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BioV 400Mycology

Handout 1

Paper Analysis/project• Some suggested topics…

– Spore dispersa l in f ungi: mode s and mechanisms– Pathogenicity mec hanisms in fung i

– Mycoreme diation: organisms and strateg ies– Modes of actions of anti-f unga l age nts– Ins ights int o the evolutionary pos ition of f ung i– Funga l species as mode l organisms

– Fung i in biotec hnology– Symbiotic a ssociation w ith fung i: getting molecular– Spore for mation during hyphal gr owth– Therape utic approac hes to fungal diseases

References• Deacon, J.W. (1997). Modern Microbiology. Third

Edition, Blackwell Science. ISBN 0-632-03077-1

• Maheshwari, R. (2005). Fungi: Experimental Methods in Biology. Mycology, volume 24, Taylor and Francis, CRC Press. ISBN 1-57444-468-9

• Ulloa, M. and Hanlin, R.T. (2001). Illustrated Dictionary of Mycology. Second Printing, APS Press. ISBN 0-89054-257-0

References• Deacon, J.W. (2006). Fungal Biology. 4th edition,

Blackwell Publishing Ltd. ISBN-13 978-1-4051-3066-0

• Dismukes, W.E., Pappas, P.G., and Sobel, J.D. (2003). Clinical Mycology. Oxford University Press, Inc. ISBN 0-19-514809-6

• Webster, J. and Weber, R.W.S. (2007). Introduction to Fungi. Third Edition, Cambridge University Press. ISBN-13 978-0-521-80739-

References• David M. (2011). 21st Century Guidebook

to Fungi. Cambridge University Press. ISBN13 978-0521186957

• Reiss, E. (2011). Fundamental Medical Mycology. John Wiley & Sons, Inc. ISBN13 978-0470177914

http://yeastwonderfulworld.wordpress.com/

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• As saprotrophs, particularly as decomposers, fungi are essential components of the carbon cycle and are among the few organisms that can break down lignin

Fairy rings

Decomposers • Fungi are important as pathogens of animals and plants – Over 70% of all plant

diseases are caused by fungi

Bioremediation• Oil spills• Cyanide in mining operations• Dioxins and pesticides• Produce organic acids, sugars • Other commercial products

What is a Fungus ?

• Eukaryotic – a true nucleus • Do not contain chlorophyll

• Have cell walls• Produce filamentous structures

• Produce spores

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Estimating the number of fungal species

• Hawksworth, D.L. (2001) The magnitude of fungal diversity: the 1.5 million species estimate revisited. Mycological Research 105 (12): 1422-1432.

• Flowering plants on British Isles described = 2,000 species

• Fungi on British Isles described = 12,000 species• Ratio of 6 fungi to each plant species• Total number of described plant species = 250,000

(most plant species are believed to be described)• 6 x 250,000=1.5 million species of fungi!• Less than 5% of which are described, and at the

current rate of description, it will take >800 years to describe all extant species

Estimating the number of fungal species

LUCA

Eucarya Archaea Bacteria

The Three-Domain System

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Extremophiles (Archea)Thermophiles Halophiles

Methanogens

Cryophiles

The sodium-potassium pump

The Kingdom Fungi• Fungi eukaryotic heterotrophs with cell walls

– Cell walls made of chitin complex carbohydrate found in insects’ exoskeletons

• Structure and Function– Multicellular (except yeasts)– Composed of hyphae thin filaments one cell thick

• Cross w alls cytoplasm and nuclei can move t hrough ope nings

• Wit hout cross walls contain many nuclei

– Secrete en zymes to digest food outside their bodies and absorb the nutrien ts

Fungi – Nutrients• Saprophytic fungi

– Live on dead organic matter = decomposers• Obligate parasites

– Only grow on living hosts (usually plants)• Facultative saprophytes

– Usually parasitic, but can live saprophytically• Facultative parasites

– Usually saprophytic, but can be parasitic when living hosts are available

Taxonomy• Medically important fungi fall in four phyla

– AscomycotaSexual reproduction is ascus ascus

– BasidiomycotaSexual reproduction in basidium basidium

– ZygomycotaSexual reproduction by gametes zygosporesZygomycota

– Mitosporic fungi (Fungi Imperfecti)No recognizable form of sexual reproduction

Hierarchical ClassificationKingdom Fungi

Phylum BasidiomycotaClass Basidiomycetes

Order AgaricalesFamily Agaricaceae

Genus AgaricusSpecies: Agaricus campestris L.

