What are fungi? Fungi belong to their own special ‘kingdom’ as they differ from both plants and animals. Fungi are eukaryotic cells. The fungal kingdom is largely hidden from our view underground and we usually only see the "fruit" of a fungus. Fungi can exist as single cells or chains of cells together. The living body of a fungus is called a mycelium and is made up of a branching network of filaments known as hyphae. Fungal mycelia are usually hidden in a food source like wood and we only know they are there when they develop mushrooms or other fruiting bodies. Some fungi only produce microscopic fruiting bodies and we never notice them. Fungal hyphae growing - contain chains of cells. Lecture notes: Objective: Introduction of fungi kingdom as distinct from plant and animal kingdoms. Fungi have similar physical characteristics to plants and are sometimes mistakenly put in the Plant Kingdom. Plants have chlorophyll that allows them to make their own food and makes them green. Fungi have no chlorophyll. Fungi break down dead organic matter or grow on other organisms to get nutrients for growth. Introduce concept of hyphae – branching chains of cells and mycelium. The genetic makeup of fungi is more similar to animal cells,the major distinction being a rigid cell wall.

Interesting fungi: One of the largest living organisms in the world is a specimen of the fungus Armillaria ostoyae which covers 1,500 acres in Washington State, USA and seems to be 400-1,000 years old. The only evidence of the fungus are clumps of golden mushrooms that pop up in autumn when it rains. Largely this fungus is seen underground and forms huge rhizomorphs (root like structure). Objective : Fungi may be hidden from view. Where found? Mycelium - groups of hyphae are the living body of fungus. This is usually underground hence not seen, until asexual growth of fruits (ie mushrooms) occurs.

How do fungi live? Fungi feed by absorbing nutrients from the organic material that they live in. They digest their food before they absorb it by secreting acids and hydrolytic enzymes. Different fungi have evolved to live on various types of organic matter, some live on plants eg.Phytopthora infestans - the potato blight fungus, as seen here; Some live on animals eg.the athlete's foot fungus and some live on insects eg.Cordyceps australis. Lecture notes: Objective: Fungi can live off dead organic material or with other living things. Great potato famine in 1845 due to potato blight fungus was responsible for death of 1 million in Ireland and emigration to the USA of many Irish. Cordyceps australis is a fungus which parasitises insects and their larvae, it can be seen growing in plant like form from the body of an infected host.

Helpful fungi: Most of us use fungi every day without knowing it. We eat mushrooms and Quorn (a vegetarian fungal protein), but we also prepare many other foods using fungi. The yeast Saccharomyces cerevisiae is used to ferment sugar to alcohol and carbon dioxide – the process used to make beer and wine and also to make bread rise. The fungi Aspergillus oryzae and Aspergillus sojae are used in the production of the oriental foods soy sauce and miso. We also use fungi to produce flavourings, vitamins and enzymes and to mature many cheeses. Fungi play an essential role in both the Nitrogen and Carbon cycle by breaking down dead organic material. Lecture notes: Objective: Fungi are used in many different types of food production. Quorn (a vegetarian protein) is made by fermenting the fungus Fusarium venenatum in vats producing the mycoprotein commonly eaten by vegetarians. An example of a major ‘commodity’ chemical manufactured by fungal (Aspergillus niger) fermentation is citric acid. Its production was introduced in 1923 in the US, decimating Italian citric acid production (lemons). It is a major commodity - approximately 1 million tons made per annum. It is used as a taste enhancer, a chelating agent and a natural acidulant. Why do we need Nitrogen? It is a necessary component of all proteins and nucleic acids. Animals can not break the covalent N2 bond to release free N.

