The cell wall is essential to nearly every aspect of the biology and pathogenicity of Candida albicans. Although it was intially considered an almost inert cellular structure that protected the protoplast against osmotic offense, more recent studies have demonstrated that it is a dynamic organelle. The major components of the cell wall are glucan and chitin, which are associated with structural rigidity, and mannoproteins. The protein component, including both mannoprotein and nonmannoproteins, comprises some 40 or more moieties. Wall proteins may differ in their expression, secretion, or topological location within the wall structure. Proteins may be modified by glycosylation (primarily addition of mannose residues), phosphorylation, and ubiquitination. Among the secreted enzymes are those that are postulated to have substrates within the cell wall and those that find substrates in the extracellular environment. Cell wall proteins have been implicated in adhesion to host tissues and ligands. Fibrinogen, complement fragments, and several extracellular matrix components are among the host proteins bound by cell wall proteins. Proteins related to the hsp70 and hsp90 families of conserved stress proteins and some glycolytic enzyme proteins are also found in the cell wall, apparently as bona fide components. In addition, the expression of some proteins is associated with the morphological growth form of the fungus and may play a role in morphogenesis. Finally, surface mannoproteins are strong immunogens that trigger and modulate the host immune response during candidiasis.

Cell Wall and Secreted Proteins of Candida albicans: Identification, Function, and Expression

https://scholarlypublications.universiteitleiden.nl/access/item%3A2865233/view

Identification of a novel acidic mammalian chitinase distinct from chitotriosidase

There has been a considerable amount of recent research aimed at elucidating the roles of chitinase in fungi and plants. In filamentous fungi and yeasts, chitinase is involved integrally in cell wall morphogenesis. Chitinase is also involved in the early events of host-parasite interactions of biotrophic and necrotrophic mycoparasites, entomopathogenic fungi and vesicular arbuscular mycorrhizal fungi. In plants, induction of chitinase and other hydrolytic enzymes is one of a coordinated, often complex and multifaceted defense mechanism triggered in response to phytopathogen attack. Chitinase induction in plants is not considered solely as an antifungal resistance mechanism. Plant chitinases can be induced by various abiotic factors as well and there is some circumstantial evidence to suggest a morphogenetic role despite the apparent absence of the substrate in plant cells. Finally, some chitinases and other chitin-binding proteins including some plant lectins share chitin-binding domains as part of their molecular structure and provide fuel for the so-called ‘lectin-chitinase’ debate and speculation for the origin of chitinase in plants.

Chitinases of fungi and plants: their involvement in morphogenesis and host‐parasite interaction

Chitin is an essential component of the fungal cell wall and its synthesis is under tight spatial and temporal regulation. The fungal human pathogen Candida albicans has a four member chitin synthase gene family comprising of CHS1 (class II), CHS2 (class I), CHS3 (class IV) and CHS8 (class I). LacZ reporters were fused to each CHS promoter to examine the transcriptional regulation of chitin synthesis. Each CHS promoter had a unique regulatory profile and responded to the addition of cell wall damaging agents, to mutations in specific CHS genes and exogenous Ca2+. The regulation of both CHS gene expression and chitin synthesis was co-ordinated by the PKC, HOG MAP kinase and Ca2+/calcineurin signalling pathways. Activation of these pathways also resulted in increased chitin synthase activity in vitro and elevated cell wall chitin content. Combinations of treatments that activated multiple pathways resulted in synergistic increases in CHS expression and in cell wall chitin content. Therefore, at least three pathways co-ordinately regulate chitin synthesis and activation of chitin synthesis operates at both transcriptional and post-transcriptional levels.

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The PKC, HOG and Ca2+ signalling pathways co-ordinately regulate chitin synthesis in Candida albicans

Synthesis and Biological Activity of Natural Aminocyclopentitol Glycosidase Inhibitors: Mannostatins, Trehazolin, Allosamidins, and Their Analogues.

Filamentous fungi with chitin as a major component of their cell walls produce chitinases at all stages of active growth, i.e. during spore germination, exponential growth and mycelial development. The roles of chitinases in these processes have been investigated by assessing the effects of treatment with the inhibitor, allosamidin. Inhibition has only been observed, however, of spore germination, of cell separation in budding yeasts and of the action of a yeast toxin, and not of apical extension or branching. This may be explained by the finding that chitinase activities in situ have different properties to those in cell homogenates, and appear to be protected from environmental stresses.

What are the roles of chitinases in the growing fungus

The effects of changes in external osmotic pressure on chitin synthase activity of a dimorphic fungus, Benjaminiella poitrasii, have been investigated. Mycelial and yeast cells incubated in medium of low osmolality (distilled water, 0 mOsm) for 10 min had 2–3-fold higher specific activities of native chitin synthase in mixed membrane preparations than cells that had been subjected to a high osmolality medium (1.2 M sorbitol in distilled water, 1612 mOsm). Cells suspended in media of different osmolalities for 10 min were also affected in the extent of germ tube formation. Germ tube formation was highest in cells incubated in low osmolality medium. The addition of protein phosphatase inhibitors (cyclosporin A, 1.2 μg/ml; cantharidin, 20 μM) abolished the effect of hypo-osmotic stress on chitin synthase activation of yeast mixed membrane preparations. The presence of protein kinase inhibitors (genistein, 40 μg/ml; H-7, 100 μM) and a Ca2+ channel blocker (verapamil, 50 μM) reduced chitin synthase activity to 50–60% of that observed in cells under hypo-osmotic shock. These inhibitors also inhibited germ tube formation. This suggests that chitin synthase activity and yeast hyphal morphogenesis are both subject to regulation by osmotic pressure, phosphorylation and calcium.

Regulation of chitin synthase activity in the dimorphic fungus Benjaminiella poitrasii by external osmotic pressure

Allosamidin, a glycoside antibiotic, is shown to be a strong, competitive inhibitor of semi-purified chitinase from yeast cells of Candida albicans. The inhibitory potency of allosamidin was pH-dependent, with IC50 values of 280 nM at pH 5.0 and 21 nM at pH 7.5. At higher, micromolar, concentrations, allosamidin inactivated this chitinase in a time- and concentration-dependent manner. Kinetic studies of this inactivation provided evidence for the formation of a reversible complex between allosamidin and chitinase, characterized by Kinact = 5 microM, followed by irreversible modification of the enzyme with velocity constant k2 = 4.6 x 10(-3) s-1. Chemical modification studies with the use of group-specific reagents suggested the presence of Glu/Asp carboxyl group(s) at or near the active site, that were important for enzyme activity. The carboxyl-specific reagent, 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide, inactivated the chitinase in a single step process, with apparent second-order rate constant of 0.014 M-1 s-1.

Raymond W. O'Donnell

Papers

What are the roles of chitinases in the growing fungus

Regulation of chitin synthase activity in the dimorphic fungus Benjaminiella poitrasii by external osmotic pressure

Chemical modification studies of the active centre of Candida albicans chitinase and its inhibition by allosamidin.