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.
- MeSH
- aspartátové endopeptidasy antagonisté a inhibitory metabolismus MeSH
- Candida parapsilosis enzymologie MeSH
- fungální proteiny antagonisté a inhibitory metabolismus MeSH
- inhibitory proteas chemická syntéza chemie farmakologie MeSH
- molekulární modely MeSH
- molekulární struktura MeSH
- peptidomimetika chemická syntéza chemie farmakologie MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
Pathogenic yeasts Candida albicans and Candida parapsilosis possess a ß-type carbonic anhydrase Nce103p, which is involved in CO2 hydration and signaling. C. albicans lacking Nce103p cannot survive in low CO2 concentrations, e.g., in atmospheric growth conditions. Candida carbonic anhydrases are orthologous to the Saccharomyces cerevisiae enzyme, which had originally been detected as a substrate of a non-classical export pathway. However, experimental evidence on localization of C. albicans and C. parapsilosis carbonic anhydrases has not been reported to date. Immunogold labeling and electron microscopy used in the present study showed that carbonic anhydrases are localized in the cell wall and plasmatic membrane of both Candida species. This localization was confirmed by Western blot and mass spectrometry analyses of isolated cell wall and plasma membrane fractions. Further analysis of C. albicans and C. parapsilosis subcellular fractions revealed presence of carbonic anhydrases also in the cytosolic and mitochondrial fractions of Candida cells cultivated in shaken liquid cultures, under the atmospheric conditions.
- MeSH
- buněčná membrána enzymologie MeSH
- buněčná stěna enzymologie MeSH
- Candida albicans enzymologie růst a vývoj MeSH
- Candida parapsilosis enzymologie růst a vývoj MeSH
- cytosol enzymologie MeSH
- elektronová mikroskopie MeSH
- fungální proteiny metabolismus MeSH
- hmotnostní spektrometrie MeSH
- karboanhydrasy metabolismus MeSH
- mitochondrie enzymologie MeSH
- techniky vsádkové kultivace MeSH
- Publikační typ
- časopisecké články MeSH
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.
- MeSH
- aspartátové endopeptidasy genetika metabolismus MeSH
- buněčné linie MeSH
- Candida parapsilosis enzymologie patogenita MeSH
- faktory virulence genetika metabolismus MeSH
- fungální proteiny genetika metabolismus MeSH
- imunitní únik MeSH
- komplement imunologie MeSH
- lidé MeSH
- makrofágy mikrobiologie MeSH
- virulence MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Most of the phosphatases of human fungal pathogens Candida albicans and C. parapsilosis have never been experimentally characterised, although dephosphorylation reactions are central to many biological processes. PHO15 genes of these yeasts have been annotated as the sequences encoding 4-nitrophenyl phosphatase, on the basis of homology to PHO13 gene from the bakers' yeast Saccharomyces cerevisiae. To examine the real function of these potential phosphatases from Candida spp., CaPho15p and CpPho15p were prepared using expression in Escherichia coli and characterised. They share the hallmark motifs of the haloacid dehalogenase superfamily, readily hydrolyse 4-nitrophenyl phosphate at pH 8-8.3 and require divalent cations (Mg2+, Mn2+ or Co2+) as cofactors. CaPho15p and CpPho15p did not dephosphorylate phosphopeptides, but rather hydrolysed molecules related to carbohydrate metabolism. The preferred substrate for the both phosphatases was 2-phosphoglycolate. Among the other molecules tested, CaPho15 showed preference for glyceraldehyde phosphate and ß-glycerol phosphate, while CpPho15 dephosphorylated mainly 1,3-dihydroxyacetone phosphate. This type of substrate specificity indicates that CaPho15 and CpPho15 may be a part of metabolic repair system of C. albicans and C. parapsilosis.
- MeSH
- aminokyselinové motivy MeSH
- biotransformace MeSH
- Candida albicans enzymologie MeSH
- Candida parapsilosis enzymologie MeSH
- Escherichia coli genetika metabolismus MeSH
- exprese genu MeSH
- fosfatasy genetika metabolismus MeSH
- fungální proteiny genetika metabolismus MeSH
- glykoláty metabolismus MeSH
- klonování DNA MeSH
- koenzymy analýza MeSH
- rekombinantní proteiny genetika metabolismus MeSH
- substrátová specifita MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: The pathogenic yeast Candida albicans can proliferate in environments with different carbon dioxide concentrations thanks to the carbonic anhydrase CaNce103p, which accelerates spontaneous conversion of carbon dioxide to bicarbonate and vice versa. Without functional CaNce103p, C. albicans cannot survive in atmospheric air. CaNce103p falls into the β-carbonic anhydrase class, along with its ortholog ScNce103p from Saccharomyces cerevisiae. The crystal structure of CaNce103p is of interest because this enzyme is a potential target for surface disinfectants. RESULTS: Recombinant CaNce103p was prepared in E. coli, and its crystal structure was determined at 2.2 Å resolution. CaNce103p forms a homotetramer organized as a dimer of dimers, in which the dimerization and tetramerization surfaces are perpendicular. Although the physiological role of CaNce103p is similar to that of ScNce103p from baker's yeast, on the structural level it more closely resembles carbonic anhydrase from the saprophytic fungus Sordaria macrospora, which is also tetrameric. Dimerization is mediated by two helices in the N-terminal domain of the subunits. The N-terminus of CaNce103p is flexible, and crystals were obtained only upon truncation of the first 29 amino acids. Analysis of CaNce103p variants truncated by 29, 48 and 61 amino acids showed that residues 30-48 are essential for dimerization. Each subunit contains a zinc atom in the active site and displays features characteristic of type I β-carbonic anhydrases. Zinc is tetrahedrally coordinated by one histidine residue, two cysteine residues and a molecule of β-mercaptoethanol originating from the crystallization buffer. The active sites are accessible via substrate tunnels, which are slightly longer and narrower than those observed in other fungal carbonic anhydrases. CONCLUSIONS: CaNce103p is a β-class homotetrameric metalloenzyme composed of two homodimers. Its structure closely resembles those of other β-type carbonic anhydrases, in particular CAS1 from Sordaria macrospora. The main differences occur in the N-terminal part and the substrate tunnel. Detailed knowledge of the CaNce103p structure and the properties of the substrate tunnel in particular will facilitate design of selective inhibitors of this enzyme.
- MeSH
- Candida albicans enzymologie MeSH
- karboanhydrasy chemie MeSH
- katalytická doména MeSH
- krystalografie rentgenová MeSH
- kvarterní struktura proteinů MeSH
- molekulární modely MeSH
- multimerizace proteinu MeSH
- sekvence aminokyselin MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
