PHA6114LEC: MICROBIOLOGY AND PARASITOLOGY SIX (6) CLASSES IN KINGDOM FUNGI

CHAPTER 4: FUNGI 1. Oomycetes

 contains mildews and water moulds
YEAST MOULDS 2. Ascomycetes
GROWTH  grow as single cells  grow as masses of
MORPHOLOGY  produce hyphae under overlapping and  contains mildews, some moulds and most yeast species
specific conditions interlinking hyphal  e.g. Saccharomyes cerevisiae
 some yeast filaments 3. Basidiomycetes
 e.g. Candida albi-
cans  contains mushrooms and bracket fungi
REPRODUCTION ASEXUAL REPRODUCTION SPORE FORMATION 4. Teliomycetes
1. Budding
2. Binary Fission
 contains rust fungi
 minority of species  rust fungi – plant pathogen
 e.g. Schizosaccha- 5. Ustomycetes
romyces pombe
SEXUAL REPRODUCTION  contains smuts
SPORE FORMATION  smuts – plant pathogen
EXAMPLES Saccharomyces cerevisiae Penicillium
 brewer’s yeast chrysogenum
6. Deuteromycetes
 produces the  e.g. Aspergillus, Fusarium and Penicillium spp.
antibiotic
penicillin
FOUR (4) PHYLA IN KINGDOM FUNGI
FUNGI
1. Chyrtridiomycota
 eukaryotic organisms
2. Zygomycota
 cells possess a nuclear membrane 3. Ascomycota
 widely distributed in nature 4. Basidiomycota
 part of the normal flora on the body of warm - blooded
animals
 decomposer of organic matter FUNGAL CELL STRUCTURE
 animal and plant pathogen
 medically important group of microbes CELL WALL
 responsible for a number of potentially fatal diseases  contains a number of structural polysaccharides
in humans  Structural Polysaccharides – account for up to 25%
 beneficial to humanity of the dry weight of the cell wall
 e.g. production of alcohol beverages, bread,  Glucan – accounts for 50 – 60%
enzymes, antibiotics and recombinant proteins  Mannan – for 15-23%
 utilized for a range of molecular biological applications  Chitin – for 1-9%
 Protein and Lipids – present in smaller amounts
FUNGAL DISEASES AND CAUSATIVE AGENTS  Thickness: vary during the life of cell
 Candida albicans – Average Thickness: 100-300 nm
TYPE OF MYCOSIS DISEASE SPECIES NAME
SUPERFICIAL Pityriasis versicolor Malassezia furfur
Glucan
(small scaly discoloured  main structural component
patches of skin)  branched polymer of glucose
White piedra Trichosporon beigelii
(rare fungal infection of
 Three (3) Forms:
the hair shaft)  β-1, 6-glucan
CUTANEOUS Tinea pedis Trichophyton rubrum  β-1, 3-glucan
(athlete’s foot)
Onychomycosis Trichophyton rubrum
 β-1, 3, β-1, 6-complexed with chitin
(nail infection) Mannan
Tinea capitis Trichophyton  polymer of mannose
(scalp ringworm) tonsurans
 found in the outer layers of cell wall
SUBCUTANEOUS Chromoblastomycosis Fonsecaea pedrosoi
(long-term fungal Chitin
infection)  concentrated in bud scars
Mycetoma Acremonium spp.  bud scars – areas of the cell from which bud has
(chronic progressively
destructive morbid detached
inflammatory disease) Proteins and Lipids
SYSTEMIC Blastomycosis Blastomyces  represent up to 30% of the cell wall contents
dermatitidis
Mannoproteins
Histoplasmosis Histoplasma
capsulatum  form a fibrillar layer
Coccidiodomycosis Coccidioides immitis  Fibrillar Layer
Paracoccidiodomycosis Paracoccidioides  radiate from an internal skeletal layer
brasiliensis
OPPORTUNISTIC Aspergillosis Aspergillus fumigatus  formed by the polysaccharide component of the
Pneumonia P neumocystis jirovecii cell wall
(carinii) Glutan and Chitin
 found in innermost layer
ECONOMICALLY IMPORTANT FUNGI  provide rigidity to the wall
 regulate cell division
FUNGAL SPECIES APPLICATION
FILAMENTOUS FUNGI
Agaricus bisporus Mushroom (edible) PERIPLASMIC SPACE
Aspergillus, Penicillium spp. Enzymes  thin region
(catalase, lipase, amylase)  lies directly below the cell wall
Aspergillus sp. + Saccharomyces sp. Sake (rice wine)
Fusarium graminearum Single Cell Protein  contains secreted proteins
Penicillium chrysogenum Penicillin Production  Proteins
Penicillium notatum Enzyme (glucose oxidase)  do not penetrate the cell wall
Penicillium roqueforti Cheese Flavouring
(Roqueforti “blue” cheese)
 location for a number of enzymes required for
YEAST processing nutrients prior to entry into the cell
Pichia sp. Gene Expression System
Saccharomyces cerevisiae Gene Expression System PLASMALEMMA (CELL MEMBRANE)
Dietary Supplement
Bakers’ Yeast (bread)
 phospholipid bilayer
Brewer’s Yeast (beer, wine, cider,  Thickness: 10 nm
etc.)  located directly below the periplasmic space
Enzyme (invertase)
 contains phospholipids, lipids, protein and sterols