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Yeasts reproduce asexually by budding

Morphology• Pathogenic fungi exist as yeasts or as

hyphae– Yeast is a unicellular organism– Hyphae are multicellular filamentous

structures

• Some fungi occur in both yeast and mycelialforms (dimorphic fungi)– Yeast (parasitic or pathogenic form)– Mycelium (saprophytic form)

Hyphae Without Cross Walls

Nuclei

Cell wall

Nuclei

Cytoplasm

Cross wall

Cell wall

Cytoplasm

Hyphae With Cross Walls

Hyphae StructureDimorphic Fungi

• Yeast Form• Parasitic form• Tissue form

• Cultured at 37ºC• Mycelial Form

• Saprophytic form• Cultured at 25ºC

The Kingdom Fungi• Reproduction

– Sexually• Two different mating typ es + (plus) and – (minus)• + and – fuse together, grow, form a diploid zygote

nucleus produce haploid spores– Asexually

• Cells or hyphae break off from a fungus and begin to grow on thei r own

• Some produce spores in sporangia which are found at the tips of speciali zed hyphae called sporangiophores

Tips fuse

Dikaryon

2 nucleiper cell

Nucleifuse

Meiosis

Dikaryoticstage(nn)

Diploidstage(2n)

Haploidstage(n)

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Spores• Sexual

• Asexual– Arthrospore– Blastospore

– Chlamydospore– Conidia• Microconidia• Macroconidia

Chlamydospores• Nuclear ploidy same as parent cell• Formed from a vegetative cell or cells• Shape may reflect cell shape• Wall may become thickened and dark

in color• Are resistant to unfavorable

environmental conditions

Intercalary hyphal chlamydospores in Mucor plumbeus vs. Terminal

chlamydospore in Pythium undulatumThe Kingdom Fungi

• How Fungi Spread– Fungal spores

• Scatter easily in the wind• Must land in favorable environment

– Temperature– Moistu re– Food

• Some are specialized to lure animals, flies– Disperse spores over long distances

The same sequence of events is common to most fungal life cycle Mitospores

Result from mitosis and cell division

No change in nuclear ploidy

Usually produced in great quantities

Resistance to unfavorable environmental conditions varies

Are important in dispersal

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Mitosporesmotile zoosporesnon-motile produced from hyphae conidianon-motile produced from

sporangia sporangiospores

Representative asexual spores of molds-overview

Conidiospores MeiosporesResult from meiosisAre haploid

Are genetically different from parent hyphae

May serve as a resistant stage

Are important in dispersal

Spore characteristicsLow water contentLarge quantities of storage compoundsWall structure different from parent

hyphaeWall differentiation

All cellular organelles are present except a vacuole

Germination factors

Water Temperature Nutrition pH (4.5-6.5) Oxygen

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Germination process Water content increases spore swells

enzymes go into solution Vacuolation increases Endoplasmic reticulum and other organelles

increase Respiration increases and mitochondria

enlarge and form more cristae

Vesicles involved in wall synthesis appearLipids disappear as they are used up as an

energy sourceAn opening on the spore wall appears or the

plug in a germ pore is removedA hyphal tube, called the germ tube, emerges

from the opening or pore

Germination process

Growth of a hypha from a spore Dimorphic fungus• Candida albicans (yeast

and hyphal stages• The yeast form produces

hyphae and pseudohyphae• Pseudohyphae give rise to

yeast cells by apical and lateral budding

• Causes candidiasis

Dimorphic fungus C. albicans

• C. albicans grows as unicellular yeast under some environmental conditions and as a filamentous fungus under other conditions

• Growth at 37°C in YPD medium

Dimorphic fungus C. albicans

• Budding yeast with septum

• Mother cell and daughter bud

• Growth at 37°C in YPD medium

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Dimorphic fungus C. albicans

• Mother and daughter cells

• Hypha-inducing conditions• Mother cell forms a germ

tube

• A septum will later form between the mother cell and the germ tube to become hypha

