1. WHAT ARE FUNGI?

 Protein & Lipids – it may represent up to

 Fungi are eukaryotic organism. Type of Mycosis Disease Species Name 30% of the cell wall.
 Penicillium chrysogenum which produces Superficial Pityriasis versicolor Malassezia furfur  Mannoproteins – a fibrillar layer that
the antibiotic penicillin are classified as White piedra Trichosporon beigelii radiates from an internal skeletal layer.
fungi. Tinea pedis (athlete’s Trichopyton rubrum (formed by polysaccharide components)
 Yeast Cells: Cutaneous foot)  Innermost layer – is rich in glucan and
o Grow as single cells which reproduce Onychomycosis (nail Trichopyton rubrum chitin that provides rigidity to the cell wall
asexually. (budding) infection) and for regulating cell division.
Tinea capitis (scalp Trichopyton tonsurans
o Some reproduce by fission. (ex.  Periplasmic Space – thin region that is
ringworm)
Schizosaccharomyces pombe) directly below the cell wall.
Subcutaneous Chromoblastomycosis Fonsecaea pedrosoi
o Many are capable of sexual o Contains secreted proteins that do
Mycetoma Acremonium spp.
reproduction and the formation of not penetrate the cell wall.
Blastomycosis Bastomyces
spores.
Systemic dermatitidis  Cell Membrane / Plasmalemma
 Moulds: Histoplasmosis Histoplasma o directly below the periplasmic space.
o Grows as masses of overlapping and capsulatum o Phospholipid bilayer
interlinking hyphal filaments. Coccidioidomycosis Coccidioides immitis o Contains lipids, proteins, sterol,
 Kingdom Fungi can subdivide: Paracoccidioidomycosis Paracoccidiodes phospholipids.
 Phylogenetic Analysis brasiliensis o Approx. 10 nm thick
o Chytridiomycota Candidosis Candida albicans o It contains Globular proteins.
o Basidiomycota (superficial/systemic) Candida glabrata  Ergosterol – the dominant sterol and
o Ascomycota Opportunistic Candida parapsilosis targeted by antifungal agent Amphotericin
o Zygomycota Aspergillosis Aspergillus fumigatus B.
Pneumonia Pneumocystis jirovecii  Sterols represent region of rigidity in the
Classes Examples (carinii) fluidity provided by phospholipid bilayer.
Oomycetes Mildews & Water  Nucleus – discrete organelle and
Moulds respiratory of the DNA that contains
Ascomycetes Mildews, Moulds, 2. STRUCTURE OF THE FUNGAL CELL
proteins (histones).
Yeast
 Yeast Cell is oval and surrounded by rigid  Cell’s Genome- it is in nucleus which
Basidiomycetes Mushrooms, Bracket
surrounded by a nuclear membrane that
Fungi cell wall.
contains pores for communication all over
Teliomycetes Rust Fungi (plant o Polysaccharides – 25%
the cell.
pathogens) o Glucan – 50-60%
o Mannan – 15-23%  Yeast chromosomes sizes from 0.2 to 6
Ustomycetes Smuts (plant o Chitin – 1-9% Mb.
pathogens)
o Proteins & Lipids – smaller amount  Yeast Cell has an extrachromosomal
Deuteromycetes Aspergillus, Fusarium, information. (plasmids)
 Glucan – main structural component of
Penicillium  Kluyveromyces lactics – killer plasmids of
fungal cell wall and a branched polymer of
glucose which are: yeast which encodes toxins.
o B-1, 6-glucan  Mitochondrion
o B-1, 3-glucan o Powerhouse of the cell.
o B-1, 3, -B-1, 6-complexed with chitin. o Tricarboxylic acid cycle is in the
 Mannan – polymer of the sugar mannose matrix.
and it is in the outer layer of the cell wall. o Possesses its own DNA
 Chitin – concentrated in bud scars. (area o Producing its own ribosomes
where bud has detached) and proteins.
  Electron Transport & Oxidative  superficial & systemic infections standard’ against which the activity of other
Phosphorylation – located in the  superficial infections include: antifungal agents is measured.
mitochondrial inner membrane. oropharyngeal and genital conditions  Nystatin was discovered in 1950; lower solubility,
 Lipid Biosynthesis – is involved outer  genital candidosis: very common and which has restricted its use to the treatment of
membrane. approx. 75% of women are affected by topical infections.
