Medically Important Fungi and Protists PDF

PHAR2921 Medically Important Fungi and Protists Andrew Holmes, School of Life & Environmental Sciences Microbiology [email protected]  COMMONWEALTH OF AUSTRALIA Copyright Regulation WARNING! This material has been reprod...

PHAR2921 Medically Important Fungi and Protists Andrew Holmes, School of Life & Environmental Sciences Microbiology [email protected]  COMMONWEALTH OF AUSTRALIA Copyright Regulation WARNING! This material has been reproduced and communicated to you by or on behalf of the University of Sydney pursuant to Part VB of the Copyright Act 1968 (the Act). The material in this communication may be subject to copyright under the Act. Any further reproduction or communication of this material by you may be the subject of copyright protection under the Act. Do not remove this notice All images sourced form Prescott’s Microbiology unless otherwise cited. 2  http://www.stacylevy.com/installations/mold_garden.php Lecture outline Eukaryote microbes and fungi What make fungi different? Importance of fungi to animal health Food spoilage fungi and mycotoxins Allergenic fungi and hypersensitivity Commensal and pathogenic fungi Diagnosing and treating fungal infections 3  Learning outcomes (LO) From this lecture students should be able to 1. Distinguish fungi from other eukaryotic microbes that may cause disease. 2. Describe basic fungal biology, reproduction, and classification. 3. Use knowledge of fungal biology to explain why fungal infections are particularly difficult to treat. 4. Describe three major categories of fungal impact on human health (Allergen, Toxins, and Infection), and able to give one fungi involved in each area. 5. For fungal infections – can distinguish superficial and invasive fungal infections, giving symptoms and causal agents as examples. 6. Use knowledge of fungal cell biology to explain the targets of current antifungal drugs, and know the names and limitations of these drugs. Each slide has a number in top left to map to these LO’s 4 LO1 Microbes: Phylogeny and medically important groups The Prokaryote ‘microbes’ – Bacteria and Archaea The ‘macrobes’ – plants and animals The Eukaryal ‘microbes’ – algae, protozoa and fungi 5 LO6 Features that are dichotomous Features that are divergent LO1 What are Protists? → Eukaryal microbes that are not fungi Algae = photosynthetic protists Euglena Protozoa = chemoorganotrophic protists Amoebae Flagellates Parasitic Protists Trypanosoma Amoeba 7 LO1 Trypanosomes: ‘Sleeping sickness’ - Africa Chagas disease - America Leishmaniasis – Worldwide Complex life cycles with arthropod vectors Antigenic variation – the glycoprotein composition is switched cyclically meaning the cell has 1 of a 1000 options at any one time – vaccines are unrealistic proposition Few drugs – available drugs target aspects of metabolism that are of greater importance to parasite cells than host cells. LO1 Apicomplexans All Apicomplexans are either inter- or intra-cellular parasites of animals. They include: Plasmodium (malaria); Toxoplasma; Cryptosporidium, and Eimeria Diagnostics of obligate parasites must rely on microscopy, serology or nucleic acids Apicomplexans share a unique collection of structures called the apical complex (designed to penetrate host cells), and in addition many have a unique plastid called the apicoplast (thought to be a relict chloroplast). Apicomplexans typically have complex life cycles that can involve multiple hosts. Baum et al, Nature Reviews Microbiology 4, 621-628, 2006 LO2 What are fungi? Eukaryotic Saprophytic heterotrophs No chlorophyll Typically aerobic Some are anaerobic (e.g Saccharomyces and Rumen fungi) Cell walls common – no peptidoglycan Chitin, beta-glucans and glycoproteins Reproduce by both asexual and sexual Single- or multi-celled ‘microbes’ Single-celled (yeast morphology) Filamentous and Independent (e.g. Typical mushrooms) Filamentous and Mutualistic (e.g. Mycorrhizae) Prescott, 2011 10 LO2 Fungal nutrition Heterotrophic = energy from organic carbon sources Absorptive (saprophytic) nutrition = soluble nutrients – Insoluble nutrient sources, such as dead plant matter, degraded outside the fungal cell by secreted enzymes Host-associations (Symbiosis spectrum) - Plant mutualisms common (70% plants need mycorrhizae) - Plant parasitism common (fungal plant pathogens) - Animal commensals (rumen fungi, skin commensals) - Animal pathogens 11 Photo Credit DR KEITH WHEELER / SCIENCE PHOTO LIBRARY LO2 Fungal morphologies In culture - macroscopic colonies Yeasts (smooth) Moulds (furry) Fungi – ‘microbes’ Hyphae can produce spores at that are typically their tips which allow multicellular and many the fungus to can grow at disperse and reproduce macroscopic scale 12 LO2 Fungi can have a large ‘foraging range’ Long-distance nutrient foraging requires big size and inter- cellular transport. Long hyphal filaments require rigid cell walls. 