Introduction

The fungi are in a Kingdom of their own and are typically eukaryotic – the nucleus and organelles are enclosed in a membrane. They are haploid (have only one chromosome copy), have a cell membrane rich in ergosterols and an outer membrane rich in chitin and 1,3 and 1,6 glucans. Fungi are heterotropic (cannot manufacture their own food) and most are saprophytic and are important decomposers of plants and animals. Thus, most are very useful and an important part of the ecosystem.

Only a very few are involved in disease, where they cause:

1. Mycosis – infection of animals tissue
2. Mycotoxicosis – ingestion of fungal toxins
3. Fungal hypersensitivities (not dealt with)

What are fungi?

1. Eukaryotic – nucleus and organelles bound in a membrane. This makes them very different to bacteria that are prokaryotes.
2. Haploid – vegetative forms carry only one set of chromosomes, other eukaryotic cells i.e. those belonging to protozoa are diploid.
3. They have rigid cell walls that are rich in glucans, mammoproteins  and chitins. They are therefore non-motile. (One exception = chytridiomycota – motile zoospores)
4. Morphologically diverse – some fungi exist as yeasts, which are unicellular and reproduce asexually by budding and some fungi occur as branching filaments (common word = mould). Some fungi known as the dimorphic fungi can produce both forms. Some of the most pathogenic fungi such as Histoplasma capsulatum, are thermal dimorphic where they are a mould at room temperature and a yeast at body temperature.  The mould phase where fine conidia are produced allows them to be easily inhaled, whereas the conversion to the yeast phase in the body allows them to overcome the host’s immune response.

5.  Heterotropic  – cannot manufacture their own food. This makes them very different to plants and a few bacteria that are autotropic. This also means that they have to be able to break down complex nutrients. For this they use extracellular enzymes. In fact some fungi are able to digest cellulose and lignin in plants which enables them to be important decomposers or saprotroph. However, sometimes these enzymes are toxic to animals resulting a mycotoxicosis. Fungi that obtain their nutrition from living cells are known as a biotroph i.e. fungi causing mycosis.

6.   They reproduce both asexually and sexually. Generally all fungi are able to undertake both lifecycles under different conditions. Asexual reproduction or the anamorphic stage is more commonly seen in clinical mycology. In this stage, yeasts reproduce by budding and filamentous fungi by the use of fruiting bodies.  Sexual reproduction (telomorphic stage) is used to classify the fungi into the different phyla. Sexual reproduction generally occurs by the fusion of two nuclei followed by meiosis. This is different to animal cells where meiosis precedes nuclear fusion.

7.   Generally grow aerobically – some pathogenic fungi will grow under anaerobic conditions. However, fruiting bodies are only produced in aerobic conditions.

8.    Environmental resistant forms – Most, if not all fungi will produce environmentally resistant spores, conidia and chlamydospores. This is because the filamentous fungi, in particular, are susceptible to drying out. The thick-walled vegetative chlamydospores will stain acid-fast and can be confused with the anamorphic fruiting bodies known as conidia.

Classification of fungi

Fungi are currently classified according to the way they sexually reproduce. This has lead in recent years to a number of changes to the genus names of fungi as many of the pathogenic fungi were originally identified by their anamorphic structures.  In fact, it is rare to observe in animal samples and fungal cultures of animal fungal pathogens the teleomorphic structures. Thus you will find that a lot of them have 2 names, the original anamorph and the newer teleomorph. The anamorph names have been retained for familiarity reasons, rather than taxonomic ones.

Below is the basic classification of the fungi, that only includes those fungal genera that are the more common animal pathogens.

You will be dealing with individual pathogenic fungi in the following workbook and will encounter these phyla when reading about the reproductive structures

Fungi and disease

mycosis (pl . mycoses)

Mycosis (plural: mycoses) is an invasive fungal infection of animals that enter the animal through skin injuries, via the inhalation of fungal spores (conidia) and occasionally via the intestinal tract.  Mycoses are less common when compared with viral, bacterial and parasitic infections. Mycoses, especially deep-seated infections tends to require prolonged antifungal therapies that are not only expensive, can cause serious adverse side-effects.

