Fungi

The fungi are a group of eukaryotic microorganisms, some of which are capable of causing superficial, cutaneous, subcutaneous, or systemic disease.

Physiology

Fungi are heterotrophic and essentially aerobic, with limited anaerobic capabilities, and can synthesize lysine by the L-α-adipic acid biosynthetic pathway.

Structure

Fungi possess chitinous cell walls, plasma membranes containing ergosterol, 80S rRNA, and microtubules composed of tubulin.

Morphology

Yeasts are single-celled forms that reproduce by budding, whereas molds form multicellular hyphae. Dimorphic fungi grow as yeasts or spherules in vivo, as well as in vitro at 37°C, but as molds at 25°C. Dimorphism is regulated by factors such as temperature, CO₂ concentration, pH, and the levels of cysteine or other sulfhydryl-containing compounds.

Propagules

Conidia are asexual propagules (reproductive units) formed in various manners. Spores may be either asexual or sexual in origin. Asexual spores are produced in sac-like cells called sporangia and are called sporangiospores. Sexual spores include ascospores, basidiospores, oospores, and zygospores, which are used to determine phylogenetic relationships.

Classification

Asexual structures are referred to as anamorphs; sexual structures are known as teleomorphs; and the whole fungus is known as the holomorph. Two independent, coexisting classification systems, one based on anamorphs and the other on teleomorphs, are used to classify fungi.

Cell Wall

The rigid cell wall of fungi (see ch. 73, Fig. 2A) is a stratified structure consisting of chitinous microfibrils embedded in a matrix of small polysaccharides, proteins, lipids, inorganic salts, and pigments that provides skeletal support and shape to the enclosed protoplast. Chitin is a (β1–4)-linked polymer of N-acetyl-D-glucosamine (GlcNAc). It is produced in the cytosol by the transfer of GlcNAc from uridine diphosphate GlcNAc into chains of chitin by chitin synthetase, which is located in the cytosol in organelles called chitosomes. The chitin microfibrils are transported to the plasmalemma and subsequently integrated into the new cell wall.

The major polysaccharides of the cell wall matrix consist of noncellulosic glucans such as glycogen-like compounds, mannans (polymers of mannose), chitosan (polymers of glucosamine), and galactans (polymers of galactose). Small amounts of fucose, rhamnose, xylose, and uronic acids may be present. Glucan refers to a large group of D-glucose polymers having glycosidic bonds. Of these, the most common glucans composing the cell wall have the β-configuration. Polymers with (β1–3)- and (β1–6)-linked glucosyl units with various proportions of 1–3 and 1–6 linkages are common. Insoluble β-glucans are apparently amorphous in the cell wall. In Paracoccidioides brasiliensis, the hyphal cell wall consists of a single, 80- to 150-nm layer composed of chitin and β-glucan. In contrast, the 200- to 600-nm-thick yeast cell wall has three layers. The inner surface is chitinous, containing some α-glucan, and the outer layer contains α-glucan. It has been suggested that the (α1–3)-glucan occurs in a microfibrillar form in P brasiliensis and Histoplasma capsulatum.

Many fungi, especially the yeasts, have soluble peptidomannans as a component of their outer cell wall in a matrix of α- and β-glucans. Mannans, galactomannans, and, less frequently, rhamnomannans are responsible for the immunologic response to the medically important yeasts and molds. Mannans are polymers of mannose or heteroglucans with α-D-mannan backbones. Structurally, mannan consists of an inner core, outer chain, and base-labile oligomannosides. The outer-chain region determines its antigenic specificity. Determination of mannan concentrations in serum from patients with disseminated candidiasis has proven a useful diagnostic technique.

Cryptococcus neoformans produces a capsular polysaccharide composed of at least three distinct polymers: glucuronoxylomannan, galactoxylomannan, and mannoprotein. On the basis of the proportion of xylose and glucuronic acid residues, the degree to which mannose has side-chain substituents, and the percentage of O-acetyl attachments of the capsular polysaccharides, isolates of C neoformans can be separated into four antigenic groups designated A, B, C, and D. The capsule is antiphagocytic, serves as a virulence factor, persists in body fluids, and allows the yeast to avoid detection by the host immune system.