13 Garcia-Rubio et al. (2020) Frontiers in Microbiology https://doi.org/10.3389/fmicb.2019.02993 LO1-3 Eukaryal phylogeny - where fungi fit with other organisms fungi fungi are closer to animals than to plants and most protists… …which helps explain why fungal diseases are so difficult to treat 14 Concept check Can fungi perform photosynthesis? Name the main difference between fungal and bacterial cell wall Which organisms cell biology processes are closest to humans – Fungi, Protists, or Bacteria ? 15 LO4 Importance of fungi to animal health 1. Allergenic fungi and hypersensitivity 2. Food spoilage fungi and mycotoxins 3. Commensal and pathogenic fungi 16 LO4 1. Allergenic fungi and hypersensitivity Main genera causing http://www.stachybotrys.com/images/WEMimage13c.j problems: ▪ Aspergillus ▪ Penicillium content/uploads/2013/03/Sick_Building_Syndro ▪ Alternaria ▪ http://imedsolutionsllc.com/wp- Cladosporium Grow under damp conditions and make allergenic spores me-3.jpg Fungal growth on damp and volatiles walls Mould and spores can be a cause of Sick Building Syndrome 17 LO4 2. Food spoilage fungi and mycotoxins Q: Fungi can grow on and damage all types of foods – is this a problem? Willey, 2009 Yes – especially if they make mycotoxins… 18 LO4 Acute toxin phenomena associated with mycotoxins 19 LO4 Mycotoxicoses Acute: moderate-high levels toxin specific, observable disease Chronic: low-moderate levels of toxin weight loss, infertility, failure to thrive Mycotoxin-associated diseases: low-very low levels of toxin general immunosuppression, increased susceptibility to infectious disease 20 LO4 3. Commensal and pathogenic fungi – the discipline of medical mycology Invasive mycoses - The “Big Three”: Candidiasis Aspergillosis Cryptococcosis Superficial, cutaneous and subcutaneous mycoses – infect skin and underlying tissue 21 Concept check What is difference between ‘mycosis” and “mycotoxicosis”? A mycosis is actual infection, and treatment is getting rid of the fungus. Mycotoxicosis is not infection but its damage caused by the toxin. Why do we distinguish mycoses as invasive, subcutaneous, cutaneous etc? 22 LO5 Superficial mycoses Limited to the outer surface of hair or skin Infection of hair shafts known as piedras Up to 20% of population may be infected; Prevented by good personal hygiene; Treated with cleansing agents Economical impact, long fungistatic treatment regime http://www.mycology.adelaide.edu Wiley et al, 2007.au/images/mal2.gif Tinea versicolor caused Black piedra on hair shaft by Malassezia furfur 23 LO5 Superficial infections – confined to surface of skin or hair Disease names  Seborrheic dermatitis (severe dandruff) Sebum → oleic acid inflammatory response  Pityriasis versicolor (flaky, discoloured skin patches)  Piedra (nodules on hair shaft)  Malasezzia folliculitis (a fungal pimple) Common fungal causes Malasezzia spp – a typical skin commensal. Trichophyton spp Treatment – good hygiene, topic antifungal ketoconazole shampoo, zinc pyrithione shampoo selenium sulfide lotion - cytostatic 24 LO5 Cutaneous mycoses: “ringworm”, tinea Disease names  Tinea corporis (ringworm of body)  Tinea capitis (ringworm on scalp)  Tinea pedis (athlete’s foot)  Tinea cruris (ringworm of groin, ‘jock itch’) Cutaneous mycoses are deeper Common fungal causes than superficial, more discomforting. Microsporum spp. Trichophyton spp. Caused by Dermatophytic fungi – ‘eat’ skin keratin Very high carriage rates of causal organisms – easily passed via infected surfaces Trichophyton Microsporum Usually treatable 25 LO5 Clinical aspects of cutaneous mycoses Blister-like lesions dry to leave scaly ring Without treatment