1 What are fungi?

Yeasts, such as brewers yeast, and moulds, such as Penicillium chrysogenum which produces the antibiotic penicillin, are classified as fungi Cells tend to grow as single cells that reproduce asexually in a process known as budding, although a minority of species (e.g. Schizosaccharomyces pombe) reproduce by fission Many yeast species are capable of sexual reproduction and the formation of spores Moulds grow as masses of overlapping and interlinking hyphal filaments and reproduce by producing masses of spores in a variety of structures. Division between yeasts and moulds based on growth morphology is not clear-cut, as some yeasts can produce hyphae under specific conditions (e.g. Candida albicans), while many normally filamentous fungi possess a yeastlike phase at some point in their life cycle Fungi are eukaryotic organisms, i.e. their cells possess a nuclear membrane, consequently there are many similarities between the biochemistry of fungal cells and human cells

Fungi (cont.)
Widely distributed in nature, occurring as part of the normal flora on

the body of warm-blooded animals, as decomposers of organic matter and as animal and plant pathogens Medically, fungi are an extremely important group of microbes, being responsible for a number of potentially fatal diseases in humans (see table 1) But a significant number of fungi are of great benefit to humanity in terms of the production of alcoholic beverages, bread and antibiotics (See Table 2). Fungi have also been utilized for a range of molecular biological applications

Taxonomic classification
Kingdom of fungi subdivided into six Classes: Class Oomycetes contains the mildews and water moulds Class Ascomycetes contains the mildews, some moulds and most yeast

species (including Saccharomyces cerevisiae),

 Class Basidiomycetes contains the mushrooms and bracket fungi Class Teliomycetes contains the rust fungi (plant pathogens) Class Ustomycetes contains the smuts (plant pathogens) Class Deuteromycetes contains species such as Aspergillus, Fusarium

and Penicillium (see Fig 3) 2 The structure of the fungal cell Typical yeast cell is oval in shape and is surrounded by a rigid cell wall, which contains a number of structural polysaccharides May account for up to 25% of the dry weight of the cell wall (See fig 4) Glucan accounts for 5060%, mannan for 1523% and chitin for 19% of the dry weight of the wall, respectively, with protein and lipids also present in smaller amounts. Thickness of the cell wall may vary during the life of the cell but the average thickness in the yeast C. albicans varies from 100 to 300 nm

Septate hyphae of Aspergillus fumigatus

Diagrammatic representation of 'typical' yeast cell.

Vacuole

Vacuolar granules

Mitochondrion

Vacuolar membrane

membrane

Bud scar

Cell membrane

 Glucan, the main structural component of the fungal cell wall, is a

branched polymer of glucose which exists in three forms in the cell, i.e. b-1,6-glucan, b-1,3-glucan and b-1,3,-b-1,6- complexed with chitin Mannan is a polymer of the sugar mannose and is found in the outer layers of the cell wall. The third principal structural component, chitin, is concentrated in bud scars which are areas of the cell from which a bud has detached Proteins and lipids are also present in the cell wall and under some conditions may represent up to 30% of the cell wall contents Mannoproteins form a fibrillar layer that radiates from an internal skeletal layer, which is formed by the polysaccharide component of the cell wall. The innermost layer is rich in glucan and chitin, which provides rigidity to the wall and is important in regulating cell division. Enzymatic or mechanical removal of the cell wall leaves an osmotically fragile protoplast that will burst if not maintained in an osmotically stabilized environment

2 The structure of the fungal cell (cont.)

Incubation of protoplasts in an osmotically stabilized agar growth

medium will allow the re-synthesis of the wall and the resumption of normal cellular functions Ability to generate protoplasts from fungi opens the possibility of fusing these under defined conditions to generate strains with novel biotechnological applications Periplasmic space is a thin region that lies directly below the cell wall It contains secreted proteins that do not penetrate the cell wall and is the location for a number of enzymes required for processing nutrients before entry into the cell. The cell membrane or plasmalemma is located directly below the periplasmic space and is a phospholipid bilayer that contains phospholipids, lipids, protein and sterols. Plasmalemma is approximately 10nm thick and in addition to being composed of phospholipids also contains globular proteins Dominant sterol in fungal cell membranes is ergosterol, which is the target of the antifungal agent amphotericin B.