Dimorphic fungus C. albicans

• 3 hours after the appearance of the germ tube

• Hypha with septa• A new germ tube

form at the distal pole of the cell

Dimorphic fungus C. albicans

• 5 hours growth under hypha-inducing conditions

• Hyphae form a mycelium

• Beginning of the formation of blastospores from hyphae

Dimorphic fungus: on 37ºC Dimorphic fungus: on 25ºC

Mycotic diseases• Hypersensitivity

Allergic reaction to molds or spores

• MycotoxicosesPoisoning by food products contaminated by

fungi

• Mushroom poisoningThe ingestion of toxin

• Infection

Hypersensitivity• FARMER’S LUNG Moldy hay

• MALT WORKER’S DISEASE Moldy barley

• CHEESE WASHER’S LUNG Moldy cheese

• WOOD TRIMMER’S DISEASE Moldy wood

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Infection caused by pathogenic fungi

• Most pathogenic fungi do not produce toxins

• But show physiologic modifications during a parasitic infection

– Increased metabolic state– Modified metabolic pathways– Modified cell wall structure

• Carbohydrate content• Lipid structure• RNA aggregates

Pathogenicity of fungi

1. Thermotolerance

2. Ability to survive in tissue environment

3. Ability to withstand host defenses

Pathogenic fungi• Normal host

– Systemic pathogens 25 species– Cutaneous pathogens 33 species– Subcutaneous pathogens 10 species

• Immunocompromised host– Opportunistic fungi 300 species

A clinician must distinguish between

• Colonization• Transient fungemia

Dissemination of fungi through the blood stream

• True infection

EYE

SKIN

UROGENITAL TRACT

ANUS

MOUTHRESPIRA TORY

TRAC T

Multiplication of an organism at a given site without harm to the host

Colonization

EYE

SKIN

UROGENITAL TRACT

ANUS

MOUTHRESPIRA TORY

TRAC T

Infection

Invasion and multiplicationof organisms in body tissue resulting inlocal cellular injury

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Revived interest in mycology

1. Increased frequency of mycotic diseases

2. Increased awareness by physicians

3. Better trained laboratory personnel

4. More invasive procedures used on patients

5. Increased use of immunosuppressive drugs

6. Increase in immunosuppressive diseases

Diagnosis• 1. Wet Mount• 2. Skin test• 3. Serology• 4. Fluorescent antibody• 5. Biopsy and histopathology• 6. Culture

Treatment Since they are eu karyoti c, fungi are biochemically similar

to the human host Thus it is difficult to develop chemotherapeuti c agents that

will destroy th e pathogenic fungus and not harm the patient

Fungal therapy Try to induce cell injury by causing the cel l me mbrane of t he

fungus to become per meable

Challenge Find an age nt that will selectively injure f unga l cells wit hout

damaging host cells

All eukaryotic cells contain sterols

•Mammalian cells cholesterol•Fungal cells ergosterol

Primary anti-fungal agents

1. Polyene derivatives– Amphotericin B– Nystatin

2. Azoles– Ketoconazole– Fluconazole– Itraconazole– Voriconazole

Amphotericin B• Mode of action Amphotericin B binds to sterols Ergosterol cell walls of fungi

Greater avidity for ergosterol Binding alters the permeability of the cell wall

and results in the leakage of the intracellular of the fungus

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AMPHOTERICIN B generates pores in the membrane Amphotericin B

• Intravenous administration• Thrombo-phlebitis

• Nephrotoxic• Fever• Chills

• Anemia• Extended administration

Acetyl-CoAAcetoacetyl-CoA

HMG-CoAMevalonic Acid

Squalene

Squalene-2,3-Epoxide

Lanosterol

ErgosterolPolyenes

Azoles

Morpholines

Allylamines

JB 5/00

Ergosterol Synthesis

3. Griseofulvin4. 5-fluorocytosine

5. Allylamines– Terbinafine (Lamisil)6. Echinocandins

– Caspofungin

Primary anti-fungal agents

Medical Mycology:The Last 50 Years

02468

101214

1950 1960 1970 1980 1990 2000

Nysta

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Gris

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5-FC MiconazoleKetoconazole

Fluc onazoleItraconazole

L-A mBABCDABLC Terbinafine

Vorico nPosacon

XMP

Sordarins

Cas

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MicafunRav ucon

Ani

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# of drugsClinical Classification

•Cutaneous•Subcutaneous

•Systemic•Opportunistic

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Opportunistic fungiUbiquitous saprophytes and

occasional pathogens that invade the tissues of those who have:

• Predisposing diseases:Diabetes, cancer, leukemia, etc.

• Predisposing conditions:Agammaglobulinemia, steroid or

antibiotic therapy