Myco Lec1 (General Characteristics of Fungi) Jan 2025 PDF

Lecture:1 Mycology / Lecture: 1 General Characteristics of Fungi Dr. Nada A. Abdelrahim 1 1. Structure & Growth 2. Classification 3. Pathogenesis 4. Fungal Toxins & Allergies 5. Laboratory Diagnosis 6. Antifungal Therapy 7. Mechanism of Action o...

Lecture:1 Mycology / Lecture: 1 General Characteristics of Fungi Dr. Nada A. Abdelrahim 1 1. Structure & Growth 2. Classification 3. Pathogenesis 4. Fungal Toxins & Allergies 5. Laboratory Diagnosis 6. Antifungal Therapy 7. Mechanism of Action of Antifungal Drugs 2 ADVANTAGES of Fungi: Fungi are used in production of important foods (e.g. bread, cheese, wine & beer) Fertilization of Soil – Production of Antibiotics – Food (e.g. mushrooms) DISADVANTAGES of Fungi: Fungi are responsible for the spoilage of certain foods; Because molds can grow in a drier, more acidic, and higher osmotic pressure environment than bacteria, they tend to be involved in the spoilage of fruits, grains, vegetables, and jams Fungi can cause disease in humans, animals & plants 3 1. Structure & Growth 4  Fungi (yeasts & molds) are Eukaryotic organisms whereas Bacteria are Prokaryotic… They differ in several fundamental respects (See Table in next slide) 2 Fungal Cell Structures are Important Medically: (1) Fungal Cell Wall consists primarily of Chitin ([Not Peptidoglycan as in Bacteria]; fungi are insensitive to antibiotics (such as penicillin, that inhibit peptidoglycan synthesis) (2) Fungal Cell Membrane contains Ergosterol (Human cell membrane contains Cholesterol) 5 6  Chitin is Polysaccharide (composed of long chains of N-acetylglucosamine) Fungal Cell Wall contains other Polysaccharides, the most important is β-glucan (a long polymer of d-glucose) β-glucan is the Site of Action of the Antifungal Drug Caspofungin Ergosterol in Fungal cell membrane WHILE Cholesterol in Human cell membrane: The selective action of Amphotericin B and Azole drugs (such as Fluconazole & Ketoconazole) on fungi is based on this difference in membrane Sterols 7  There are 2 Types of Fungi: ❖ Yeasts ❖ Molds Yeasts grow as Single Cells that reproduce by Asexual Budding Molds grow as Long Filaments (Hyphae) and form a mat (Mycelium) Some Hyphae form Transverse Walls (Septate Hyphae) Others Do Not (Nonseptate Hyphae) Nonseptate Hyphae are Multinucleated (Coenocytic) Growth of hyphae occurs by extension of tip of hypha, NOT by cell division all along the filament 8 ❖ Yeasts 9 Examples: C. albicans, C. dubliniensis, Many isolates of C. tropicalis 10 ❖ Molds 11 12 13 14 15  Structure (or anatomy) of Fungi True Yeast (Single-celled, uses Also known as Dimorphic Fungi (or Black Yeasts). Are diverse group, organic compounds for energy, can change from Yeast to Mycelial form in response to doesn't require sunlight to grow, can environmental factors. Some species can cause human infections reproduce asexually by budding or (including: Candida albicans, C. parapsilosis, N. glabratus, C. binary fission) tropicalis, and Pichia kudriavzevii) 1) Yeast & Yeast-Like Aerial Hyphae (Mycelium) 2) Filamentous Fungi Vegetative Hyphae (Mycelium) 3) Dimorphic Fungi Can exist as (1) Or as (2) 16 17  Several medically important fungi are Thermally Dimorphic (form different structures at different temperatures): - They exist as Molds in the environment at ambient temperature - and as Yeasts (or other structures) in human tissues at body temperature Most fungi are Obligate Aerobes; Some are Facultative Anaerobes; but None are Obligate Anaerobes All fungi require Preformed Organic Source of Carbon (hence frequently associated with decaying matter) --- Heterotrophic organisms (NOT Autotrophs) Fungi are eukaryotic, non-vascular, non-motile, unicellular or filamentous Natural Habitat of most fungi is, therefore, the environment Important exception is Candida albicans (it is part of the normal human flora) - Heterotrophs are dependent on organic compounds for nutrition (cannot produce their own) and use cellular respiration to convert their food into energy 18  Some fungi reproduce Sexually by mating and forming Sexual Spores (e.g., Zygospores, Ascospores & Basidiospores): ❖ Zygospores are single large spores with thick walls ❖ Ascospores are formed in a sac called ascus ❖ Basidiospores are formed externally on the tip of a pedestal called a basidium Classification of these fungi is based on their Sexual Spores Fungi that Do Not form Sexual