Primary Substrate Specificities of Secreted Aspartic Proteases of Candida albicans

ABSTRACT The secretion of aspartic proteases (Saps) has long been recognized as a virulence-associated trait of the pathogenic yeast Candida albicans. In this study, we report that different recombinant Saps, including Sap1, Sap2, Sap3, and Sap6, have differing abilities to induce secretion of proinflammatory cytokines by human monocytes. In particular Sap1, Sap2, and Sap6 significantly induced interleukin-1β (IL-1β), tumor necrosis factor alpha (TNF-α), and IL-6 production. Sap3 was able to stimulate the secretion of IL-1β and TNF-α. All Saps tested were able to induce Ca2+ influx in monocytes. Treatment of these Saps with pepstatin A did not have any effect on cytokine secretion, indicating that their stimulatory potential was independent from their proteolytic activity. The capacity of Saps to induce inflammatory cytokine production was also independent from protease-activated receptor (PAR) activation and from the optimal pH for individual Sap activity. The interaction of Saps with monocytes induced Akt activation and phosphorylation of IκBα, which mediates translocation of NF-κB into the nucleus. Overall, these results suggest that individual Sap proteins can induce an inflammatory response and that this phenomenon is independent from the pH of a specific host niche and from Sap enzymatic activity. The inflammatory response is partially dependent on Sap denaturation and is triggered by the Akt/NF-κB activation pathway. Our data suggest a novel, activity-independent aspect of Saps during interactions of C. albicans with the host.

Download Full-text

Inactivation of α1-proteinase inhibitor by Candida albicans aspartic proteases favors the epithelial and endothelial cell colonization in the presence of neutrophil extracellular traps.

Acta Biochimica Polonica ◽

10.18388/abp.2015_1163 ◽

2015 ◽

Vol 63 (1) ◽

Cited By ~ 4

Author(s):

Mariusz Gogol ◽

Dominika Ostrowska ◽

Kinga Klaga ◽

Oliwia Bochenska ◽

Natalia Wolak ◽

...

Keyword(s):

Candida Albicans ◽

Proteinase Inhibitor ◽

Proteolytic Enzymes ◽

Fungal Infections ◽

Critical Role ◽

Neutrophil Extracellular Traps ◽

Host Cells ◽

Inhibition Mechanism ◽

Aspartic Proteases ◽

Extracellular Traps

Candida albicans, a causative agent of opportunistic fungal infections in immunocompromised patients, uses ten secreted aspartic proteases (SAPs) to deregulate the homeostasis of the host organism on many levels. One of these deregulation mechanisms involves a SAP-dependent disturbance of the control over proteolytic enzymes of the host by a system of dedicated proteinase inhibitors, with one important example being the neutrophil elastase and alpha1-proteinase inhibitor (A1PI). In this study, we found that soluble SAPs 1-4 and the cell membrane-anchored SAP9 efficiently cleaved A1PI, with the major cleavage points located at the C-terminal part of A1PI in a close vicinity to the reactive-site loop that plays a critical role in the inhibition mechanism. Elastase is released by neutrophils to the environment during fungal infection through two major processes, a degranulation or formation of neutrophil extracellular traps (NET). Both, free and NET-embedded elastase forms, were found to be controlled by A1PI. A local acidosis, resulting from the neutrophil activity at the infection sites, favors A1PI degradation by SAPs. The deregulation of NET-connected elastase affected a NET-dependent damage of epithelial and endothelial cells, resulting in the increased susceptibility of these host cells to candidal colonization. Moreover, the SAP-catalyzed cleavage of A1PI was found to decrease its binding affinity to a proinflammatory cytokine, interleukin-8. The findings presented here suggest a novel strategy used by C. albicans for the colonization of host tissues and overcoming the host defense.

Download Full-text

The yeast Candida albicans evades human complement attack by secretion of aspartic proteases

Molecular Immunology ◽

10.1016/j.molimm.2009.08.019 ◽

2009 ◽

Vol 47 (2-3) ◽

pp. 465-475 ◽

Cited By ~ 88

Author(s):

Katharina Gropp ◽

Lydia Schild ◽

Susann Schindler ◽

Bernhard Hube ◽

Peter F. Zipfel ◽

...

Keyword(s):

Candida Albicans ◽

Human Complement ◽

Aspartic Proteases

Download Full-text

Role of secreted aspartic proteases in Candida albicans pathogenesis

10.5353/th_b5576756 ◽

2015 ◽

Author(s):

Haiping Yang

Keyword(s):

Candida Albicans ◽

Aspartic Proteases

Download Full-text

Enzymic characteristics of secreted aspartic proteases of Candida albicans

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology ◽

10.1016/s0167-4838(00)00068-6 ◽

2000 ◽

Vol 1480 (1-2) ◽

pp. 117-131 ◽

Cited By ~ 59

Author(s):

Gerald Koelsch ◽

Jordan Tang ◽

Jeffrey A Loy ◽

Michel Monod ◽

Kenneth Jackson ◽

...