 contains globular proteins
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Ergosterol  arises when a weakened or immature immune system is
 dominant sterol in fungal cell membranes present
 target of antifungal agent amphotericin B 2. Genital
Sterols  Genital Candidosis
 important components of plasmalemma  very common
 represent regions of rigidity in the fluidity provided by the  Vulvovaginal Candidosis (VVC) – affects 75% of
phospholipid bilayer women during their life with a further 5 – 12%
suffering from recurring bouts of infection over a
NUCLEUS prolonged period of time
 repository of the DNA
 concentrated most of the cell’s genome ASPERGILLUS FUMIGATUS
 surrounded by a nuclear membrane  mould
 contains proteins in the form of histones  dominant fungal pulmonary pathogen of humans
Nuclear Membrane  presents as a problem in those with pre-existing lung
 contains pores disease or damage
 pores – allow communication with the rest of the cell Site of Infections
Chromosomes  depends upon level of immunocompromise of the
 vary in size individual
 Size: 0.6-6 Mb 1. Pulmonary
 vary in number 2. Brain
 S. cerevisiae – as many as 16 3. Kidney
 Sch. pombe – as few as 3 4. Sinuses
Groups Susceptible to Colonization
PLASMIDS  patients with:
 extrachromosomal information 1. cavities due to tuberculosis
2µm Plasmid 2. asthma or cystic fibrosis
3. profound immunosuppression due to leukemia
 present in S. cerevisiae
 Leukaemia – neutropenia
 function is unclear
Aspergillosis
Killer Plasmids
 presents as a serious problem in patients
 present in Kluyveromyces lactis
immunosuppressed in advance of organ transplantation
 encode a toxin
DERMATOPHYTE
MITOCHONDRION
 range of fungi capable of colonizing the skin, nails or hair
 “powerhouse” of the cell
Principal Dermatophytic Fungi
 semi -independent organelle 1. Tricophyton
 possesses its own DNA 2. Microsporum
 produces its own proteins on its own ribosomes 3. Epidermophyton
 Mitoribosomes – ribosomes of mitochondria Most Commonly Encountered Dermatophytic Infections
1. Athlete’s Foot
PART OF COMPOSITION/FUNCTION
MITOCHONDRION
 infection of the foot
Matrix where enzymes of Kreb’s cycle 2. Ringworm
(tricarboxylic acid cycle) are located  infection of the scalp or skin
Inner Membrane electron transport and oxidative and
phosphorylation
Outer Membrane where enzymes of lipid biosynthesis are
located ANTIFUNGAL THERAPY

RIBOSOMES POLYENE ANTIFUNGALS

 present in the form of polysomes  large macrolide ring of carbon atoms closed by the
 Polysomes – lines of ribosomes strung together by formation of an internal ester or lactone
a strand of mRNA  large number of hydroxyl groups distributed along the
 site of protein biosynthesis macrolide ring on alternate carbon atoms
 combination of highly polar and non – polar regions
GOLGI APPARATUS, ENDOPLASMIC RETICULUM AND  Polyenes – amphiphatic
PLASMALEMMA  Amphiphatic
 membranous compartments  hydrophobic and hydrophilic regions in the one
 mediates the export of proteins from the cell molecule
 assists solubility in lipid membranes
VACUOLE Principal Polyenes
 “storage space” 1. Amphotericin B
 where nutrients, hydrolytic enzymes or metabolic 2. Nystatin
intermediates are retained until required Amphotericin B
 produced by the bacterium Streptomyces nodosus
 binds ergosterol
MEDICAL SIGNIFICANCE OF FUNGI  Ergosterol – dominant sterol in fungal cell
membranes
THREE (3) CLASSES OF MOST COMMON HUMAN  consequently increases membrane permeability by the
FUNGAL PATHOGENS formation of pores
1. Yeasts  relies on the formation of pores
2. Moulds  Pores – intracellular contents can escape from cell
3. Dermatophytes  lead to renal damage during prolonged antifungal
therapy
C. ALBICANS  “gold standard”
 yeast  active against a broad range of fungal pathogens
 most frequently encountered human fungal pathogen  against which the activity of other antifungal agents is
 responsible for a wide range of superficial and systemic measured
infections  Renal Toxicity
Superficial Infections
 tends to be reserved for severe cases of systemic
1. Oropharyngeal
fungal disease
 occurs predominantly in HIV - positive individuals,  encapsulated within liposomes – reduced toxicity
geriatric patients and premature infants
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Nystatin  fungicidal effects to some species
 discovered in 1950  e.g. Aspergillus spp.