Classification of mycoses

Invasive fungal infections are classified according to where in or on the body lesions occur:

1. Superficial = on the surface of the skin
2. Cutaneous = in the skin
3. Subcutaneous = in the subcutis, including the cutaneous lymphatics
4. Systemic/Deep = In the body, lesions can be localised or disseminated (in multiple sites). Generally the prognosis for the treatment of fungal infections within the body (systemic/deepand especially it there are multiple granulomas in different sites is guarded

mycotoxicosis

Mycotoxicosis is the consequence of ingestion of feed or water containing toxic metabolites produced by certain saprophytic fungi. These toxins are classified according the the organ system they affect i.e. the liver = hepatotoxin and the brain = neurotoxin.

Note that sick building syndrome as a consequence of air-borne fungal exposure is usually due to a hypersensitivity (allergic) response to the fungi. The linked URL will provide you with more information on building-related illnesses. I will not test you on this knowledge.

morphology and functionS of fungi associated with disease

rigid outer cell membrane

The fungal cell wall/membrane is rigid like plants and bacteria. Thus it confers shape to the fungus and assists in the morphological identification of the fungi. It is also able to protect the fungus from external destruction and support the higher internal turgor pressure of the fungus. Note that the wall of is less protective from drying out than spores and conidia produced by fungi. 20% of the fungus genome is dedicated to cell wall manufacture.

Mannoproteins: These proteins link to either B-1,6 glucan or B-1,3-glucan and assist in the adherence of fungi to surfaces. They also are involved in cell wall porosity. They are antigenic and are recognized by TLR 2 and TLR 4 pattern receptors on macrophages, neutrophils and dendritic cells as foreign.

Beta-1,6-glucan is found in the cell wall of both the fungi and oomycetes (see below).

Beta 1,3-glucan comprises 60% of fungal wall. Found in fungi, oomycetes and plants. It is detected by Dectin 1 on macrophages which will then result in inflammation. Inflammation of the endothelium of blood vessels leads to thrombi formation in the arterioles with a drop in blood supply to the tissues, leading to ischaemic necrosis. (Refer to General pathology Notes). Some pathogenic fungi like Candida albicans are able to hide their Beta 1,3 glucan from the immune system. Formation is inhibited by the antifungal echinocandins – only administered IV.

Chitin imparts rigidity to the cell wall and is found in fungi and the exoskeleton of insects. At one time antichitin (anti-flea) drugs were thought to work against fungi. However, the fungus does not transport these drugs internally and thus they are ineffective.  Can convert to chitosan – more flexible

Melanins (brown pigment) are dispersed in the outer cell wall layer by some fungi such as Cryptococcus neoformans and the dark pigmented fungi. Those fungi will only add pigment to their fruiting bodies i.e. Aspergillus fumigatus has melanin pigmented conidia.  Melanins, which are potent anti-oxidants, assist in 1) Environmental survival – Resists high temperatures and ionising radiation (i.e. UV); 2) Protection against drugs – binds to heavy metals and some antifungal drugs; and 3) Resistance to killing after phagocytosis.

The cell membrane is unique in that the cholesterol found in animal cells is replaced by ergosterol. Ergosterol is a Vitamin D2 precursor and stabilises the cell membrane. Commonly used antifungals i.e. azoles and terbinafine prevent ergosterol synthesis and the polyenes bind to ergosterol causing the cell membrane to leak.

Protozoal fungal impersonators

The oomycetes also known as water moulds are protista that have a permanent life cycle in water. Both Pythium insidiosum and Lagenidium species can infect though the water macerated or otherwise damaged skin of animals causing “kunkers”. This is a rapidly developing subcutaneous granulomatous disease common in the Tropics.

Oomycetes have chitin like the exoskeleton of insects, beta 1,3 and beta 1,6 glucan (75%) , like fungi and cellulose (20%) like plants. The outer membrane is rich in the unique protein, hydroxyproline. The chromosomes of the vegetative hyphae are diploid rather than haploid

The taxonomy of the animal pathogenic oomycetes are shown in the Flow Chart below.