Helpful fungi: We get some important drugs from fungi such as the antibiotic penicillin and cyclosporin A - a drug that stops organ rejection after transplantation. Research scientists use several fungi to investigate basic functions that occur in all cells because they are simple and easy to grow; some cancer research is done using fungi. Fungi are responsible for breaking down dead organic matter which allows nutrients to be cycled through the ecosystem. Lecture notes: Apart from penicillin, the most important antibiotics from fungi are the cephalosporins (beta-lactams with similar mode of action to penicillin, but with less allergenicity) and griseofulvin (from Penicillium griseofulvum and related species) which is used to treat athlete's foot and related fungal infections of the skin. Lovastatin a cholesterol lowering drug is produced by Aspergillus terreus.

Ringworm infection on leg Importance of fungi: Without fungi we would not have bread, beer, wine or antibiotics, but more importantly without the nutrient recycling and plant nutrition provided by fungi - we probably could not survive at all. In humans, fungi cause skin infections such as ringworm and athlete's foot, but they also cause several deadly diseases which can be hard to treat. Fungi that can cause life-threatening infections in people include Aspergillus fumigatus, Candida albicans and Cryptococcus neoformans – they are called pathogens. Ringworm infection on leg Lecture notes: Objective: Some fungi are essential for nutrient recycling and providing plant nutrition. Not all fungi are this useful – some can cause deadly diseases and are therefore pathogens. For example: Aspergillus fumigatus can cause pneumonia in patients with leukaemia and AIDS; Candida can cause bloodstream infections in intensive care patients; Cryptococcus neoformans can cause meningitis in AIDS patients. All these infections are increasing in frequency because treatment of AIDS and cancer are more effective and hence patients at risk survive longer.

Importance of fungi? Most patients with deadly fungal diseases do not have a fully functional immune system. They may have leukaemia or AIDS or they may be taking drugs to suppress their immune system because of organ transplantation. Although there are drugs to treat fungal infections these drugs can have some nasty side-effects because they are often toxic to people as well as to fungi. There is a desperate need for new and better anti-fungal agents. Aspergillus fumigatus spore forming head (Electron micrograph) Lecture notes: Objective: People with fully functional immune systems are very unlikely to suffer severe fungal disease. White blood cells are important for attacking fungal infections. Antibodies however are not useful in fighting most fungal infections. Four drugs may be effective against different types of fungal infection - Amphotericin B, Itraconazole, Fluconazole, and Caspofungin. Amphotericin B (broad spectrum) is given intravenously but can cause renal failure; fluconazole, given intravenously and orally is effective against candidiasis and Cryptococcus but not aspergillosis; itraconazole (broad spectrum) can be given intravenously or orally, is not very effective against Aspergillus; caspofungin given intravenously is effective against Aspergillus and Candida but not Cryptococcus.

What is Aspergillus? It is a genus of around 200 fungi (moulds) found worldwide. Fungi are identified in the lab by their structure and appearance. They may appear as round single cells like yeast, or made of chains of cells called hyphae. Aspergillus is a filamentous fungus as opposed to yeast which is single celled. Fungi reproduce by forming tiny spores which can easily be airborne. Conidial head or fruiting body of Aspergillus - producing spores Yeast Aspergillus hyphae Objective: Aspergillus is a commonly occurring genus. NB. The term mould is used to describe any filamentous fungus. Aspergillus forms hyphae since it is a filamentous fungi and can be dispersed by airborne spores. Yeast on the other hand are single cells which reproduce by budding. Candida, another type of fungus can be filamentous or single cellular it is therefore termed dimorphic. Useful link: http://www.herb.lsa.umich.edu/kidpage/factindx.htm

When was aspergillus first identified? In 1729 Aspergillus was first catalogued by an Italian biologist - P Micheli. The first known case of infection was in a jackdaw in 1815 and in a human in 1842. In the 19th century it was an occupational hazard amongst wig combers when it caused allergic disease of the lungs. Objective: In the 19th century professional wig combers were among the first diagnosed cases of allergic aspergillosis. Repeated exposure to Aspergillus spores can lead to hypersensitivity and an allergic form of the disease aspergillosis - ABPA (allergic bronchopulmonary aspergillosis). The disease has been reported to occur mainly as a complication in patients with asthma and cystic fibrosis, in whom the incidence may be as high as 10%. Useful link: www.medscape.com/viewarticle/408747_9 (you will need to register - free of charge - with medscape to view this).