 Fungal Cells contains number of vulvovaginal candidosis (VVC)  Use has been overtaken by the introduction
ribosomes in the form of polysomes-lines.  mould Aspergillus fumigatus of azole antifungal drugs (oral and vaginal
(strung by mRNA)  dominant fungal pulmonary pathogen candidosis)
 Ribosomes- site of protein biosynthesis.
 present problems in those with pre- 4.2 Azole antifungals
 Membranous Compartment:
existing lung disease or damage  first generation of azole antifungals -treatment of
o Golgi Apparatus
 brain, kidney, and sinuses may also be mucosal and invasive fungal infections
o Endoplasmic Reticulum
o Plasmalemma
affected  The azole derivatives are classified as imidazoles
o Vacuole (Nutrient, hydrolytic  aspergillosis presents as a serious or triazoles (two or three nitrogen atoms in the
enzymes/metabolic problem in patients immunosuppressed five- membered azole ring)
intermediates are retained) in advance of organ transplantation  The azoles in current clinical use are clotrimazole,
 dermatophyte miconazole, econazole and ketoconazole; newer
Fungal Species Application  capable of colonizing skin, nails, and hair drugs such as itraconazole, fluconazole and
Filamentous fungi  principal dermatophytic fungi: voriconazole- treatment of systemic infections.
Agaricus bisporus Edible Mushroom
Trychophyton, Microsporum,  Azoles interfere with ergosterol biosynthesis-
Aspergillus, Enzymes (catalase, lipase, amylase)
Epidermotophyton species binding to the cytochrome P-450 mediated
Penicillium spp.
Aspergillus sp. + Sake (rice wine)  most commonly encountered infection: enzyme known as 14-α-demethylase (P-450 DM)-
Saccharomyces sp. athlete's foot and ringworm blocks the formation of ergosterol by preventing
Fusarium Single cell protein 4 ANTIFUNGAL THERAPY the methylation of lanosterol (a precursor of
graminearum 4.1 Polyene antifungals ergosterol)
Penicillum Penicillin production  Polyene antifungals- large macrolide ring of  result in a reduction in the amount of
chrysogenum carbon atoms closed by the formation of an ergosterol in the fungal cell membrane which
Penicillum notatum Enzyme (glucose oxidase)
internal ester or lactone leads to membrane instability, growth
Penicillum Cheese flavoring (Roqueforti ‘blue’
roqueforti cheese)  amphipathic, hydrophobic and hydrophilic regions inhibition and cell death.
Yeast in the one molecule, solubility in lipid membranes.  An additional consequence- build-up of toxic
Pichia sp. Gene expression system  principal polyenes- amphotericin B and nystatin. intermediates which can prove fatal to the
Bakers’ yeast – bread  Amphotericin B- by Streptomyces nodosus- bind fungal cell
Brewers’ yeast – beer, wine, cider, ergosterol, (dominant sterol), increases  Azoles exhibit a broad spectrum of activity in vitro,
Saccharomyces etc. membrane permeability by the formation of pores being capable of inhibiting the growth of most
cervisiae Enzyme (invetase) (intracellular contents can escape) Candida, Cryptococcus and Aspergillus species,
Gene expression system
 renal damage during prolonged antifungal and dermatophytes.
Dietary supplement
therapy, reserved for severe cases of  Miconazole- first azole used to treat systemic
3 MEDICAL SIGNIFICANCE OF FUNGI
systemic fungal disease but recent fungal infections; number of toxic side
formulations in which the drug is effects.
 most common fungal pathogens of humans:
encapsulated within liposomes have been  Ketoconazole produced high serum
yeasts, moulds, dermatophytes
shown to have reduced toxicity. concentrations upon oral administration;
 yeast C. albicans
 active against a broad range of fungal poor activity against aspergillosis; range of
 most frequently encountered human
pathogens and is considered the ‘gold side effects which limited its applicability.
fungal pathogen
  Fluconazole was introduced for clinical use in
1990, water soluble, good penetration and
deposition into the pulmonary tissues; it also
reaches high levels in the cerebrospinal fluid
and the peritoneal fluids.
 Fluconazole highly effective in the treatment
of infections caused by C. albicans but shows
limited activity against Aspergillus.
 Itraconazole became available for clinical use
in the late 1980s and was the first azole with
proven efficacy against Aspergillus; effective
in treating severe Aspergillus infections and
exhibits both fungicidal and fungistatic
effects.
 Upon ingestion, itraconazole undergoes
extensive hepatic metabolism which yields
up to 30 metabolites, a number of which
retain antifungal activity.
 Itraconazole is currently available as an
intravenous formulation and is widely used
for the treatment of severe Aspergillus
infection in this form.
 Fluconazole and Itraconazole demonstrate
significantly reduced side effects compared
to ketoconazole.
