Analysis of the 3H8 antigen of Candida albicans reveals new aspects of the organization of fungal cell wall proteins

The fungal cell wall is essential in maintaining cellular integrity and plays key roles in the interplay between fungal pathogens and their hosts. The PGA59 and PGA62 genes encode two short and related glycosylphosphatidylinositol-anchored cell wall proteins and their expression has been previously shown to be strongly upregulated when the human pathogen Candida albicans grows as biofilms. Using GFP fusion proteins, we have shown that Pga59 and Pga62 are cell-wall-located, N- and O-glycosylated proteins. The characterization of C. albicans pga59Δ/pga59Δ, pga62Δ/pga62Δ and pga59Δ/pga59Δ pga62Δ/pga62Δ mutants suggested a minor role of these two proteins in hyphal morphogenesis and that they are not critical to biofilm formation. Importantly, the sensitivity to different cell-wall-perturbing agents was altered in these mutants. In particular, simultaneous inactivation of PGA59 and PGA62 resulted in high sensitivity to Calcofluor white, Congo red and nikkomicin Z and in resistance to caspofungin. Furthermore, cell wall composition and observation by transmission electron microscopy indicated an altered cell wall structure in the mutant strains. Collectively, these data suggest that the cell wall proteins Pga59 and Pga62 contribute to cell wall stability and structure.

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A Whole-Cell Candida albicans Assay for the Detection of Inhibitors towards Fungal Cell Wall Synthesis and Assembly.

The Journal of Antibiotics ◽

10.7164/antibiotics.48.306 ◽

1995 ◽

Vol 48 (4) ◽

pp. 306-310 ◽

Cited By ~ 81

Author(s):

DAVID J. FROST ◽

KIM D. BRANDT ◽

DAVID CUGIER ◽

ROBERT GOLDMAN

Keyword(s):

Cell Wall ◽

Candida Albicans ◽

Fungal Cell Wall ◽

Cell Wall Synthesis ◽

Fungal Cell ◽

Whole Cell

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The Viscoelastic Properties of the Fungal Cell Wall Allow Traffic of AmBisome as Intact Liposome Vesicles

mBio ◽

10.1128/mbio.02383-17 ◽

2018 ◽

Vol 9 (1) ◽

Cited By ~ 55

Author(s):

Louise Walker ◽

Prashant Sood ◽

Megan D. Lenardon ◽

Gillian Milne ◽

Jon Olson ◽

...

Keyword(s):

Cell Wall ◽

Candida Albicans ◽

Cell Membrane ◽

Amphotericin B ◽

Mode Of Action ◽

Viscoelastic Properties ◽

Fungal Cell Wall ◽

Fungal Cell ◽

Pass Through ◽

Cell Wall Porosity

ABSTRACT The fungal cell wall is a critically important structure that represents a permeability barrier and protective shield. We probed Candida albicans and Cryptococcus neoformans with liposomes containing amphotericin B (AmBisome), with or without 15-nm colloidal gold particles. The liposomes have a diameter of 60 to 80 nm, and yet their mode of action requires them to penetrate the fungal cell wall to deliver amphotericin B to the cell membrane, where it binds to ergosterol. Surprisingly, using cryofixation techniques with electron microscopy, we observed that the liposomes remained intact during transit through the cell wall of both yeast species, even though the predicted porosity of the cell wall (pore size, ~5.8 nm) is theoretically too small to allow these liposomes to pass through intact. C. albicans mutants with altered cell wall thickness and composition were similar in both their in vitro AmBisome susceptibility and the ability of liposomes to penetrate the cell wall. AmBisome exposed to ergosterol-deficient C. albicans failed to penetrate beyond the mannoprotein-rich outer cell wall layer. Melanization of C. neoformans and the absence of amphotericin B in the liposomes were also associated with a significant reduction in liposome penetration. Therefore, AmBisome can reach cell membranes intact, implying that fungal cell wall viscoelastic properties are permissive to vesicular structures. The fact that AmBisome can transit through chemically diverse cell wall matrices when these liposomes are larger than the theoretical cell wall porosity suggests that the wall is capable of rapid remodeling, which may also be the mechanism for release of extracellular vesicles. IMPORTANCE AmBisome is a broad-spectrum fungicidal antifungal agent in which the hydrophobic polyene antibiotic amphotericin B is packaged within a 60- to 80-nm liposome. The mode of action involves perturbation of the fungal cell membrane by selectively binding to ergosterol, thereby disrupting membrane function. We report that the AmBisome liposome transits through the cell walls of both Candida albicans and Cryptococcus neoformans intact, despite the fact that the liposome is larger than the theoretical cell wall porosity. This implies that the cell wall has deformable, viscoelastic properties that are permissive to transwall vesicular traffic. These observations help explain the low toxicity of AmBisome, which can deliver its payload directly to the cell membrane without unloading the polyene in the cell wall. In addition, these findings suggest that extracellular vesicles may also be able to pass through the cell wall to deliver soluble and membrane-bound effectors and other molecules to the extracellular space.