Why is Aspergillus important Why is Aspergillus important? The following slides focus on Aspergillus because this fungus illustrates a spectrum of positive and negative aspects of fungi with respect to the environment and disease. Some Aspergillus species cause serious disease in humans and animals – it is pathogenic. Some Aspergillus species produce enzymes which have important industrial applications. Aspergillus can produce mycotoxins – these are often found in contaminated foodstuff and are hazardous to the consumer. Objective: Some species of Aspergillus can cause serious disease in a susceptible individual. It can also produce toxins which can contaminate foods and are harmful to both animals and man. Useful links: http://www.lfra.co.uk/eman/fsheet1.htm#top and http://www.mold-help.org/fungi.mycotoxins.currentresearch.htm In contrast Aspergillus is useful in industry, for instance, the enzymes in biological washing powders and enzymes for maturing cheeses are derived from Aspergillus species.

Where is Aspergillus found? Its natural habitat is in hay and compost. Aspergillus spores are easily airborne and we normally breathe in 100-200 spores daily. Some species withstand heat eg; Aspergillus fumigatus (pathogenic type) these are commonly found in compost. Objective: Aspergillus fumigatus is thermotolerant and grows at temperatures ranging from 15°C to 53°C. Although it is ubiquitous in nature, present both indoors and outdoors, it is commonly found in large numbers around construction sites where digging occurs. Aspergillus fumigatus is also common in compost piles, potting soil, mulches, sewage facilities, and bird excreta. The spores of A. fumigatus are produced on specialized conidiophores ranging from 1.0 to 3.0 micrometer in diameter and deposited in the lower respiratory tract. The hyphae of A. fumigatus are 3 to 5 micrometer in diameter, septate, and often branch typically at a 45° angle.

The life cycle of Aspergillus Germination Spores inhaled Hyphal elongation and branching Mass of hyphae (plateau phase) Short movie clips showing germination of spores and growth of hyphae can be viewed on this website at the following link: http://www.aspergillus.man.ac.uk/secure/educationsection/movies/af65hyphae.html

Air; spores may be inhaled Water / storage tanks in hospitals etc Food Sources of Infection? Aspergillus species are found in : Soil Air; spores may be inhaled Water / storage tanks in hospitals etc Food Compost and decaying vegetation Fire proofing materials Bedding, pillows Ventilation and air conditioning systems Computer fans Aspergillus spores Objective: Aspergillus spores are widespread and are readily inhaled.

Which species of Aspergillus are pathogens? The most common causing invasive disease are Aspergillus fumigatus and Aspergillus flavus. The most common causing allergic disease are Aspergillus fumigatus and Aspergillus clavatus. EM of Aspergillus clavatus Objective: Name pathogenic species. Aspergillus fumigatus causes all manifestations of aspergillosis which is why it is one of the most important from a disease point of view. Aspergillus flavus causes invasive disease (particularly sinusitis and plant disease) and produces aflatoxin. Aspergillus clavatus is common in hay barns and causes allergy - probably because it does not grow well at 37C, it does not cause invasive disease.

Aspergillus as a pathogen in man- Aspergillosis is a group of diseases caused by Aspergillus. The symptoms – fever, a cough, chest pain or breathlessness occur in many other illnesses so diagnosis can be hard. Usually only patients with already weakened immune systems or who suffer other lung conditions are susceptible. In man the major forms of disease are: Allergic aspergillosis (affects asthma, cystic fibrosis and sinusitis patients). Acute invasive aspergillosis (risk increases if patient has weakened immunity such as some cancer patients and those on chemotherapy). Disseminated invasive aspergillosis (widespread through body). Aspergillus keratitis (eye) Objective : Aspergillus species can cause a variety of illnesses in man. Useful link: http://www.doctorfungus.org/mycoses/human/aspergillus/aspergillosis.htm

An example of invasive aspergillosis Aspergillus bone and soft tissue infection in a patient with the inherited condition chronic granulomatous disease (CGD). Aspergillus as a pathogen in man- Objective: An example of invasive aspergillosis affecting bone and soft tissue in an individual with an inherited disease which results in a defective immunity, characterised by grossly reduced neutrophil and macrophage killing. Arrows indicate abnormal thickening of first metatarsal.