may get hair loss, change of hair colour, local inflammatory reactions Can be highly adapted to humans or transmitted from animals or via soil Willey et al 2007 Treat by changing environment or by topical antifungal agents such as miconazole, terbinafine, clotrimazole, ketoconazole, or tolnaftate applied twice daily until symptoms resolve — usually within one or two weeks (except nail infections) Cat with tinea infection Warning: graphic image on next slide 26 LO5 Subcutaneous mycoses Willey, 2007 Deep tissue Caused by numerous soil fungi, especially black moulds; introduced via puncture wounds; not spread from person-to-person Subcutaneous fungal Disease develops very slowly with fungi infection http://www.mycetoma.org/images/F4.jpg spreading along lymphatic channels – Fungal tumor Diagnosis is important as some bacterial infections mimic subcutaneous mycoses Treatment: Generally difficult; - some respond “Fungal tumor” to antifungals; may require surgical excision 27 Fungal Skin tissue Source Fungi disease involved responsible Superficial Outermost layers mycoses of skin and hair Cutaneous Epidermis, mycoses = deeper invasion tinea/ringworm of hair and nails Subcutaneous Dermis, mycoses subcutaneous tissue, muscle and fascia 28 Summary of superficial, cutaneous and subcutaneous mycoses Fungal Skin tissue Source Fungi disease involved responsible Superficial Outermost layers Normal Melassezia, mycoses of skin and hair flora/ubiquitous Piedraia, fungi Trichosporon Cutaneous Epidermis, Other people, “Dermatophytes” mycoses = deeper invasion animals and soil – eg. tinea/ringworm of hair and nails - direct or Trichophyton, indirect contact Microsporum Subcutaneous Dermis, Soil - implanted Soil fungi, mycoses subcutaneous by trauma especially black tissue, muscle moulds and fascia 29 LO5 Invasive Fungal Infections (IFIs) Few fungi can cause IFIs and these are mostly rare or geographically confined The Big Three of global importance are: Species Location Life- Mortality rate threatening infections/yr Aspergillus Worldwide, esp. >200,000 30-95% developed countries Candida Worldwide, esp. >400,000 46-74% developed countries Cryptococcus Worldwide, esp. sub- >250,000 20-70% Saharan Africa Willey, 2011 ***All have increased because of increasing numbers of immunocompromised hosts*** 30 From Brown et al, 2012 www.ScienceTranslationalMedicine.org LO5 IFIs cause big problems in sick people http://www.kepro.com/media/1022/infection_mortality_rate_graph.jpg http://www.cdc.gov/fungal/images/cryptococcal_meningitis- 2.jpg = with fungal infection = no fungal infection HSCT/BMT = hematopoietic stem cell transplantation or bone marrow transplantation, SOT = solid-organ 31 transplantation, COPD = chronic obstructive pulmonary disease, HIV = human immunodeficiency virus. LO5 Aspergillus - Aspergillosis Primarily Aspergillus fumigatus (~85%) and Aspergillus flavus (~5%); other Aspergillus spp. ~5% Acquired from the environment as Aspergillus makes millions of tiny spores Aspergillus spore heads A plate of Aspergillus. The green colour is due to pigmented spores 32 LO5 www.clarian.org/ADAM/doc/graphics/images/en/1 Aspergillus - Aspergillosis Aspergillosis is an opportunistic infection – gets more serious with the level of 7263.jpg immunocompromise of the host – Aspergilloma (fungus ball) (TB) www.ispub.com/ispub/ijpa/volume_7_number – Invasive pulmonary aspergillosis Similar to bacterial pneumonia – Disseminated aspergillosis _2_15 Crossing blood-brain barrier Aspergillus dissemination in the brain 33 LO5 IFIs: Candidiasis Candida albicans and other Candida species cause candidiasis http://www.studyblue.com/notes/note/n/introduction-to-mycology/deck/1875859 Single celled yeast that often forms pseudohyphae A common cause of bloodstream infections in hospitals Candida producing pseudohyphae Many Candida spp are commensal so: can be acquired endogenously, or can be passed from person-to-person 34 LO5 Candidiasis takes many forms Candidiasis of the vagina = vulvocandidiasis, vulvovaginitis. Candidiasis of the mouth = oral candidiasis or thrush Candidiasis of skin = fingers (paronychia) and nails (onychomycosis) Disseminated candidiasis following immuno- suppression, surgery, severe burns, drug abuse 35 LO5 Cryptococcosis Caused by Cryptococcus species in