Keyword(s):

Candida Albicans ◽

Aspartic Proteases

Download Full-text

Inactivation of the Antifungal and Immunomodulatory Properties of Human Cathelicidin LL-37 by Aspartic Proteases Produced by the Pathogenic Yeast Candida albicans

Infection and Immunity ◽

10.1128/iai.00023-15 ◽

2015 ◽

Vol 83 (6) ◽

pp. 2518-2530 ◽

Cited By ~ 39

Author(s):

Maria Rapala-Kozik ◽

Oliwia Bochenska ◽

Marcin Zawrotniak ◽

Natalia Wolak ◽

Grzegorz Trebacz ◽

...

Keyword(s):

Candida Albicans ◽

Phorbol Esters ◽

Proteolytic Processing ◽

Yeast Cells ◽

Calcium Flux ◽

Fungal Colonization ◽

Pathogenic Yeast ◽

Aspartic Proteases ◽

Content Type ◽

Immunomodulatory Properties

Constant cross talk betweenCandida albicansyeast cells and their human host determines the outcome of fungal colonization and, eventually, the progress of infectious disease (candidiasis). An effective weapon used byC. albicansto cope with the host defense system is the release of 10 distinct secreted aspartic proteases (SAPs). Here, we validate a hypothesis that neutrophils and epithelial cells use the antimicrobial peptide LL-37 to inactivateC. albicansat sites of candidal infection and thatC. albicansuses SAPs to effectively degrade LL-37. LL-37 is cleaved into multiple products by SAP1 to -4, SAP8, and SAP9, and this proteolytic processing is correlated with the gradual decrease in the antifungal activity of LL-37. Moreover, a major intermediate of LL-37 cleavage—the LL-25 peptide—is antifungal but devoid of the immunomodulatory properties of LL-37. In contrast to LL-37, LL-25 did not affect the generation of reactive oxygen species by neutrophils upon treatment with phorbol esters. Stimulating neutrophils with LL-25 (rather than LL-37) significantly decreased calcium flux and interleukin-8 production, resulting in lower chemotactic activity of the peptide against neutrophils, which may decrease the recruitment of neutrophils to infection foci. LL-25 also lost the function of LL-37 as an inhibitor of neutrophil apoptosis, thereby reducing the life span of these defense cells. This study indicates thatC. albicanscan effectively use aspartic proteases to destroy the antimicrobial and immunomodulatory properties of LL-37, thus enabling the pathogen to survive and propagate.

Download Full-text

Structure-based specificity mapping of secreted aspartic proteases of Candida parapsilosis, Candida albicans, and Candida tropicalis using peptidomimetic inhibitors and homology modeling

Biological Chemistry ◽

10.1515/bc.2006.154 ◽

2006 ◽

Vol 387 (9) ◽

pp. 1247-1254 ◽

Cited By ~ 7

Author(s):

Filip Majer ◽

Libuše Pavlíčková ◽

Pavel Majer ◽

Martin Hradilek ◽

Elena Dolejší ◽

...

Keyword(s):

Crystal Structure ◽

Candida Albicans ◽

Homology Modeling ◽

Candida Tropicalis ◽

Disulfide Bonds ◽

Candida Parapsilosis ◽

Aspartic Proteases ◽

Hydrophobic Residues ◽

Structural Differences ◽

Peptidomimetic Inhibitors

AbstractSecreted aspartic proteases (Saps) of pathogenicCandidaspp. represent a specific target for antifungal drug development. We synthesized a series of peptidomimetic inhibitors with different isosteric groups and modifications at individual positions and tested them with purified Saps fromC. albicans(Sap2p),C. tropicalis(Sapt1p), andC. parapsilosis(Sapp1p). The kinetic parameters indicated that all three proteases prefer binding of inhibitors containing bulky hydrophobic residues between positions P3 and P3′. The most divergent specificity was found for Sapp1p. The sequence alignment of Sap2p, Sapt1p, and Sapp1p, and homology modeling of Sapp1p with the crystal structure of Sapt1p and the complex of Sap2p with a peptidomimetic inhibitor showed that the overall folds of Sap2p, Sapt1p, and Sapp1p are similar. However, the N- and C-terminal loops formed by disulfide bonds between residues 47–53 and 258–292 are significantly shorter in Sapp1p, and a unique insertion following Tyr 129 in Sapp1p results in the formation of a loop that can interact with inhibitor residues. These Sapp1p structural differences might lead to its altered susceptibility to inhibition.

Download Full-text