Relative risk of Aspergillus infection Aspergillus as a pathogen in man- Patients whose immune system is already weakened are most susceptible. Those most at risk include some cancer and leukaemia patients, those on chemotherapy and transplant patients. Acute invasive aspergillosis Allergic aspergillosis Allergic sinusitis Frequency of aspergillosis Frequency of aspergillosis Objective: The risk of developing invasive aspergillosis increases with increasing levels of immune deficiency. In contrast the risk of allergic aspergillosis is enhanced in individuals whose immune system is hyper-reactive. The two chest X-rays show examples of acute invasive and allergic pulmonary aspergillosis. Aspergilloma - This is a very different disease also caused by the Aspergillus mould where the fungus exploits an existing weakness. The fungus grows within a cavity of the lung, which was previously damaged during an illness such as tuberculosis or sarcoidosis. Any lung disease which causes cavities can leave a person open to developing an aspergilloma. The spores penetrate the cavity and germinate, forming a fungal ball within the cavity. The fungus secretes toxic and allergic products which may make the person feel ill. The picture shows the fungal ball (aspergilloma) which was removed from a lung and measures about 6cm diameter. Aspergilloma Normal immune function Immune malfunction Immune hyper-reactivity

Aspergillus as a pathogen in animals All domestic mammals, birds and numerous wild species can get aspergillosis. Birds such as penguins and falcons when stressed by malnutrition or capture are particularly susceptible to aspergillosis. Spores of Aspergillus fumigatus cause lung infections – leading to death. Aspergillus fumigatus spores are often present on the surface of eggs after laying. The spores may penetrate the shell pores and contaminate newly hatched chicks. Objective: Animals are also susceptible to aspergillosis. (Chinstrap penguin shown). Birds seem particularly prone especially in a weakened condition, this may be due to malnutrition, breeding, virus infection, overcrowding or capture.

Aspergillus species secrete a number of enzymes with important industrial uses – Genetically modified A. oryzae is used for the large scale production of lipases used in biological washing powders. A.niger is used in the commercial production of citric acid, which is widely used in the food industry. Fermentation of genetically modified A.oryzae is the major source of recombinant chymogen which is used to curdle milk to make hard cheeses. Objective : useful functions of Aspergillus species. Useful link: http://fungus.org.uk/nwfg/fungbiot.htm for fungal biotechnology and enzymes of commercial importance. The use of fungal enzymes in cheese production replaces enzymes scraped from the stomach lining of animals - making these cheeses available to strict vegetarians.

Aspergillus Mycotoxins Mycotoxins are chemical products of fungi that have the capacity to damage animal health and contaminate crops. Repeated aflatoxin ingestion in man has been linked to liver cancer. Mycotoxins (aflatoxins) produced by Aspergillus parasiticus and A. flavus are commonly found to contaminate corn, peanuts, and other crops used for animal feedstuff. High temperature and humidity increase chances of contamination. Turkey ‘X’ syndrome - in 1960, 100,000 turkeys died in the South of England, from liver damage after consumption of peanuts contaminated with A. flavus. Objective: What is the significance of crop contamination and aflatoxin ingestion? Aflatoxin is produced by A. flavus and A. parasiticus and many others. How many other mycotoxins are there? Examples: Ochratoxin A - secreted by Aspergillus ochraceus and Penicillium species, is a nephrotoxin found often in cereal grains from tropical countries. Ergot is a neurotoxin produced by Claviceps purpurea and causes gangrene if taken in large quantities. Links: http://www.fao.org/agrippa/publications/4DMellonew-05.htm