Spores are termed “imperfect” and are classified as Fungi imperfecti 19 20 Sexual Spores 21 Sexual Spores 22 Sexual Spores 23 Sexual Spores 24  Most fungi of medical interest propagate Asexually by forming Conidia (Asexual Spores) from the sides or ends of specialized structures (see next slide) Shape, Color & Arrangement of Conidia aid in the identification of fungi ❖ Some important Conidia are: (1) Arthrospores: arise by fragmentation of ends of hyphae and are the mode of transmission of Coccidioides immitis (2) Chlamydospores: are rounded, thick-walled, and quite resistant (terminal chlamydospores of C. albicans aid in its identification) (3) Blastospores: are formed by budding by which yeasts reproduce asexually (some yeasts [C. albicans] can form multiple buds that do not detach, producing sausage-like chains called pseudohyphae, used for identification) (4) Sporangiospores: formed within a sac (sporangium) on a stalk by molds such as Rhizopus and Mucor 25 26 2. Classification 27 Based on Mode of Nutrition On the Basis of Nutrition, Kingdom Fungi can be Classified into 3 Groups: 1. Saprophytic – fungi obtain nutrition by feeding on dead organic substance Examples: Rhizopus, Penicillium & Aspergillus 2. Parasitic – fungi obtain nutrition by living on other living organisms (plants or animals) and absorb nutrients from their host. Examples: Taphrina & Puccinia 3. Symbiotic – fungi live by having interdependent relationship with other species (both are mutually benefited). Examples: Lichens & Mycorrhiza ❖ Lichens are the symbiotic association between algae and fungi. Here both algae and fungi are mutually benefited (fungi provide shelter for algae & algae synthesis carbohydrates for fungi) ❖ Mycorrhiza is the symbiotic association present between fungi and plants (fungi improve nutrient uptake by plants, whereas, plants provides organic molecules like sugar to the fungus) 28 Based on Spore Formation Based on the formation of spores, Kingdom Fungi are classified into: 1. Zygomycetes – formed by fusion of two different cells. Sexual spores are known as Zygospores, while Asexual spores are known as Sporangiospores. Hyphae are without septa. Example: Mucor 2. Ascomycetes – also called Sac Fungi. They can be coprophilous, decomposers, parasitic or saprophytic. Sexual spores are called Ascospores. Asexual reproduction occurs by Conidiospores. Example: Saccharomyces 3. Basidiomycetes – Mushrooms are the most commonly found basidiomycetes and mostly live as parasites. Sexual reproduction occurs by Basidiospores. Asexual reproduction occurs by Conidia, Budding or Fragmentation. Example: Agaricus 4. Deuteromycetes – They are otherwise called imperfect fungi as they do not follow the regular reproduction cycle as other fungi. They do not reproduce sexually. Asexual reproduction occurs by Conidia. Example: Trichoderma 29 3. Pathogenesis 30  Response to infection with many fungi is the formation of Granulomas Granulomas are produced in Major Systemic Fungal Diseases (e.g., Coccidioidomycosis, Histoplasmosis & Blastomycosis, as well as several others) Cell-mediated immune response is involved in Granuloma formation Acute Suppuration (characterized by the presence of neutrophils in the exudate) also occurs in certain fungal diseases such as Aspergillosis and Sporotrichosis Fungi do not have endotoxin in their cell walls Do not produce bacterial-type exotoxins 31  Activation of cell-mediated immune system results in Delayed Hypersensitivity Skin Test Response to certain fungal antigens injected intradermally Positive skin test indicates exposure to fungal antigen (It does not imply current infection, because exposure may have occurred in the past) Negative skin test makes the diagnosis unlikely unless the patient is immunocompromised Because most people carry Candida as part of the normal flora, skin testing with Candida antigens can be used to determine whether cell-mediated immunity is normal 32  Transmission and geographic locations of some important fungi are shown in Table in next slide Intact skin is an effective host defense against certain fungi (e.g., Candida, Dermatophytes), but if the skin is damaged, organisms can become established Fatty acids in skin inhibit Dermatophyte growth Hormone-associated skin changes at puberty Limit Ringworm of scalp caused by Trichophyton Normal flora of skin and mucous membranes suppress fungi - when normal flora is inhibited (e.g., by antibiotics) overgrowth of fungi (C. albicans) can occur 33 34  In respiratory tract, important host defenses are Mucous Membranes of Nasopharynx (trap inhaled fungal spores) and Alveolar Macrophages Circulating IgG and IgM are produced in response to fungal infection (their role in protection from disease is uncertain!) Cell-mediated immune response is Protective; (Its suppression can lead to reactivation and dissemination of asymptomatic fungal infections and to disease caused by opportunistic fungi) 35 In Human Host, Fungi Can Cause: 1. Allergies 2. Mycotoxicosis 3. Mycosis 1) Superficial (Cutaneous) Mycosis Next 2) Subcutaneous Mycosis Lectures 3) Deep or Systemic Mycosis 36 4. Fungal Toxins & Allergies 37  In addition to Mycotic Infections, there are 2 other kinds of Fungal Disease: (1) Mycotoxicoses: caused by ingested toxins, and (2) Allergies: to fungal spores The best-known Mycotoxicosis occurs after eating Amanita mushrooms - These fungi produce 5 toxins, two (Amanitin and Phalloidin) are among the most potent Hepatotoxins - Toxicity of Amanitin is based on its ability to inhibit cellular RNA polymerase, which prevents mRNA synthesis Another Mycotoxicosis (Ergotism) is caused by the mold Claviceps purpurea, which infects grains and produces alkaloids (e.g., ergotamine and lysergic acid diethylamide [LSD]) that cause pronounced vascular and neurologic effects 38 The death cap (Amanita phalloides) - A Poisonous Wild Mushroom 39  Other ingested toxins (Aflatoxins) are Coumarin Derivatives produced by Aspergillus flavus that cause liver damage and tumors in animals and are suspected of causing Hepatic Carcinoma in Humans Aflatoxins are ingested with spoiled grains and peanuts and are metabolized by the liver to the epoxide (a potent carcinogen) Aflatoxin B1 induces a mutation in the p53 tumor suppressor gene, leading to loss of p53 protein and consequent loss of growth control in hepatocytes 40  Allergies to fungal spores (particularly those of Aspergillus) are manifested primarily by: ❖ Asthmatic Reaction (rapid bronchoconstriction mediated by IgE), ❖ Eosinophilia, and a ❖ “Wheal & Flare” skin test reaction These clinical findings are caused by an Immediate Hypersensitivity Response to fungal spores 41 5. Laboratory Diagnosis 42  4 approaches to laboratory diagnosis of fungal diseases: (1) Direct Microscopic Examination (2) Culture of the Organism (3) DNA Probe Tests (4) Serologic Tests (1) Direct microscopic examination of clinical specimens such as sputum, lung biopsy material, and skin scrapings depends on finding characteristic Asexual Spores, Hyphae, or Yeasts in light microscope 43  Specimen is either treated with 10% potassium hydroxide (KOH) to dissolve tissue material (leaving the alkali-resistant fungi intact) or stained with special fungal stains Some examples of diagnostically important findings made by direct examination are: (1) Spherules of C. immitis (2) Wide capsule of Cryptococcus neoformans (seen in India ink preparations of spinal fluid) Calcofluor white (fluorescent dye that binds to fungal cell walls) is useful in the identification of fungi in tissue specimens Methenamine silver stain is also useful in microscopic diagnosis of fungi in tissue 44  1\ Coccidioidomycosis Coccidioides immitis Spherules filled with endospores Coccidioides immitis large Spherule in are typically seen in infected tissues (e.g., Lung) lung (Grocott methenamine silver stain)45  2\ Cryptococcosis CSF Gram stain with multiple encapsulated, rounded Cryptococcus neoformans yeast cells surrounded by halo Wide Capsule of Cryptococcus neoformans (India ink preparations of CSF) 46 Morphological characteristics of hyphae & conidiophores by light microscope (400×) of some endophytic fungi iden- 47 tified belonged to genera: Alternaria (1), Fusarium (2), Mucor (3), Trichoderma (4),Penicillium (5), Paecilomyces (6) (2) Fungi are frequently Cultured on Sabouraud’s Agar This medium facilitates appearance of Slow-Growing Fungi by Inhibiting Growth of Bacteria in specimen Inhibition of bacterial growth is by: - Low pH of Medium, and - Chloramphenicol & Cycloheximide Appearance of Mycelium and Nature of Asexual Spores are frequently sufficient to identify the organism 48  Sterile medium of Saborauds Dextrose Agar Penicillium notatum on SDA Aspergillus niger on SDA 49  Trichophyton terrestre Sporothrix schenckii on SDA on SDA Trichophyton rubrum on SDA 50  Penicillium