2 The structure of the fungal cell (cont.)

Sterols are important components of the plasmalemma and represent

regions of rigidity in the fluidity provided by the phospholipid bilayer. Most of the cells genome is concentrated in the nucleus, which is surrounded by a nuclear membrane that contains pores to allow communication with the rest of the cell Nucleus is a discrete organelle and, in addition to being the repository of the DNA, also contains proteins in the form of histones. Yeast chromosomes vary in size from 0.2 to 6 Mb and the number per yeast is also variable, with S. cerevisiae having as many as 16 while the fission yeast Sch. pombe has as few as 3 In addition to the genetic material in the nucleus the yeast cell often has extrachromosomal information in the form of plasmids For example, the 2-mm plasmid is present in S. cerevisiae, although its function is unclear, and killer plasmids in the yeast Kluyveromyces lactis which encode a toxin Actively respiring fungal cells possess a distinct mitochondrion, which has been described as the power-house of the cell

2 The structure of the fungal cell (cont.)

Enzymes of the tricarboxylic acid cycle (Krebs cycle) are located in the

matrix of the mitochondrion, while electron transport and oxidative phosphorylation occur in the mitochondrial inner membrane Outer membrane contains enzymes involved in lipid biosynthesis The mitochondrion is a semi independent organelle as it possesses its own DNA and is capable of producing its own proteins on its own ribosomes, which are referred to as mitoribosomes. Fungal cell contains a vast number of ribosomes, which are usually present in the form of polysomes lines of ribosomes strung together by a strand of mRNA Ribosomes are the site of protein biosynthesis The system that mediates the export of proteins from the cell involves a number of membranous compartments including the Golgi apparatus, the endoplasmic reticulum and the plasmalemma. In addition, the vacuole is employed as a storage space where nutrients, hydrolytic enzymes or metabolic intermediates are retained until required.

Table 1 Examples of fungal diseases and selected causative agents

Type of mycosis Superficial Cutaneous Disease Pityriasis versicolor White piedra Tinea pedis (athletes foot) Onychomycosis (nail infection) Tinea capitis (scalp ringworm) Chromoblastomycosis Mycetoma Species name Malassezia furfur White piedra Trichosporon beigelii Trichophyton rubrum Onychomycosis (nail infection) Trichophyton rubrum Tinea capitis (scalp ringworm) Trichophyton tonsurans Fonsecaea pedrosoi Acremonium spp.

Subcutaneous

Systemic

Blastomycosis, Histoplasmosis Coccidioidomycosis, Paracoccidioidomycosis

Candidosis (superficial/systemic) Aspergillosis Pneumonia

Blastomyces dermatitidis Histoplasma capsulatum Coccidioides immitis Paracoccidioides brasiliensis

Candida albicans Candida glabrata Candida parapsilosis Aspergillus fumigatus Pneumocystis carinii

Opportunistic

Table 2 Examples of economically important fungi

Fungal species
Filamentous fungi Agaricus bisporus Aspergillus, Penicillium spp. Aspergillus sp. + Saccharomyces sp. Fusarium graminearum Penicillium chrysogenum Penicillium notatum Penicillium roqueforti Yeasts Pichia sp. Saccharomyces cerevisiae

Application
Edible mushroom Enzymes (catalase, lipase, amylase) Sake (rice wine) Single cell protein Penicillin production Enzyme (glucose oxidase) Cheese flavouring (Roquefort blue cheese) Gene expression system Bakers yeastbread Brewers yeastbeer, wine, cider, etc. Enzyme (invertase) Gene expression system Dietary supplement

3 Medical significance of fungi

Can cause a range of diseases in humans. In the right set of conditions a normally non-pathogenic fungus can

cause a clinically relevant problem In a case of a profoundly immunocompromised individual, a wide range of fungi can present as capable of inducing disease. Most common fungal pathogens of humans can be divided into three broad classes: The yeasts The moulds The dermatophytes The yeast C. albicans is the most frequently encountered human fungal pathogen, being responsible for a wide range of superficial and systemic infections The superficial infections include oropharyngeal and genital conditions, the former occur predominantly in HIV-positive individuals, geriatric people and premature infants and may arise when a weakened or immature immune system is present.