 exhibits the same mode of action as amphotericin B 1. Voriconazole
 tends to have lower solubility 2. Posaconazole
 restricted its use to the treatment of topical infections 3. Ravuconazole
Voriconazole
AZOLE ANTIFUNGALS  one of the newest second-generation triazole antifungal
First Generation of Azole Antifungals drugs
 revolutionized the treatment of mucosal and invasive  shows
fungal infections  good activity against pulmonary aspergillosis and
Azoles cerebral aspergillosis
 most widely used group of antifungal agents
Mode of Action ECHINOCANDINS
 interferes with ergosterol biosynthesis  relatively new group of antifungal drug
 binds to 14 - α – demethylase (P-450DM)  semisynthetic lipopeptides comprising a cyclic
 14-α-Demethylase – cytochrome P-450 hexapeptide core connected to a lateral fatty acid chain
mediated enzyme Mode of Action
 blocks the formation of ergosterol by preventing the  targets the synthesis of β-1,3-glucan
methylation of lanosterol  β-1,3-Glucan – major polymer of the fungal cell wall
 Lanosterol – precursor of ergosterol  Cell Wall
 reduces amount of ergosterol  provides physical protection
 leads to membrane instability, growth inhibition,  maintains osmotic stability
cell death and build-up of toxic intermediates  regulates cell shape
 Build-up of Toxic Intermediates – fatal to  acts as a scaffold for proteins
fungal cell  mediates cell-cell communication
 exhibits broad spectrum of activity in vitro  site of a number of enzymatic reactions
 inhibits the growth of most Candida, Cryptococcus  disrupts the structure of the growing cell wall
and Aspergillus species, and dermatophytes  results in osmotic instability and ballooning out of
Azole Derivatives the intracellular contents due to high osmotic
 imidazoles or triazoles pressure
 two or three nitrogen atoms in the five- membered azole  ends in cell lysis
ring  offers safer alternative to conventional antifungal
Azoles in Current Use therapies
1. Clotrimazole  e.g. Polyenes, Azoles
2. Miconazole  displays a unique mode of action which results in defects
3. Econazole in cell wall morphology and osmotic instability
4. Ketoconazole  results in the efficient destruction of the fungal cell as the
Newer Azoles cell wall is an essential component for stability and
 important applications in the treatment of systemic ultimately virulence
infections Echinocandins in Current Use
1. Itraconazole 1. Caspofungin
2. Fluconazole 2. Micafungin
3. Voriconazole 3. Anidulafungin
Miconazole Caspofungin
 first azole used to treat systemic fungal infections  demonstrated in vitro antifungal activity against various
 demonstrated a number of toxic side effects filamentous fungi and yeasts
Ketoconazole  activity against different Aspergillus species
 produced high serum concentrations upon oral  e.g. A. fumigatus, A. flavus, A. niger and A. terreus
administration  considered to be more fungistatic than fungicidal
 poor activity against aspergillosis  particularly fungicidal against a range of Candida
 associated with a range of side effects which limited its species:
applicability  resistant to azoles
Fluconazole  e.g. C. krusei
 introduced for clinical use in 1990  less susceptible to azoles
 water-soluble  e.g. C. dubliniensis, C. glabrata
 shows good penetration and deposition into the  resistant to amphotericin B
pulmonary tissues
 reaches high levels in the cerebrospinal fluid and the SYNTHETIC ANTIFUNGAL AGENTS
peritoneal fluids Flucytosine
 proved highly effective in the treatment of infections  synthetic fluorinated pyrimidine
caused by C. albicans  used as an oral antifungal agent
 showed limited activity against Aspergillus  demonstrated good activity against a range of yeast
Itraconazole species and moderate levels of activity against
 introduced for clinical use in the late 1980’s Aspergillus species
 first azole with proven efficacy against Aspergillus  Yeast Cells
 effective in treating severe Aspergillus infections  increase in size when exposed to levels of flucytosine
 exhibits both fungicidal and fungistatic effects lower than the minimum inhibitory concentration
 undergoes extensive hepatic metabolism which yields up (MIC)
to 30 metabolites  display alterations in their surface morphology
 30 metabolites – number of which retain antifungal  Fungi
activity  develop resistance after a relatively short exposure
 available as an intravenous formulation  attributed to alteration of cytosine deaminase
 Intravenous – widely used for the treatment of  Cytosine Deaminase – enzyme required to
severe Aspergillus infection in this form process flucytosine once inside the cell or to an
Fluconazole and Itraconazole elevation in the amount of pyrimidine synthesis
 demonstrated significantly reduced side effects  Problem of Resistance
compared to ketoconazole  limited the use of flucytosine
Novel Azole Drugs  used in combination with an antifungal agent