notatum on SDA 51 Candida albicans on SDA 52 (3) Tests involving DNA Probes Can identify colonies growing in culture at earlier stage of growth than can tests based on visual detection of colonies ---- Its more rapid At present, DNA Probe Tests are Available for: - Coccidioides - Histoplasma - Blastomyces - Cryptococcus 53  Examples of some of the Molecular techniques that can be used for fungus identification and detection: For more details, see attached paper in pdf 54 1\ Sequencing Independent Methods: 1. Fluorescent in situ hybridisation Examples of fluorescence in situ hybridisation A. Clavariopsis aquatica growing on aquatic leaf litter (probed with 18 rRNA-targeted MY1574 domain specific probe). B. Accessibility of Tetracladium marchalianum conidia for FISH 28SrRNA-targeted species-specific probe TmarchB10 55 1\ Sequencing Independent Methods: 2. DNA array hybridization DNA macroarray hybridization results. The x-ray film is overlaid with the oligonucleotide spotting pattern on DNA array membrane. The top left pair of spots and bottom right pair of spots are oligonucleotides which served as positive controls. This array is showing positive signal for Phyllosticta, Coleophoma, Epicoccum, Godronia, Alternaria, Pestalotia, and Pilidium. 56 1\ Sequencing Independent Methods: 3. Multiplex tandem PCR Schematic representation of the multiplex tandem—PCR procedure illustrating specific detection and identification from a blood sample containing Candida albicans and C. glabrata 57 2\ Isothermal Systems: 1. Padlock probe technology and rolling circle amplification Schematic overview of the padlock principle combined with OpenArray®, Technology for multiplex detection of three different targets 58 Schematic representation of the steps in padlock probe technology coupled with Hyperbranched Rolling Circle Amplification (H-RCA) for SNPs detection. 1. The hybridization of padlock probes (containing the complementary sequences at the 5′ and 3′ ends) to the target templates. 2. During a perfect match, the probe forms a circular molecular with the aid of DNA ligase; while in the case of mismatch, no circular molecules formed. 3. Non-hybridized template will be removed during the exonucleolysis reactions (digestion by exonucleases I and III). 4. H-RCA is performed using two pre-designed primers and Bst polymerase, but no amplification will take place in the absence of a circular molecular. 5. The accumulation of dsDNA products during isothermal rolling circle amplification of DNA minicircles is monitored in a real time PCR thermocycler with the addition of SYBR green. 59 2\ Isothermal Systems: 2. Loop mediated isothermal amplification Schematic representation of the mechanism of LAMP. A. General location of the LAMP primer set in relation to defined regions of the target DNA. Forward (F3) and backward (B3) outer primers and forward (FIP) and backward (BIP) inner primers are indicated. B./C. Basic principle and amplification steps in LAMP. In general new DNA strands are synthesizes from the F3 and B3 primers, and these strands are recognized by FIP and BIP to start loop- mediated autocycling amplifications. The final products are stem-loop DNAs with several inverted repeats of the target DNA, and cauliflower-like 60 structures bearing multiple loops  Next-Generation Sequencing & Pyrosequencing Approaches also provide promising ways of enlarging scope of molecular-detection of fungi 61 (4) Tests for the Presence of Antibodies in patient’s serum or spinal fluid are useful in diagnosing Systemic Mycoses (but less useful in diagnosing other fungal infections) Significant rise in antibody titer must be observed to confirm the diagnosis (as is the case for bacterial and viral serologic testing) Complement Fixation Test is most frequently used in suspected cases of Coccidioidomycosis, Histoplasmosis, and Blastomycosis In Cryptococcal Meningitis, presence of polysaccharide capsular antigens of C. neoformans in CSF can be detected by Latex Agglutination Test 62 6. Antifungal Therapy 63  Drugs used to treat bacterial diseases have no effect on fungal diseases Example: Penicillins & Aminoglycosides inhibit growth of many bacteria but do not affect growth of fungi This is because of the presence of specific bacterial structures (e.g., Peptidoglycan & 70S Ribosomes) that are absent in fungi 64  Most effective Antifungal Drugs (Amphotericin B & various Azoles) targets Ergosterol in