3 Medical significance of fungi (cont.)

Genital candidiasis is very common and approximately 75% of

women are affected by vulvovaginal candidiasis (VVC) during their life, with a further 512% suffering from recurring bouts of infection over a prolonged period of time. Genital candidiasis in men is comparatively rare, although it is occasionally seen in alcoholics and diabetics Candidiasis of the skin and fingernails (onychomycosis) may be present in up to 10% of cancer patients Continually moist, macerated or burnt skin is an ideal environment for the development of cutaneous candidiasis. C. albicans is also responsible for a range of systemic infections and many of these can commence as superficial infections Infection of the gastrointestinal tract is seen in diabetics, cancer and AIDS patients and the oesophagus is a common site of infection, rendering swallowing difficult Candida infection of the respiratory tract is seen in those with underlying disease (e.g. cancer) or following the insertion of a silicone voice prosthesis.

3 Medical significance of fungi (cont.)

The urinary tract can also be the site of candidiasis, which may be

due to renal infection, other underlying disease(s) or cystitis The presence of an indwelling urinary catheter may also predispose to Candida infection. A range of factors is capable of predisposing the individual to superficial or systemic infections Factors which impair the hosts immune system such as the presence of underlying disease (AIDS, cancer, diabetes), the use of immunosuppressive therapy during organ transplantation and broadspectrum antibiotic therapy can leave the individual susceptible to candidiasis Different stages of life may also predispose to specific types of infection For example, pregnant women have a higher incidence of vaginal candidiasis and premature infants and geriatric individuals are susceptible to oral candidiasis due to either immature or faltering immune systems, respectively.

3 Medical significance of fungi (cont.)

Other factors that may predispose to Candida infection include the

presence of indwelling catheters and skin damage as a result of burns or other trauma. The mould Aspergillus fumigatus is the dominant fungal pulmonary pathogen of humans and generally presents as a problem in those with preexisting lung disease or damage In addition to pulmonary infection other sites may be affected including the brain, kidneys and sinuses, depending on the level of immunocompromisation of the individual. Groups particularly susceptible to colonization by Aspergillus species include those with cavities as a result of tuberculosis, patients affected with asthma or cystic fibrosis and those with profound immunosuppression due to leukaemia (neutropenia). Aspergillosis presents as a serious problem in patients immunosuppressed in advance of organ transplantation. Dermatophyte is the term applied to a range of fungi capable of colonizing the skin, nails or hair. The principal dermatophytic fungi are Trichophyton, Microsporum and Epidermophyton species.

3 Medical significance of fungi (cont.)

The most commonly encountered dermatophytic infections are

athletes foot (infection of the foot) and ringworm (fungal infection of the scalp or skin).

4 Fungal species identification methods The first step in identifying a fungus in clinical samples is to observe the tissue sample microscopically, often in conjunction with specific staining procedures. For instance, addition of potassium hydroxide to the specimen facilitates any fungal elements present to be distinguished from host structures. Tissue samples routinely tested for fungi in the diagnostic laboratory include blood, cerebrospinal fluid, bronchial aspirates and biopsy material, all of which are usually sterile While microscopic analysis of samples is certainly rapid and inexpensive it is not very sensitive Consequently, in the diagnostic laboratory samples are routinely inoculated onto agar plates that specifically allow the growth of fungi.

