ALGAE, FUNGI, PROTOZOA and
MULTICELL PARASITES
Marlia Singgih Wibowo
Questions
• What is eucaryotic microorganisms?
• What is the difference between procaryotic cells and eucaryotic cells?
• Unicellular? Multicellular?
• What is “FUNGI”?
• What is “saphrophyte” ?
• What is “Biotransformation”?
• Primary metabolites and secondary metabolites of fungi?
• What is “fermentation”?
• Spores vs mycelium?
• Sexual and Asexual reproduction?
• What is Mycotoxin?
• What is “Budding”?
Outline
• Introduction : eucaryotic mikroorganisms
• FUNGI : Classification, growth, and reproduction, role of fungi in nature and pharmacy fields • ALGAE : Classification, growth, and reproduction, role of fungi in nature and pharmacy fields • PROTOZOA : Classification, growth, and reproduction, role in nature and pharmacy fields • MULTICELLULAR PARASITES : Classification, growth, and reproduction, role in nature and pharmacy fields References
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Farmakope Indonesia edisi IV, 1995.
USP 27, 2004.
TGA, Australia, 2002.
Denyer SP, Baird RM, Guide to Microbiological Control in Pharmaceuticals, Ellis Horwood, New York, 1990.
Denyer S.P., Hodges N.A., Gorman S.P., (Eds.), Hugo and Russell’s Pharmaceutical Microbiology, 7th ed., Blackwell Science Ltd., Massachusetts, 2004.
Baird R.M., Hodges N.A., Denyer S.P., (Eds.), Handbook of Microbiological Quality Control : Pharmaceuticals and Medical Devices, CRC Press, Boca Raton, 2000.
Barnett, Microbiology Laboratory Exercise, Wm.C.Brown Publ., Dubuque, 1992
Aszalos, A., Modern Analysis of Antibiotics, Marcel Dekker Inc., New York, 1986.
Baird R M., Handbook of Microbiological Quality Control : Pharmaceuticals and Medical Devices, CRC Press, London, 2000.
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Neidleman S.I, and Laskin A.I., Advances in Applied Microbiology, Academic Press, New York, 1997.
Stanbury PF, Whitaker A, Principles of Fermentation Technology, Pergamon Press, Oxford, 1995.
Ward OP, Fermentation Biotechnology : Principles, Process and Product, John Wiley & Sons, Chichester, 1989.
Demain A.L., and Solomon N.A.(Eds.), Biology of Industrial Microorganisms, The Benjamin/Cumming Publ.Co.,Inc., London, 1985.
Demain A.L., and Solomon N.A.(Eds.), Manual of Industrial Microbiology and Biotechnology, American Soc. For Microbiology, Washington DC, 1986.
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Pelczar, M.J., et al., Microbiology, Concepts and Applications, McGraw Hill,Inc., New York, 1993.
Madigan, M.T., et al., Brock Biology of Microorganisms, 8th ed., Prentice Hall Int.Inc., 1997 and new edition.
Prave, P. et al.(ed), Basic Biotechnology : a Student’s guide, VCH, New York, 1987
Neidleman S.I, and Laskin A.I., Advances in Applied Microbiology, Academic Press, New York, 1997.
Deacon J.W, Modern Mycology, 3rd ed., Blackwell Science, Edinburgh, 1997
Dan lain‐lain
Deacon J.W, Modern Mycology, 3rd ed., Blackwell Science, Edinburgh, 1997
Introduction
• Type of microorganism : eucaryotes cells
• What the difference between eucaryotic and procaryotic cells?
• Domain of eukarya including : animals, plants, slime moulds, protozoa, parasites
• Classification is based on : morphology, DNA sequence analysis, ribosomal RNA sequence analysis, etc.
Eucaryotic Microorganisms
• Unicellular (bersel tunggal) dan Multicellular (bersel banyak)
• FUNGI
• ALGAE
• PROTOZOA
• PARASITES ( MULTICELLULAR)
Three Domains of Life
Prokaryotes
Bacteria
Archaea
Green non-sulfur
bacteria
Mitochondrion
Proteobacteria
Chloroplast
Cyanobacteria
Flavobacteria
Thermotoga
Aquifex
Eucarya
(Eukaryote)
Crenarchaeota
Euryarchaeota
Methanosarcina
Gram
Methanobacterium
positivesThermoproteus
Methanococcus
Pyrodictium
Thermococcus
Animals
Entamoebae Slime
molds
Extreme
Halophiles
Thermoplasma
Methanopyrus
Fungi
Plants
Ciliates
Flagellates
Trichomonads
Korarchaeota
Microsporidia
Diplomonads
Universal phylogenetic tree as determined from
comparative sequencing of 16S or 18S rRNA
(Wheelis et al. 1992).
Characteristic of eukaryotic microorganisms
• Definite cell walls (except for aquatic fungi
and cellular slime moulds)
• Nuclei with membranes • Genetic Information inside the nucleus and contains more than one chromosome
• Respiration in mitochondria
• Chloroplast for photosynthesis
• Movement by cilia atau flagella (stop kul 2)
FUNGI
FUNGI
• Terms : Fungus (singular)
Fungi (plural) Jamur (Ind.)