Fungal Cell Membranes that is Not Found in Bacterial or Human Cell Membranes Amphotericin B (Fungizone): disrupts fungal cell membranes at the site of ergosterol and Azole drugs (Imidazoles [Ketoconazole] or Triazoles [Fluconazole]): inhibit synthesis of ergosterol (is an essential component of fungal membranes) Caspofungin (Cancidas): inhibits synthesis of β-glucan (is found in fungal cell walls but not in bacterial cell walls. Human cells do not have a cell wall) 65  Table in next slide summarizes mode of action and important adverse effects of major antifungal drugs Clinically significant resistance to antifungal drugs is Uncommon Resistance to Azole drugs is Rare but is increasing 66 67 7. Mechanism of Action of Antifungal Drugs 68 1\ Inhibition of Fungal Cell Wall Synthesis Echinocandins (such as Caspofungin [Cancidas] & Micafungin [Mycamine]): lipopeptides; block fungal cell wall synthesis by inhibiting β-glucan synthase (enzyme that synthesizes β-glucan) Caspofungin inhibits growth of Aspergillus and Candida but not Cryptococcus or Mucor Caspofungin is used for treatment of disseminated candidiasis and for treatment of invasive aspergillosis that does not respond to Amphotericin B Micafungin is approved for treatment of esophageal candidiasis and the prophylaxis of invasive Candida infections in bone marrow transplant patients Anidulafungin (another Echinocandin) is approved for the treatment of esophageal candidiasis and other serious Candida infections 69 2\ Alteration of Fungal Cell Membranes ❖ Amphotericin B & Nystatin Amphotericin B (most important antifungal drug) is used in treatment of variety of disseminated fungal diseases It is a Polyene (with series of 7 unsaturated double bonds in its macrolide ring) It Disrupts Cell Membrane of Fungi because of its Affinity for Ergosterol Fungi resistant to Amphotericin B are uncommon Nystatin is another Polyene antifungal agent It is used topically (because of its toxicity) for infections caused by Candida yeast 70 ❖ Azoles & Terbinafine Azoles are antifungal drugs that act by inhibiting ergosterol synthesis [They block cytochrome P450-dependent demethylation of lanosterol (the precursor of ergosterol)] 1) Fluconazole, Ketoconazole, Voriconazole, Posaconazole, Itraconazole, & Isavuconazonium (a prodrug of isavuconazole) are used to treat systemic fungal diseases :- Ketoconazole is useful in treatment of blastomycosis, chronic mucocutaneous candidiasis, coccidioidomycosis & skin infections caused by dermatophytes Fluconazole is useful in the treatment of candidal & cryptococcal infections Itraconazole is used to treat histoplasmosis & blastomycosis Isavuconazonium is used to treat invasive aspergillosis 71  Posaconazole is used for treatment of oropharyngeal candidiasis and the prevention of Candida and Aspergillus infections in immunocompromised individuals 2) Clotrimazole & Miconazole are used only topically because they are too toxic to be given systemically :- Miconazole & Clotrimazole (two other Imidazoles) are useful for topical therapy of Candida infections and Dermatophytoses ❖ Fungi resistant to the azole drugs are an increasing problem Terbinafine blocks ergosterol synthesis by inhibiting squalene epoxidase - It is used in treatment of dermatophyte infections of skin, fingernails & toenails 72 3\ Inhibition of Fungal DNA Synthesis ❖ Flucytosine Flucytosine (5-fluorocytosine, 5-FC) is antifungal drug that inhibits DNA synthesis Is a nucleoside analogue that is metabolized to fluorouracil (inhibits thymidylate synthetase) --- limit the supply of thymidine Is used in combination with Amphotericin B in treatment of disseminated cryptococcal or candidal infections, especially cryptococcal meningitis ❖ It is not used alone because resistant mutants emerge very rapidly 73  4\ Additional Antifungal Drug Mechanisms ❖Griseofulvin is an antifungal drug that is useful in treatment of hair and nail infections caused by dermatophytes It binds to tubulin in microtubules and may act by preventing formation of the mitotic spindle ❖Pentamidine is active against fungi and protozoa It is widely used to prevent or treat pneumonia caused by Pneumocystis jiroveci It inhibits DNA synthesis by unknown mechanism 74 Model of typical fungal cell showing sites of action of important antifungal drugs: 75 Mechanism of Resistance to Antifungal Drugs Common mechanisms are listed on the table: 76 Questions? 77 Source of Lecture 78