 A wide variety of media have been developed for the selective

growth of fungal species On these media filamentous fungi are often identifiable to the species level based on their colony morphology following macroscopic and microscopic analysis, with many species having recognizable characteristic hyphal and conidial structures As C. albicans and the recently discovered closely related species C. dubliniensis can grow in the yeast or hyphal form, the ability of these species to produce germ-tubes (i.e. hyphae) can easily be tested in the laboratory by incubation of the cells in serum. Similarly these species are also the only members of the genus Candida to produce large spore-like structures called chlamydospores on specific media Identification of other clinically important Candida species has been greatly facilitated by the recent introduction of commercially available chromogenic media, which allow the Candida species to be distinguished from each other on the basis of colony colour

4 Fungal species identification methods (cont.)

In addition, Candida species can also be identified on the basis of their

different nutrient (especially carbohydrate) requirements, which can be assessed using commercial kits Furthermore, as in the case of direct microscopic analysis of samples, the levels of fungal elements present in samples taken are often very low and are not present at sufficiently high levels to be detected using culture-based methods Therefore, in many cases the identity of the agent responsible for causing a fungal infection is only determined once the infection has been cured using empirical treatment. Indeed sometimes the cause of disease is only identified at autopsy Clearly, to improve our ability to treat fungal diseases effectively there is a great need to develop techniques that allow the detection and identification of infecting fungal species more quickly Currently, two areas of research are endeavouring to facilitate this goal. Firstly, it has been proposed that rather than detecting the infecting species it may be more appropriate to monitor the host immune response to specific pathogens

4 Fungal species identification methods (cont.)

To this end enzyme-linked immunosorben assay (ELISA)-based tests

have been developed to detect antibodies to specific antigens belonging to fungal species In another recent development molecular biologists are attempting to adapt polymerase chain reaction (PCR)-based methods to detect the nucleic acids of specific fungal species in normally sterile sites This method in tandem with genome-based microchip technology offers great promise for automated diagnosis While both ELISA-based and PCR-based technologies have the potential to revolutionize the diagnosis of fungal infections as they are very rapid and accurate, they are still at the experimental stage and are only in use in specialized laboratories. 5 Antifungal therapy Choice and dose of an antifungal agent will depend on the nature of the condition, whether there are any underlying diseases, the health of the patient and whether antifungal resistance has been identified as compromising therapy

 Part of the difficulty in designing effective antifungal agents lies in the

fact that fungi are eukaryotic organisms, so agents that will kill fungi may also have a deleterious effect on human tissue The ideal antifungal drug should target a pathway or process specific to the fungal cell, so reducing the possibility of damaging tissue and inducing unwanted side-effects 5.1 Polyene antifungal agents Polyene antifungal agents are characterized by having a large macrolide ring of carbon atoms closed by the formation of an internal ester or lactone (see fig below) In addition, polyenes have a large number of hydroxyl groups distributed along the macrolide ring on alternate carbon atoms This combination of highly polar and non-polar regions within the molecule render the polyenes amphiphatic, i.e. having hydrophobic and hydrophilic regions in the one molecule which assists solubility in lipid membranes. The principal polyenes are amphotericin B and nystatin.

5.1 Polyene antifungal agents

Amphotericin B is produced by the bacterium Streptomyces nodosus

and its activity is due to the ability to bind ergosterol, which is the dominant sterol in fungal cell membranes, and consequently increases membrane permeability by the formation of pores (Fig. ) The action of amphotericin B seems to rely on the formation of pores through which intracellular contents can escape from the cell Amphotericin B can lead to renal damage during prolonged antifungal therapy. Amphotericin B is active against a broad range of fungal pathogens and is considered the gold standard against which the activity of other antifungal agents is measured. Due to its renal toxicity amphotericin B tends to be reserved for severe cases of systemic fungal disease, but recent formulations in which the drug is encapsulated within liposomes have been shown to be of reduced toxicity. Nystatin was discovered in 1950 and exhibits the same mode of action as amphotericin B but tends to be of lower solubility, which has restricted its use to the treatment of topical infections While Nystatin was effective for the treatment of conditions such as oral and vaginal candidiasis its use has been overtaken by the introduction of azole antifungal drugs

Fig. 4.3 Structures of polyene (amphotericin B) and azole (itraconazole, fluconazole, miconazole and ketoconazole) antifungal agents

Itraconazole
Amphotericin B

Fig. 4.3 Structures of polyene (amphotericin B) and azole (itraconazole, fluconazole, miconazole and ketoconazole) antifungal
agents (CONT)

Fluconazole

Miconazole

Fig. 4.3 Structures of polyene (amphotericin B) and azole (itraconazole, fluconazole, miconazole and ketoconazole) antifungal agents (CONT).