In addition to chitin, glucan, and mannan, cell walls may contain lipid, protein, chitosan, acid phosphatase, α-amylase, protease, melanin, and inorganic ions such as phosphorus, calcium, and magnesium. The outer cell wall of dermatophytes contains glycopeptides that may evoke both immediate and delayed cutaneous hypersensitivity. In the yeast Candida albicans, for example, the cell wall contains approximately 30 to 60 percent glucan, 25 to 50 percent mannan (mannoprotein), 1 to 2 percent chitin (located primarily at the bud scars in the parent yeast cell wall), 2 to 14 percent lipid, and 5 to 15 percent protein. The proportions of these components vary greatly from fungus to fungus. Table-M1 summarizes the relationship between cell wall composition and taxonomic grouping of the fungi.

Table

Cell Wall Composition and Taxonomic Classification of Representative Medically Important Fungi.

Plasma Membrane

Fungal plasma membranes are similar to mammalian plasma membranes, differing in having the nonpolar sterol ergosterol, rather than cholesterol, as the principal sterol. The plasma membrane regulates the passage of materials into and out of the cell by being selectively permeable. Membrane sterols provide structure, modulation of membrane fluidity, and possibly control of some physiologic events.

The plasma membrane contains primarily lipids and protein, along with small quantities of carbohydrates. The major lipids are the amphipathic phospholipids and sphingolipids that form the lipid bilayer. The hydrophilic heads are toward the surface, and the hydrophobic tails are buried in the interior of the membrane. Proteins are interspersed in the bilayer, with peripheral proteins being weakly bound to the membrane. In contrast, integral proteins are tightly bound. The lipoprotein structure of the membrane provides an effective barrier to many types of molecules. Molecules cross the membrane by either diffusion or active transport. The site of interaction for most antifungal agents is the ergosterol in the membrane or its biosynthetic pathway. Polyene antifungal agents such as amphotericin B bind to ergosterol to form complexes that permit the rapid leakage of the cellular potassium, other ions, and small molecules. The loss of potassium results in the inhibition of glycolysis and respiration.

Several antifungal agents interfere with ergosterol synthesis. The first step in the synthesis of both ergosterol and cholesterol is demethylation of lanosterol. The necessary enzymes are associated with fungal microsomes, which contain an electron transport system analogous to the one in liver microsomes. Cytochrome P₄₅₀ catalyzes the 1 4-α-demethylation of lanosterol, an essential step in the synthesis of ergosterol. The imidazole and triazole antifungal agents interfere with cytochrome P₄₅₀-dependent 1 4-α-demethylase, which inhibits the formation of ergosterol. This results in plasma membrane permeability changes and inhibition of growth. Ergosterol may also be involved in regulating chitin synthesis. Inhibition of ergosterol synthesis by antifungal agents can result in a general activation of chitin synthetase zymogen, leading to excessive chitin production and abnormal growth.

Microtubules

Fungi possess microtubules composed of the protein tubulin. This protein consists of a dimer composed of two protein subunits. Microtubules are long, hollow cylinders approximately 25 nm in diameter that occur in the cytoplasm as a component of larger structures. These structures are involved in the movement of organelles, chromosomes, nuclei, and Golgi vesicles containing cell wall precursors.

Microtubules are the principal components of the spindle fibers, which assist in the movement of chromosomes during mitosis and meiosis. When cells are exposed to antimicrotubule agents, the movement of nuclei, mitochondria, vacuoles, and apical vesicles is disrupted. Griseofulvin, which is used to treat dermatophyte infections, binds with microtubule-associated proteins involved in the assembly of the tubulin dimers. By interfering with tubulin polymerization, griseofulvin stops mitosis at metaphase. The destruction of cytoplasmic microtubules interferes with the transport of secretory materials to the cell periphery, which may inhibit cell wall synthesis.

The fungal nucleus is bounded by a double nuclear envelope and contains chromatin and a nucleolus. Fungal nuclei are variable in size, shape, and number. The DNA and associated proteins occur as long filaments of chromatin, which condenses during nuclear division. The number of chromosomes varies with the particular fungus. Within the cell, 80 to 99 percent of the genetic material occurs in chromosomes as chromatin, and approximately 1 to 20 percent in the mitochondria. In some isolates of Saccharomyces cerevisiae, up to 5 percent of their DNA can be found in nuclear plasmids. When the DNA helix unwinds, one strand serves as the template for the synthesis of rRNA, tRNA, and mRNA. mRNA passes into the cytoplasm and attaches to one of the ribosomes, which are complexes of RNA and protein that serve as sites for the synthesis of protein.