A mould compared with a yeast

A yeast is defined as a microscopic unicellular fungus, 4 – 6 um in diameter. Yeasts divide by budding and will grow on artificial media as creamy bacterial-like colonies. The two best known pathogenic yeasts belong to the genera Candida (an ascomycete) and Cryptococcus (a basidiomycete). Many of the pathogenic yeasts are dimorphic i.e. they will shift between a mould and a yeast. Pathogenic Candida species often switch to a mould form in host tissue. This is done to mask the beta-1,3-glucans in the cell wall and thus reduce the inflammatory response. In the laboratory changing the pH of their environment from acidic to alkaline will stimulate the conversion of a yeast to a mould form. They will also produce hyphae when coming into contact with a surface. The thermally dimorphic yeasts such as Sporothrix schenckii and Histoplasma capsulatum will convert from a mould, which they were in the cooler external environment, to a yeast in tissue which is at a higher temperature. This allows the revealed beta-1,6-glucan at their scars to stimulate macrophages the ingest them. They will then grow and bud in the cytoplasm of the macrophages. Thus they maintain an intracellular lifestyle. Yeasts will also produce pseudohyphae. These are a chain of yeast cells that have constrictions but no cross walls that can easily be disrupted.

A mould is the multicellular form of a fungus, where branching filaments or hyphae will develop in the nutrient substrate and aerial fruiting bodies will develop either asexually or sexually. The variety of structures on moulds allows them to be identified by morphological examination.

The importance of hyphae

Hyphae (hyphus singular) are multinucleate branching structures found in multicellular fungi and oomycetes. Filamentous bacteria also have hyphae, however, the bacterial hyphus is very different structurally as they are 2-4um wide (thinner) and there are no nuclei or organelles. Hyphae are vegetative structures that are used to anchor the fungus to their substrate and also to provide nutrition to the entire fungus. A mass of fungi is known as a mycelium.

The tip of a hyphus is usually anchored in the substrate and will produce and excrete powerful enzymes that breakdown the substrates into simple nutrients that can be absorbed and transported throughout the fungus. The tip is the only part of the hyphus that grows by extension, a process known as apical growth. In higher fungi, this growth is mediated by an organelle at the tip of the hyphus known as a Spitzenkorper. These enzymes in some fungi can also when ingested cause serious disease known as a mycotoxicosis.

Hyphae of oomycetes and the primitive fungi as as the Mucoramycota (bread moulds) and Chytrid fungi,  tend grow as continuous wide branching structures with very few septa whereas the higher fungi belonging to the basidiomyceota and ascomycota have a number of septa in their cell wall. Thus damage to a hyphus of the more primitive fungi can be more destructive to the fungus overall as they tend to “bleed” from the cut surface. This fact is important when the mycologist is trying to grow these fungi from a sample. Even though the higher fungi have a large number of septa that divide the hyphus, they have pores in the septa that allow fluids and nutrients to be transported from the hyphal tips to the rest of the fungus.

fungal reproduction

All fungi when they come into contact with air or the environment will reproduce asexually (anamorphically) and sexually (teleomorphically) to produce environmentally resistant structures. These fruiting structures are very distinctive allowing us to morphologically identify the fungus to genus and even species level. It is unusual to observe teleomorphic structures in diagnostic fungal cultures. Most of the time they are anamorphic. This is for two reasons, the primary being that only one mating type is present in animal tissue, the other being that the artificial growth conditions in the laboratory  favour asexual reproduction.

Teleomorphic Reproduction

Vegetative fungi are usually haploid (n). Sexual reproduction is initiated when the hyphae of two different mating types fuse (somatogamy). This puts the different nuclei in contact with each other. At this stage nuclear mitosis can occur without any nuclear fusion (karyogamy)  happening or the nuclear fusion occurs immediately. The spores that form are now diploid (2n). They may undergo meiosis (n) or precede meiosis with a few rounds of mitosis (2n).

The teleomorphic life cycle of the mucormycota

Two different hyphae align, develop protuberances, wall off their nuclei into a coenogamete (n), fuse into a zygote (2n), undergo meiosis (n) and develop into a resistant, resting zygospore.

The teleomorphic and only life cycle of a mushroom, a basidiomycete

The most well known fungi in this phylum are the mushrooms. They are not known to cause mycoses, however, some mushrooms can produce potent neurotoxins that cause a mycotoxicosis in animals that ingest them.