• Mould (Kapang)
• Yeast (Ragi= khamir)
• Mushroom (Jamur besar)
FUNGI
FUNGI
Saccharomyces cereviseae
Candida albicans
FUNGI
Fusarium sp.
Aspergillus sp.
Penicillium sp.
Taxonomy of Fungi (Alexopoulos, 1996)
Recent names
Previous names
Oomycota
Oomycetes
ChytridiomycotaChytridiomycetes Lower fungi
Zygomycota
Zygomycetes
Ascomycota
Ascomycetes
Deuteromycota
Deuteromycetes, Deuteromycotina, Higher fungi
Fungi Imperfecti
Basidiomycota
Basidiomycetes, Basidiomycotina
Role of Fungi •
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Positive Role :
Industrial Microorganisms : drug raw material, growth promoter, enzymes for food industry, etc.
Saprophyte : decomposer Analytical tools : mikroorganisms for test
Biotransformation (jasa) : hormon
Research : bioinformatic
Negative Role:
• Parasites on plants, animals, human, (cause of diseases)
• Deterioration of many products (food, drinks, households, cosmetics, etc.)
How to use Fungi?
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Biomasa Primary Metabolites Secondary Metabolites Enzymes
Bioinformatics
Biotransformation
Some fungi in Industry
Field
Pharmacy
Product
Penicillin
Griseofulvin
Cyclosporin
Source
Usage
P.chrysogenum
antibacteria
P.griseofulvumantifungi
Trich.polysporum
immunosuppresant
Agriculture
Zealarenone Gibberella zeae
Gibberellins G.fujikuroi
growth promoter
plants hormon Industry
Amylase
Asp.oryzae, niger
Lipase
Mucor, Aspergillus
degradation of amilum
degradation of lipid
Morphology
• Filamentous fungi = moulds
‐ Hyphae mycelium
‐ Producing spores (sexual and asexual)
‐ Growth on tips (apical) of hyphae
• Yeast = Ragi (Ind.)
‐ Form “budding”
‐ Self deviding reproduction
Characteristic of Fungi
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Nutrition : Heterotroph
No photosynthesis (saprophyte or parasite)
Cell wall : contains chitin (kitin) dan chitosan (kitosan)
Nucleus : mostly haploid, some are diploid
Histone : Histone 2B (similar to animal)
Lysin : Syntesized by AAA biosynthesis pathway
Mitochondria : disc‐shape Storage compounds : glicogen, lipid, trihalose
Sterol : ergosterol
Ribosome : 18s rRNA
Differences between mould and yeast Characteristic
Mould
Yeast
1. Cell size
5 – 30 m
 4 m(e.g Candida)
2. Growth form
Form hyphae
/mycelium
Budding system
3. Cell wall component
chitin, glucan
chitin and glucan in
various conc.
4. Spores
with Pigment
mostly colourless
5. Protein content
10%
6%
6. Glucosamine content
28%
33%
7. Nucleus
mostly haploid
can be haploid/diploid
Moulds‐Filamentous Fungi (Kapang)
• Hyphae with protoplasmic cylinder form surrounded by cell walls • Diameter of hyphae : 2 – 18 μm
• Hyphae consists of several compartments separated by septa (septum), but some have no septa
• Inside one compartment : consists of one or two nucleus, dan aktivitas protoplasma terpusat pada daerah ujung hifa (disebut daerah apex = apical region)
Moulds‐Filamentous Fungi (Kapang)
• Apical end with 50 nm thickness , cell‐walls with range up to 125 nm thickness.
• Apical end contains numerous of organell, and at tip area have a lot of AVC = apical vesicle cluster, which have an important role in mould growth • If the old hyphae lysed (lysis), pores of septa will be filled by such proteins called Woronin bodies and the septa will be rigidly closed
Structure of Moulds
Hyphae structure
Hyphae under electron
microscope
Yeast (Ragi)
• Unicellular
• Some yeast have Dimorf properties (the morphology changed due to environment condition, such as temperature, etc.)
• Each cell has one nuclues and several organell
• Big Vacuola • Reproduction with “budding” system : bipolar or multipolar
Structure of Yeast
Budding of Yeast
Fungi as eucaryotic cells
• NUCLEUS : contains the genome of cells
• DNA within the nucleus is wound around histone to form nucleosomes and then chromosomes
• Nucleus is enclosed by a pair of membranes, separated by a space. The inner membrane is a simple sac, while the outer is in many places continuous with the ER. Eukaryotic cell
• The nuclear membrane also contains pores, formed from holes where the inner and outer membrane are joined
• The pore’s function : transport proteins and nucleic acids into and out of the nucleus (called : nuclear transport)
• Nucleolus : inside the nucleus, is the site of ribosomal RNA synthesis
• Ribosomal proteins synthesized in cytoplasm and transported into nucleolus and combined with ribosomal RNA to form the small and large subunits of eukaryotic ribosome
Nucleus of yeast cell under electron microscope
Mitochondria
• Mitochondrion (singular) is energy‐generating organelles of eukaryotic cells.