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Iron alters the cell wall composition and intracellular lactate to affect Candida albicans susceptibility to antifungals and host immune response

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.013413 ◽

2020 ◽

Vol 295 (29) ◽

pp. 10032-10044 ◽

Cited By ~ 3

Author(s):

Aparna Tripathi ◽

Elisabetta Liverani ◽

Alexander Y. Tsygankov ◽

Sumant Puri

Keyword(s):

Immune Response ◽

Cell Wall ◽

Candida Albicans ◽

Signaling Pathways ◽

Host Immune Response ◽

Fungal Cell Wall ◽

Mitogen Activated Protein Kinase ◽

Fungal Cell ◽

High Iron ◽

Cell Wall Architecture

Fungal pathogen Candida albicans has a complex cell wall consisting of an outer layer of mannans and an inner layer of β-glucans and chitin. The fungal cell wall is the primary target for antifungals and is recognized by host immune cells. Environmental conditions such as carbon sources, pH, temperature, and oxygen tension can modulate the fungal cell wall architecture. Cellular signaling pathways, including the mitogen-activated protein kinase (MAPK) pathways, are responsible for sensing environmental cues and mediating cell wall alterations. Although iron has recently been shown to affect β-1,3-glucan exposure on the cell wall, we report here that iron changes the composition of all major C. albicans cell wall components. Specifically, high iron decreased the levels of mannans (including phosphomannans) and chitin; and increased β-1,3-glucan levels. These changes increased the resistance of C. albicans to cell wall-perturbing antifungals. Moreover, high iron cells exhibited adequate mitochondrial functioning; leading to a reduction in accumulation of lactate that signals through the transcription factor Crz1 to induce β-1,3-glucan masking in C. albicans. We show here that iron-induced changes in β-1,3-glucan exposure are lactate-dependent; and high iron causes β-1,3-glucan exposure by preventing lactate-induced, Crz1-mediated inhibition of activation of the fungal MAPK Cek1. Furthermore, despite exhibiting enhanced antifungal resistance, high iron C. albicans cells had reduced survival upon phagocytosis by macrophages. Our results underscore the role of iron as an environmental signal in multiple signaling pathways that alter cell wall architecture in C. albicans, thereby affecting its survival upon exposure to antifungals and host immune response.

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Effect of cilofungin (LY121019), a fungal cell wall synthesis inhibitor, on interactions of Candida albicans with human neutrophils

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/24.5.741 ◽

1989 ◽

Vol 24 (5) ◽

pp. 741-745 ◽

Cited By ~ 6

Author(s):

Tova Meshulam ◽

Stuart M. Levitz ◽

Richard D. Diamond ◽

Alan M. Sugar

Keyword(s):

Cell Wall ◽

Candida Albicans ◽

Fungal Cell Wall ◽

Human Neutrophils ◽

Cell Wall Synthesis ◽

Synthesis Inhibitor ◽

Fungal Cell ◽

Cell Wall Synthesis Inhibitor

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Proteolytic Cleavage of Covalently Linked Cell Wall Proteins by Candida albicans Sap9 and Sap10

Eukaryotic Cell ◽

10.1128/ec.00210-10 ◽

2010 ◽

Vol 10 (1) ◽

pp. 98-109 ◽

Cited By ~ 57

Author(s):

Lydia Schild ◽

Antje Heyken ◽

Piet W. J. de Groot ◽

Ekkehard Hiller ◽

Marlen Mock ◽

...

Keyword(s):

Cell Wall ◽

Candida Albicans ◽

Cell Surface ◽

Pathogenic Fungus ◽

Proteolytic Cleavage ◽

Cell Wall Proteins ◽

Ph Optimum ◽

Fungal Cell ◽

Content Type ◽

Covalently Linked

ABSTRACT The cell wall of the human-pathogenic fungus Candida albicans is a robust but also dynamic structure which mediates adaptation to changing environmental conditions during infection. Sap9 and Sap10 are cell surface-associated proteases which function in C. albicans cell wall integrity and interaction with human epithelial cells and neutrophils. In this study, we have analyzed the enzymatic properties of Sap9 and Sap10 and investigated whether these proteases cleave proteins on the fungal cell surface. We show that Sap9 and Sap10, in contrast to other aspartic proteases, exhibit a near-neutral pH optimum of proteolytic activity and prefer the processing of peptides containing basic or dibasic residues. However, both proteases also cleaved at nonbasic sites, and not all tested peptides with dibasic residues were processed. By digesting isolated cell walls with Sap9 or Sap10, we identified the covalently linked cell wall proteins (CWPs) Cht2, Ywp1, Als2, Rhd3, Rbt5, Ecm33, and Pga4 as in vitro protease substrates. Proteolytic cleavage of the chitinase Cht2 and the glucan-cross-linking protein Pir1 by Sap9 was verified using hemagglutinin (HA) epitope-tagged versions of both proteins. Deletion of the SAP9 and SAP10 genes resulted in a reduction of cell-associated chitinase activity similar to that upon deletion of CHT2 , suggesting a direct influence of Sap9 and Sap10 on Cht2 function. In contrast, cell surface changes elicited by SAP9 and SAP10 deletion had no major impact on the phagocytosis and killing of C. albicans by human macrophages. We propose that Sap9 and Sap10 influence distinct cell wall functions by proteolytic cleavage of covalently linked cell wall proteins.

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