Mushrooms only have a teleomorphic life cycle. The basidium forms part of the outer layer of the gills. When compatible nuclei fuse (2n) – 4 daughter cells (basidiospores = n) are produced on a stalk by meiosis . Meiosis allows fungi to repair DNA damaged by oxidation.

The teleomorphic life cycle of the ascomycota

Ascomycota is the biggest phylum and contains most of the invasive and toxic fungi. The ubiquitous genus Aspergillus is in this phylum.

Cells of different mating types fuse (somatogamy) then divide by mitosis and form sacs known as an asci. The nuclei fuse (2n) = zygote and then meiosis to produce 4 spores (n) and then mitosis occurs in each sac to produce 8 ascospores which are released from the sac.

Anamorphic/asexual reproduction

Most vegetative fungi are haploid. Asexual reproduction usually occurs via nuclear mitosis. Filamentous fungi or moulds are multinucleate thus fungal mitosis occurs within the nucleus using a spindle apparatus. It occurs independent of hyphal branching. E.g. nuclei have to be positioned by cytoplasmic tubules to ensure that budding can take place in yeasts. It is the most common form of reproduction observed in pathogenic fungi.

You will be introduced to a number of pathogenic fungi that have a variety of anamorphic fruiting structures all aimed at producing a resistant dormant structure, that can be dispersed by the wind or animals to a new location to germinate and continue growing. Reproduction occurs in a few ways:

1. By budding in yeasts
2. In a sac known as a sporangium = spore
3. External on a conidiophore = conidium
4. By segmentation of a specialised hyphus = arthroconidia (aleuroconidia)

1. The yeasts

Yeasts are single celled fungi that belong to either the basidiomycota or ascomycota. They reproduce asexually by budding.

the moulds

You will be introduced to a number of pathogenic fungi that have a variety of anamorphic fruiting structures all aimed at producing a resistant dormant structure, that can be dispersed by the wind or animals to a new location to germinate and continue growing.

2. Spore production

Spores are always produced within a sac. The sac will often rupture explosively releasing the spores far and wide.

3. Conidia production

Conidia are asexual reproductive structures that are produced on specialised adaption of hyphae known as conidiophores. They will detach from the conidiophore.

4. Hyphae segmentation

Hyphae will segment themselves into arthroconidia

Identification of the pathogenic fungi INVOLVED IN MYCOSES

The identification of fungal infections is technically more difficult than bacterial infections. This is for the following reasons:

1. Less common, so diagnosticians have less experience in their diagnosis
2. Lesions caused by fungi mimic other diseases including neoplasia
3. Some fungi stain poorly and take on unusual morphologies when encountering the immune system of the host.
4. May require specialised media and a prolonged incubation period to detect them.

The pathway to the diagnosis of fungal infections is the following:

1. Suspect infections: Lesions typical for superficial infections; Granulomas present in sub-cutaneous infections; Space-occupying lesions evident in diagnostic imaging
2. Cytology: Fungi – moulds or yeasts observed in cytology of fine needle aspirates, fluids (urine, respiratory secretions, nasal flushes, joint fluid), hair examination and impression smears of lesions and histology of biopsies. Special stains such as Periodic acid-Schiff (PAS) or Gomorii silver stains may have to be sued to visualise the fungi. Some fungi like Cryptococcus and Malassezia can be identified cytologically, others not.
3. Fungal culture. Fungi will grow on most media. Antibacterials can be added to the media to reduce the growth of contaminate bacteria. Fungi are encouraged to produce anamorphic fruiting bodies in cultures as morphological identification is still important in the identification of most fungi. Since non-pathogenic fungi can be isolated as sample contaminants. repeat culturing of the same fungus from the same disease disorder may be required.
4. Phenotypic tests. Done for the yeasts when there are very few tests to distinguish them.
5. DNA sequencing or microarray systems – Can be used to identify the fungus to species level. More reliable as not affected by polymorphism of the fungi.
6. MALDITOF: Mass spectrophotometry pattern with consultation of a database to find a reference specimen with the same pattern.
7. Serological and antigen detection tests: Serious fungal diseases such as Aspergillosis and Cryptococcosis have tests that either detect antibodies to these fungi or detect an antigenic component in the blood of the target fungi.

END OF CHAPTER