Ketoconazole

Ergosterol biosynthetic pathway. Steps at which various antifungal agents exert their inhibitory activities are shown. TERB, terbinafine; FLU, fluconazole; ITRA, itraconazole; VOR, voriconazole

Mechanisms by which microbial cells might develop resistance. 1, The target enzyme is overproduced, so that the drug does not inhibit the biochemical reaction completely. 2, The drug target is altered so that the drug cannot bind to the target. 3

Biochemical basis of azole resistance

Mechanism Alteration in drug target (14-demethylase) Caused by: Mutations which alter drug binding but not binding of the endogenous substrate Comments Target is active (i.e., can catalyze demethylation) but has a reduced affinity towards azoles

Alteration in sterol biosynthesis

Reduction in the intercellular concentration of target enzyme Overexpression of antifungal drug target

Lesions in the 5(6)desaturase

Results in accumulation of 14-methyl fecosterol instead of ergosterol

Change in membrane Poor penetration across lipid and sterols; the fungal membrane; overexpression of specific active drug efflux drug efflux pumps (CDR1, PDR5, and BENr) Increased copy number of Results in increased the target enzyme ergosterol synthesis; contributes to crossresistance between fluconazole and itraconazole

5.2 Azole antifungal agents

The first generation of azole antifungal agents revolutionized the

treatment of mucosal and invasive fungal infections and azoles are still the most widely used group of antifungal agents. The azole derivatives are classified as imidazoles or triazoles on the basis of whether they have two or three nitrogen atoms in the fivemembered azole ring (Fig) The azoles in current and long-standing clinical use are clotrimazole, miconazole, econazole and ketoconazole, while newer drugs such as itraconazole, fluconazole and voriconazole have important applications in the treatment of systemic infections. Azoles function by interfering with ergosterol biosynthesis by binding to the cytochrome P-450-mediated enzyme known as 14-a-demethylase (P-450 ). This blocks the formation of ergosterol by preventing the methylation of lanosterol (a precursor of ergosterol) (Fig.), resulting in a reduction in the amount of ergosterol in the fungal cell membrane, which leads to membrane instability, growth inhibition and cell death in some cases An additional consequence of the block in ergosterol biosynthesis is the build-up of toxic intermediates, which can prove fatal to the cell
DM

5.2 Azole antifungal agents (CONT.)

Azoles exhibit a broad spectrum of activity in vitro, being capable of

inhibiting the growth of most Candida, Cryptococcus and Aspergillus species and dermatophytes Miconazole was the first azole used to treat systemic fungal infections but demonstrated a number of toxic side-effects Ketoconazole produced high serum concentrations upon oral administration but had poor activity against aspergillosis In addition, ketoconazole was associated with a range of side-effects which limited its applicability. Newer triazoles such as fluconazole and itraconazole have increased the options for dealing with fungal infections Fluconazole is water-soluble and shows good penetration and deposition into the pulmonary tissues; it also reaches high levels in the cerebrospinal and peritoneal fluids Fluconazole has proved highly effective in the treatment of infections caused by C. albicans but shows limited activity against Aspergillus. Itraconazole has proven efficacy against Aspergillus.