two medically important species complexes: - Cryptococcus neoformans species complex - Cryptococcus gattii species complex Encapsulated Cryptococcus cells Grows as a yeast with a large capsule, which is an important virulence factor http://www.mycology.adelaide.edu.au – Thick polysaccharide coat (chitosan) – No longer virulent if removed. Cryptococcosis – cryptococcal pneumonia – cryptococcal meningitis Cryptococcus lesions in the brain 36 LO5 Process of cryptococcal infection Acquired from the environment (tree hollows, soil, guano) The first site of infection is the lungs From there it can disseminate to other parts of the body, particularly the brain – lipid rich http://www.bmolchem.wisc.edu/labs/hull/research.html People most at risk are patients with relatively severe immune defects 37  Concept check Aspergillus, Candida, Cryptococcus What is meant by endogenous infections? Which of the above are least likely to cause endogenous infection? Which of the above is associated with the widest range of disease presentations? Why might this be so? 38 Fungal Tissue/Organ Source Fungi disease involved responsible Candidiasis Candida albicans and other Candida species Aspergillosis Aspergillus fumigatus, Aspergillus flavus and some others Cryptococcosis Cryptococcus neoformans and Cryptococcus gattii species complexes 39 Summary of Big Three IFIs Fungal Tissue/Organ Source Fungi disease involved responsible Candidiasis Mucosal Self, other Candida albicans surfaces; can people and other Candida disseminate species Aspergillosis Lungs; can Environment; Aspergillus disseminate in spread by fumigatus, immunocom- spores Aspergillus flavus promised and some others Cryptococcosis Starts in lungs, Environment; Cryptococcus can go to brain Especially neoformans and pigeon guano Cryptococcus and decaying gattii species wood complexes 40 LO5 Diagnosis and treatment of fungal infections Early and correct diagnosis and proper treatment are essential Fungi not harmed by antibacterials or antivirals 41 LO6 Diagnosis and treatment of fungal infections http://www.vet.uga.edu/vpp/clerk/ritter/fig07.jpg Diagnosis done by culture and microscopy Today immunological and molecular techniques frequently used to speed up diagnosis – eg Antigen dip-sticks, PCR with fungus-specific primers, MALDI-TOF Eg. Cryptococcal Antigen Lateral Flow assay (CrAg® LFA) for point-of-care India ink stain of CSF shows diagnosis of cryptococcosis presence of Cryptococcus cells http://www.mycology.adelaide.edu.au/i mages/mala1.gif Agar plate showing yeast colonies 42 http://www.hain-lifescience.co.za/products/cryptococcus/crag-lfa.html LO6 Treatment with antifungal drugs There are few effective anti-fungal drugs Current drugs are grouped into 4 categories: 1. Nucleic acid synthesis inhibitors (5-flucytosine) 2. Ergosterol biosynthesis inhibitors (azoles, terbinifine) 3. Polyenes (amphotericin B, nystatin) - fungicidal 4. Echinocandins (eg. caspofungin) 43 LO6 Treatment: Most antifungals target cell wall or membrane Fungal cell Fungal cell membrane and cell wall - - Squalene Ergosterol Amphotericin B, 5-flucytosine nystatin - - Terbinafine - Azoles Echinocandins 44 Squalene Squalene epoxide epoxide Lanosterol LO6 Problems with antifungal drugs There are few effective anti-fungal drugs Current drugs are grouped into 4 categories: 1. Nucleic acid synthesis inhibitors (5-flucytosine) 2. Ergosterol biosynthesis inhibitors (azoles, terbinifine) 3. Polyenes (amphotericin B, nystatin) 4. Echinocandins (eg. caspofungin) Problems include: toxicity limited spectrum poor bioavailability resistance cost 45 Concept check 1. Nucleic acid synthesis inhibitors 2. Ergosterol biosynthesis inhibitors 3. Polyenes 4. Echinocandins Most effective: Most cytotoxic: NOT targeting cell wall: Targets beta glucan synthesis: 46 Summary of medically important fungi Fungi can cause a variety of clinical issues, ranging from hypersensitivity and toxicoses to severe, invasive infections Fungal infections are usually not severe but are highly prevalent Severity generally increases in immunocompromised hosts Fungi are difficult to treat: there are few good targets, and all current antifungals have limitations Superficial and cutaneous infections easier to treat – topic application less risk of toxic side-effects. 47