Yeasts

Yeasts are fungi that grow as solitary cells that reproduce by budding (see ch. 73Fig. 4 and 5). Yeast taxa are distinguished on the basis of the presence or absence of capsules, the size and shape of the yeast cells, the mechanism of daughter cell formation (conidiogenesis), the formation of pseudohyphae and true hyphae, and the presence of sexual spores, in conjunction with physiologic data. Morphology is used primarily to distinguish yeasts at the genus level, whereas the ability to assimilate and ferment various carbon sources and to utilize nitrate as a source of nitrogen are used in conjunction with morphology to identify species.

Yeasts such as C albicans and Cryptococcus neoformans produce budded cells known as blastoconidia. The formation of blastoconidia involves three basic steps: bud emergence, bud growth, and conidium separation. During bud emergence, the outer cell wall of the parent cell thins. Concurrently, new inner cell wall material and plasma membrane are synthesized at the site where new growth is occurring. New cell wall material is formed locally by activation of the polysaccharide synthetase zymogen. The process of bud emergence is regulated by the synthesis of these cellular components as well as by the turgor pressure in the parent cell. Mitosis occurs, as the bud grows, and both the developing conidium and the parent cell will contain a single nucleus. A ring of chitin forms between the developing blastoconidium and its parent yeast cell. This ring grows in to form a septum. Separation of the two cells leaves a bud scar on the parent cell wall. The bud scar contains much more chitin than does the rest of the parent cell wall. When the production of blastoconidia continues without separation of the conidia from each other, a pseudohypha, consisting of a filament of attached blastoconidia, is formed. In addition to budding yeast cells and pseudohyphae, yeasts such as C albicans may form true hyphae.

Molds

Molds are characterized by the development of hyphae (see ch. 73, Fig. 3), which result in the colony characteristics seen in the laboratory. Hyphae elongate by a process known as apical elongation, which requires a careful balance between cell wall lysis and new cell wall synthesis. Because molds are often differentiated on the basis of conidiogenesis, structures such as conidiophores and conidiogenous cells must be carefully evaluated. Some molds produce special sac-like cells called sporangia, the entire protoplasm of which becomes cleaved into spores called sporangiospores. Sporangia are typically formed on special hyphae called sporangiophores.

Dimorphism

A number of medically important fungi express themselves phenotypically as two different morphologic forms, which correlate with the saprophytic and parasitic modes of growth. Such fungi are called dimorphic fungi. Some researchers restrict the term to pathogens that grow as a mold at room temperature in the laboratory and as a budding yeast or as spherules either in tissue or at 37°C. In contrast, others use dimorphic for any fungus that can exist as two different phenotypes, regardless of whether it is pathogenic. We prefer to use the term “dimorphic” to describe fungi that typically grow as a mold in vitro and as either yeast cells or spherules in vivo (Table-M2). Examples of medically important dimorphic fungi include Blastomyces dermatitidis (hyphae and yeast cells) and Coccidioides immitis (hyphae and spherules).

Table

Overview of Major Medically Important Fungi.

A number of external factors contribute to the expression of dimorphism. Increased incubation temperature is the single most important factor. Increased carbon dioxide concentration, which probably affects the oxidation-reduction potential, enhances the conversion of the mycelial form to the tissue form in C immitis and Sporothrix schenckii. pH affects the development of the yeast form in some fungi, and cysteine or other sulfhydryl-containing compounds affects it in others. Some fungi require a combination of these factors to induced dimorphism.

Coccidioides immitis

Coccidioides immitis is a unique dimorphic fungus because it produces spherules containing endospores in tissue, and hyphae at 25°C. Increased temperature, nutrition, and increased carbon dioxide are important for the production of sporulating spherules. A uninucleate arthroconidium begins to swell and undergo mitosis to produce additional nuclei. Once mitosis stops, initiation of spherule septation occurs. The spherule is segmented into peripheral compartments with a persistent central cavity. Uninucleate endospores occurring in packets enclosed by a thin membranous layer differentiate within the compartments. As the endospores enlarge and mature, the wall of the spherule ruptures to release the endospores (Fig. M1 ) . Pairs of closely appressed endospores that have not completely separated from each other may resemble the budding yeast cells of B dermatitidis.

Figure M1

Development of the spherule of Coccidioides immitis from an arthroconidium. (From Cole GT, Kendrick B: Biology of Conidial Fungi. Academic Press, San Diego, 1981, with permission.)