 increased ability to inhibit the fungal 14-α demethylase  e.g. Amphotericin B
 wider spectrum of activity than fluconazole  potentiates effect of second agent
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Two (2) Modes of Action
1. disruption of protein synthesis by the inhibition of
DNA synthesis
2. depletion in the amino acid pools within the cell as a
result of inhibition of protein synthesis
 allow yeast to exploit microniches in the body and
alters a variety of factors in addition to the actual
phenotype
 e.g. antifungal drug resistance, adherence,
extracellular enzyme production

MEDICALLY IMPORTANT HUMAN FUNGAL PATHOGENS Process of Tissue Colonization and Penetration
1. Adherence
CANDIDA ALBICANS  initial step in the process
C. albicans 2. Enzymes (Phospholipase and Proteinase)
 yeast  facilitate adherence by damaging or degrading cell
 opportunistic fungal pathogen membranes and extracellular proteins
 present as a normal part of the body’s microflora 3. Hyphae
Systemic Infections  penetrate layers of cells using thigmotropism to find the
 start as superficial infections line of least resistance
1. Gastrointestinal Tract 4. Extracellular Enzymes
 e.g. diabetics, cancer patients and people with AIDS  aid passage through cells
 Oesophagus 5. Enzymes
 common site of infection  assist in the degradation of tissue and allow the yeast to
 render swallowing difficult enter the host’s blood stream once endothelial cells are
2. Urinary Tract reached
 e.g. renal infection, other underlying disease(s) or 6. Phenotypic Switching or Coating
cystitis  evade the immune system
Predisposing Factors 7. Haemolysin
 factors which impair the host’s immune system  burst blood cells and release iron which is essential for
1. Presence of Underlying Disease growth
2. Use of Immunosuppressive Therapy
 e.g. organ transplantation, broad-spectrum antibiotic ASPERGILLUS FUMIGATUS
therapy A. fumigatus
3. Presence of Indwelling Catheters  saprophyte
4. Presence of Skin Damage  opportunistic pathogen
 e.g. burns, other trauma  present in decaying vegetation and damp surfaces
Virulence Factors  commonest etiological agent of pulmonary aspergillosis
1. Ability to adhere to host tissue using a variety of  Pulmonary Aspergillosis
mechanisms (Adherence)  Cases: 80-90%
 ability to adhere to various mucosal surfaces and to  Incidence: < less than Candida
withstand forces that may lead to its removal from the  Mortality Rates: > greater than Candida
body Virulence Factors
 e.g. bathing/washing action of body fluids 1. Extracellular Enzyme Production
 C. albicans and C. tropicalis  facilitate growth in the lung and dissemination through
 more common etiological agents of candidosis the body
 more adherent to tissue in vitro  Phospholipase
 C. krusei and C. guilliermondii  Optimal Production: 37°C
 less adherent non-pathogenic species  plays critical role in tissue degradation and may
2. Existence of two morphologically distinct forms facilitate exit of the fungus from the lung into the
(Budding Blastophores and Hyphae) bloodstream
 ability to switch between each form and is usually 2. Protease Production
encountered in tissue samples in both morphological  degrade tissue
forms  neutralize immune system
 Hyphae  allow the fungus to degrade animal and plant material
 capable of thigmotropism  Elastin
 Thigmotropism  constitutes 30% of lung tissue
 contact sensing  incapability of elastinolytic activity = ↓ reduced
 aid in finding the line of least resistance between virulence
and through layers of cells in tissue  Serine Protease and Metalloproteinase
3. Extracellular Enzyme Production  elastinases produced by A. fumigatus
 facilitate adherence and/or tissue penetration  Serine Protease
 Phospholipases A, B, C and Lysophospholipase  functions as allergen
 damage host cell membranes and facilitate invasion  important in the induction and persistence of
4. Acid Proteinase Production allergic aspergillosis
 aid adherence and invasion  Local Inflammation
 play an important role in the degradation of the  due to presence of proteases
immunoglobulins IgG and IgA  results in airway damage
 Secreted Aspartic Family  induce epithelial cell detachment from basement
 10 members membranes
 low pH optimum
 assist in the colonization of the vagina
5. Haemolysin Production
 allow the yeast to access iron released from ruptured red
blood cells
6. Ability to bind to platelets via fibrinogen-binding ligands
 surround fungal cell in cluster of platelets
7. Capability to give rise to variety of interconvertible
phenotypes
 provide extra dimension to the existing virulence factors
 Phenotypic Switching
 switching system
 “dominant” or “controlling virulence factor”
 compensate for the lack of variation achieved in other
organisms that utilize sexual reproduction

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