Why sequence the Aspergillus genome? Aspergillus contains about 10,000 genes compared to the possible 33,000 genes or more found in humans – how many of these genes are shared with humans? 50% of the fungal genes identified so far are completely new to science, implying they are unique to fungi. So far genetic analysis shows fungi may contain many unique coding sequences – do these encode unique genes which may be useful to mankind? Can we identify fungal genes which also function or malfunction in man? Yes - Aspergillus nidulans has been a successful genetic model for the identification of genes responsible for alkaptonuria – a metabolic disorder. Objective: Fungi on the other hand appear to contain many unique coding sequences. What does this mean? Do these sequences encode unique genes? Are these genes useful to mankind, given that filamentous fungi have produced many useful enzymes and products of commercial use. Useful link: http://www.microbeworld.org/htm/aboutmicro/tools/genetic.htm Aspergillus nidulans has proved a sucessful model for unravelling the metabolic disorder alkaptonuria – where the breakdown of phenylalanine and tyrosine is incomplete and results in this debilitating disease. This enzyme defect (and others) has now been identified – see slide presentation ‘Alkaptonuria’.

Genomic Size of various species Comparison of the size of different genomes Aspergillus as a pathogen in man- Why sequence a genome? Species Approx. Size Type Human 3,300x106 Mammal Aspergillus fumigatus 30x106 Multi-cellular Mycobacterium tuberculosis 4x106 Single cellular (complex) Mycoplasma pneumoniae 400,000 Smallest independent life form Haemophilus influenzae 1.2x106 Single cellular Malaria Single and multi-cellular forms Worm 100 x 106 Objective: The relative size of the genomes illustrated poses interesting questions. Where species are closely related they may share many common sequences? Mycoplasma pneumoniae, is a human pathogenic bacterium causing tracheobronchitis and atypical pneumonia however it lacks a normal bacterial cell wall, is very small in size and is one of the smallestr independent life forms. Useful link : http://www.zmbh.uni-heidelberg.de/M_pneumoniae/genome/Introduction.html Haemophilus influenzae is a bacterium which can cause meningitis or pneumonia. Useful link: http://www.cdc.gov/ncidod/dbmd/diseaseinfo/haeminfluserob_t.htm

How will the sequence of Aspergillus be useful? Genome sequencing of a harmful pathogen allows us to compare DNA sequences with other Aspergillus species which are not pathogenic. That information will enable an understanding of why Aspergillus fumigatus can cause infection resulting in allergic or invasive disease. New drug targets will emerge for use in medicine and agriculture. New diagnostic tools will be developed - early detection of infection is critical for a better outcome for the patient. Objective: Key reasons for sequencing a genome, especially A. fumigatus. Examples include: A whole genome approach asks what functions are encoded by this organism and what is it capable of? Can we identify fungal genes which also function or malfunction in man? The recent use of Aspergillus nidulans genetics to identify the gene responsible for alkaptonuria supports this. Methods for detecting aspergillosis? Development of new diagnostics will improve early diagnosis and patient outcome.

Aspergillus is a remarkable member of the fungal kingdom, with a wide diversity of uses and effects on mankind. In the environment it plays a role in both the Carbon and Nitrogen cycles and in the breakdown of organic material into compost. It is a pathogen and allergen in humans and animals. Aspergillus nidulans has played a crucial role as a genetic model including identifying the genes responsible for alkaptonuria. The biotechnology industry has harnessed it’s potentially useful enzymes for the food industry and commercial uses. The future understanding of these fungal genomes will hopefully pave the way for understanding the role of aspergillus species as pathogens and to enable the development of effective and perhaps less toxic medicines for the treatment of aspergillosis. Objective: Summary slide - relevance of Aspergillus species in a nutshell.