• For respiration and oxidative phosphorylation
• Can be rod‐shape or shperical
• Animal contain over 1000 mitochondria, yeast have fewer • Mitochondria are surrounded by two membranes. The outer membrane composed of an equal mixture of protein and lipid, relatively permeable. The inner membrane is more protein and less permeable
• Lack of sterols, so are much less rigid
• Posses a series of folded internal membranes called CRISTAE, sites of enzymes involved in respiration and ATP production
Mitochondria
• Some anaerobic eukaryotic microorganisms lack mitochondria, instead : Hydrogenosome.
• Examples : Trichomonas (parasites)
• Function : Oxidation of pyruvate to H2, CO2
and acetate
Hydrogenosome
Endoplasmic Reticulum and Golgi • ER is a network of membranes continuous with nuclear membrane
• Rough ER attached ribosomes, Smooth ER for lipid synthesis and carbohydrate metabolism
• Golgi complex consists of a stack of membranes distinct from ER. Product of ER are chemically modified and sorted to be secreted (for example: glycosylation)
Golgi complex of Toxoplasma gondii
Lysosome and Peroxisomes
• Lysosomes contain various digestive‐enzymes to digest macromolecules, such as proteins, fats and polysaccharides
• Peroxisomes produce hydrogen peroxide (H2O2) from the reduction of O2 by various hydrogen donors. Reproduction system
• Asexual
‐ yeast forms spores in sporangiospores
while moulds forms spores in conidia. Function: for dispersal purposes (penyebaran)
• Sexual
‐ Sexual Reproduction occurs when haploid gamet fused to form zygote
‐ Zygote enter into meiosis (pembelahan) process to form
sexual haploid spores. Function: for survival in ‘dormant’ form
Conidia (spores)
Conidiophore
Asexual Spores
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Non‐motile : Zygomycota
Move with one flagella : Chytridiomycota
Move with two flagella : Oomycota
Ascomycota, Deuteromycota and Basidiomycota never form sporangium, and have no flagella. They reproduce by budding system, fragmentation, etc
Sexual spores
• ‘Lower fungi’ usually have clear sexual organ while in ‘higher fungi’ their sexual organ are not definite, only fusion of somatic (non‐
reproductive) hyphae
• Sexual Spores play a role on ‘dormant’ form for survival (oospores, zygospores, ascospores)
Yeast cells
Oomycota
(Water molds)
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Phytophtora (a)
Saprolegnia (b), (c)
Pythium (d)
Habitat : Aquatic
Sexual spores : oospore
Phytophtora infestans
Is a pathogenic fungi for
potatoes
(a)
(d)
(b)
(c)
Chytridiomycota
• Allomyces (a)
• Rhizophlyctis (b)
• Olpidium (c)
Mostly saprophyte
Zygomycota
(Bread molds)
Mucor (a)
Entomophthora (b)
Pilobolus (c)
Erynia (b)
Piptochepalis (d)
Rhizopus
Habitat : soil, decaying plant material
• Sexual spores : zygospore
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(a)
(d)
(b)
(c)
Ascomycota
(Sac fungi)
Neurospora (a)
Eurotium (d)
Ascobolus (c)
Saccharomyces
Pneumocystis
Habitat : soil, decaying
plant material
• Sexual spores: ascospores •
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(a)
(b)
(c)
(d)
Deuteromycota
(Fungi imperfecti)
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Alternaria, Aspergillus
Cladosporium
Geotrichum, Humicola
Penicillium, Phomopsis
Pesotum, Fusarium
Trichoderma, Candida
Habitat : soil, surface of
animal bodies
Basidiomycota
(mushroom)
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Agaricus (a)
Lycoperdon (b)
Ganoderma (c)
Puccinia (d)
Amanita
Pathogenic Fungi
 Fungi causes diseases pada skin surface or topical (Superficial mycoses)
 Fungi causes diseases on subkutan layer (Subcutaneous mycoses)
 Fungi causes diseases inside the human body or sistemic system (Deep or sistemic mycoses)
Mycotoxin (Mikotoksin)
• Mycotoxin is fungal secondary metabolites which is secreted for survival purposes against other microorganisms or other organisms • Main entrance for pathogenic fungi attacking human is though oral cavity or inhalation or through mucose adhesion or lesion contact.
Oportunist Fungi
• Fungi that caused serious diseases due to other diseases • Candida albicans is one sample of oportunist
fungi, in certain condition
• Cryptococcus neoformans
• Aspergillus flavus, parasiticus
Detection of toxigenic fungi
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Identification by microscope
Microbiological analysis
Immunochemistry
Molecular methods
Some methods for
pathogenic fungi detection
• Microsporon, Trichophyton, Epidermophyton,
Sporotrichum, can be cultured on Sabouraud
medium agar
• Candida albicans on object glass , add 10%
KOH, observed under microscope : mycelium
produced
• To differentiate Candida with other nonpathogenic yeasts : use selective media (cornmeal agar)