5.2 Azole antifungal agents (CONT.)

Voriconazole is one of the newest second generation triazole

antifungal drugs and it shows good activity against pulmonary aspergillosis and cerebral aspergillosis 5.3 Synthetic antifungal agents Flucytosine is a synthetic fluorinated pyrimidine that has been used as an oral antifungal agent and demonstrates good activity against a range of yeast species and moderate levels of activity against Aspergillus species Two modes of action have been proposed for flucytosine One involves the disruption of protein synthesis by the inhibition of DNA synthesis, while the other possible mode of action is the depletion in the amino acid pools within the cell as a result of inhibition of protein synthesis. In general yeast cells increase in size when exposed to sub-MIC levels of flucytosine and display alterations in their surface morphology, both of which can be interpreted as a result of an imbalance in the control of cellular growth

5.2 Azole antifungal agents (CONT.)

Itraconazole is effective in treating severe Aspergillus infections and

exhibits both fungicidal and fungistatic effects Upon ingestion itraconazole undergoes extensive hepatic metabolism which yields up to 30 metabolites a number of which retain antifungal activity. Itraconazole is currently available as an intravenous formulation and is widely used for the treatment of severe Aspergillus infection in this form. Fluconazole and itraconazole demonstrate significantly reduced side-effects compared with ketoconazole Novel azole drugs with increased ability to inhibit the fungal 14ademethylase are also becoming available These agents, which include voriconazole, posaconazole and ravuconazole, have a wider spectrum of activity than fluconazole and it has been suggested that some of them show fungicidal effects with some species (e.g. Aspergillus spp.).

5.3 Synthetic antifungal agents

Many fungi are inherently resistant to flucytosine or develop

resistance after a relatively short exposure and resistance has been attributed to alteration in the enzyme (cytosine deaminase) required to process flucytosine once inside the cell or to an elevation in the amount of pyrimidine synthesis. The problem of resistance has limited the use of flucytosine so that now it is generally used in combination with an antifungal agent (e.g. amphotericin B) where it can potentiate the effect of the second agent. 6 Mechanisms of antifungal drug resistance Resistance to antifungal drugs is becoming an increasingly perplexing problem This is particularly the case with one of the most widely used antifungal agents, fluconazole. Fluconazole is a fungistatic agent that acts by inhibiting the activity of cytochrome P-450 (lanosterol -demethylase), an enzyme in the pathway responsible for the synthesis of ergosterol

6 Mechanisms of antifungal drug resistance (cont.)

Inhibition of this enzyme results in decreased ergosterol levels and a

build-up of toxic intermediary products which subsequently leads to aberrant membrane function and stasis of cell growth and division Since the widespread introduction of fluconazole during the early 1990s, there have been increasing reports of Candida strains that have decreased susceptibility to azole drugs and of infections that are recalcitrant to treatment In addition, the prevalence of infections caused by Candida species that are inherently less susceptible to azoles has increased over the same time period One of the most important mechanisms of resistance is the overexpression of efflux proteins which act by pumping the drug out of the cell at a rate faster than the drug enters the cell (see fig) Therefore the intracellular concentration of the drug does not reach a level high enough to inhibit the target enzyme. Another resistance mechanism that has been identified is mutation within the gene encoding the cytochrome P-450

6 Mechanisms of antifungal drug resistance (cont.)

These amino acid substitutions do not affect the activity of the

enzyme, but the conformation of the enzyme is sufficiently altered to decrease the affinity of the enzyme for the drug, thus the levels of membrane ergosterol are not altered significantly.
An alternative resistance mechanism is the overexpression of the

gene encoding the target cytochrome enzyme, this results in increased levels of the cytochrome inside the cell, thus countering the effects of the drug Another resistance mechanism is mutation in genes that encode other proteins involved in the biosynthesis of ergosterol These mutations prevent the build-up of toxic byproducts, thus the inhibitory effects of the azoles are decreased Very often in azole-resistant clinical isolates of C. albicans multiple resistance mechanisms have been identified as contributing to the resistance phenotype, thus it is difficult to determine which are the most important mechanisms (see fig.)

6 Mechanisms of antifungal drug resistance (cont.)

Resistance to other antifungal agents such as amphotericin B has

been observed less frequently in clinical fungal isolates; however, the molecular basis of this resistance is not currently well understood To solve the problems associated with antifungal resistance a number of novel azole drugs (e.g. voriconazole) and novel classes of drug (e.g. echinocandins) have been developed