C. parapsilosis genome. Of them, SAPP1 and SAPP2 genes have been extensively characterized, but only now do we report that two SAPP2 homologs sharing 91.5 % identity occur in C. parapsilosis genome. Existence of SAPP2 homologs points to unexpected complexity of the SAPP gene family.

• MeSH
• Aspartic Acid Proteases chemistry   genetics   metabolism   MeSH
• Candida chemistry   enzymology   genetics   MeSH
• Fungal Proteins chemistry   genetics   metabolism   MeSH
• Genome, Fungal * MeSH
• Molecular Sequence Data MeSH
• Amino Acid Sequence MeSH
• Sequence Alignment MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Aspartic Acid Proteases MeSH
• Fungal Proteins MeSH

Secreted aspartic proteinase Sapp1p of Candida parapsilosis represents one of the factors contributing to the pathogenicity of the fungus. The proteinase is synthesized as an inactive pre-pro-enzyme, but only processed Sapp1p is secreted into extracellular space. We constructed a plasmid containing the SAPP1 coding sequence under control of the ScGAL1 promoter and used it for proteinase expression in a Saccharomyces cerevisiae kex2Δ mutant. Because Sapp1p maturation depends on cleavage by Kex2p proteinase, the kex2Δ mutant secreted only the pro-form of Sapp1p. Characterization of this secreted proteinase form revealed that the Sapp1p signal peptide consists of 23 amino acids. Additionally, we prepared a plasmid with the SAPP1 coding sequence under control of its authentic CpSAPP1 promoter, which contains two GATAA motifs. While in C. parapsilosis SAPP1 expression is repressed by good low molecular weight nitrogen sources (e.g., ammonium ions), S. cerevisiae cells harboring this plasmid secreted a low concentration of active proteinase regardless of the type of nitrogen source used. Quantitative real-time PCR analysis of a set of genes related to nitrogen metabolism and uptake (GAT1, GLN3, STP2, GAP1, OPT1, and PTR2) obtained from S. cerevisiae cells transformed with either plasmid encoding SAPP1 under control of its own promoter or empty vector and cultivated in media containing various nitrogen sources also suggested that SAPP1 expression can be connected with the S. cerevisiae regulatory network. However, this regulation occurs in a different manner than in C. parapsilosis.

• MeSH
• Candida enzymology   genetics   MeSH
• Nitrogen metabolism   MeSH
• Endopeptidases metabolism   MeSH
• Real-Time Polymerase Chain Reaction MeSH
• Saccharomyces cerevisiae enzymology   genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Nitrogen MeSH
• Endopeptidases MeSH

Pathogenic yeasts of the genus Candida produce secreted aspartic proteinases, which are known to enhance virulence. We focused on Sapp1p proteinase secreted by Candida parapsilosis and studied the final stage of its passage through the cell wall and release into the extracellular environment. We found that Sapp1p displays enzyme activity prior to secretion, and therefore, it is probably fully folded within the upper layer of the cell wall. The positioning of cell surface-associated Sapp1p was detected by cell wall protein labeling using biotinylation agents, extraction of cell wall proteins by β-mercaptoethanol, immunochemical detection, and mass spectrometry analysis. All lysine residues present in the structure of soluble, purified Sapp1p were labeled with biotin. In contrast, the accessibility of individual lysines in cell wall-associated Sapp1p varied with the exception of four lysine residues that were biotinylated in all experiments performed, suggesting that Sapp1p has a preferred orientation in the cell wall. As the molecular weight of this partially labeled Sapp1p did not differ among the experiments, we can assume that the retaining of Sapp1p in the cell wall is not a totally random process and that pathogenic yeasts might use this cell-associated proteinase activity to enhance degradation of appropriate substrates.

• MeSH
• Aspartic Acid Proteases analysis   chemistry   metabolism   MeSH
• Biotinylation MeSH
• Cell Wall chemistry   enzymology   MeSH
• Candida chemistry   enzymology   MeSH
• Models, Molecular MeSH
• Molecular Sequence Data MeSH
• Proteolysis MeSH
• Amino Acid Sequence MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Aspartic Acid Proteases MeSH