 Novel azole drugs with increased ability to inhibit
the fungal 14-α-demethylase are also becoming
available. These agents, which include
voriconazole, posaconazole and ravuconazole,
have a wider spectrum of activity than fluconazole
and it has been suggested that some of them show
fungicidal effects to some species (e.g. Aspergillus
spp.).
4.3 Echinocandins
 The echinocandins- new group of antifungal drug
and are semisynthetic lipopeptides comprising a
cyclic hexapeptide core connected to a lateral
fatty acid chain. Three compounds of this group
are currently in use: caspofungin, micafungin and
anidulafungin
 echinocandins target the synthesis of β-1,3-
glucan, the major polymer of the fungal cell wall.
The cell wall provides physical protection,
maintains osmotic stability, regulates cell shape,
acts as a scaffold for proteins, mediates cell–cell
communication and is the site of a number of
enzymatic reactions.
 Inhibition of β-1,3-glucan synthesis disrupts the
structure of the growing cell wall, resulting in
osmotic instability and ballooning out of the
intracellular contents as a result of high osmotic
pressure, and ultimately ends in cell lysis.
 Caspofungin has demonstrated in vitro antifungal
activity against various filamentous fungi and
yeasts; activity against different Aspergillus
species including A. fumigatus, A. flavus, A. niger
and A. terreus; more fungistatic than fungicidal.
 caspofungin is particularly fungicidal against a
range of Candida species including species that
are resistant (e.g. C. krusei) or isolates that are less
susceptible (e.g. C. dubliniensis, C. glabrata) to
azoles, or resistant to amphotericin B.
 fungal cell wall represents an attractive target and
the echinocandins- safer alternative to
conventional antifungal therapies. (i.e. polyenes
and azoles).
 Echinocandins- unique mode of action which
results in defects in cell wall morphology and
osmotic instability. The targeting of the wall by
echinocandins results in the efficient destruction
of the fungal cell
4.4 Synthetic antifungal agents
 Flucytosine- synthetic fluorinated pyrimidine
which has been used as an oral antifungal agent
and good activity against a range of yeast species
and moderate levels of activity against Aspergillus
species.
 Two modes of action have been proposed for
flucytosine: disruption of protein synthesis by the
inhibition of DNA synthesis & the depletion in the
amino acid pools within the cell as a result of
inhibition of protein synthesis.
 yeast cells increase in size when exposed to levels
of flucytosine lower than the minimum inhibitory
concentration (MIC) and display alterations in
their surface morphology: a result of an imbalance
in the control of cellular growth.
 Many fungi are inherently resistant to flucytosine
or develop resistance after a relatively short
exposure and resistance has been attributed to
alteration in the enzyme (cytosine deaminase)
required to process flucytosine once inside the cell
or to an elevation in the amount of pyrimidine
synthesis.
 The problem of resistance has limited the use
of flucytosine so that now it is generally in
combination with an antifungal agent
(e.g.amphotericin B) where it can potentiate
the effect of the second agent.
5 MEDICALLY IMPORTANT FUNGAL PATHOGENS OF
HUMANS
5.1 Candida albicans
- opportunistic fungal pathogen
- normal part of the body’s microflora
- responsible for a range of systemic infections,
starting off as superficial infections
- has a variety of virulence factors that aid
colonization and persistence in the body:
o ability to adhere to host tissue (e.g.
mucosal surfaces) to withstand forces
that may lead to its removal
- can exist in two morphologically distinct forms:
o budding plastophores
o hyphae - capable of thigmotrophism
(contact sensing), aid in finding the line
of least resistance between and
through layers of cells
- capable of giving rise to interconvertible
phenotypes
Infection of gastrointestinal tracts is seen in:
 - diabetics
- cancer patients
- people with AIDS
Oesophagus – common site of infection; renderng
swallowing difficult
Urinary Tract – site of candidosis, due to renal infection,
other underlying issues, or cystitis
Factors which impair the host’s immune system:
- presence of underlying diseases (AIDS, cancer,
diabetes)
- use of immunosuppressive therapy during organ
transplantation
- broad-spectrum antibiotic therapy
- presence of indwelling catheters and skin
damage (predispose to Candida infection)
More common etiological agents of candidosis
- C. albicans
- Candida tropicalis
- More adherent to host tissue in-vitro
Phospholipases A, B, C, and lysophospholipase
- may function to damage to host cell membranes
and facilitate invasion
- extracellular enzyme produced by C. albicans
Acid proteinases
- aid adherence and invasion but also play a
significant role in the degradation of the
immunoglobulins IgA and IgG.
Haemolysin production of C. albicans
- important in allowing the yeast access iron
released from ruptured red blood cells.
Important immune invasion tactic of C. albicans
- ability to bind to platelets via fibrinogen binding
ligands, resulting to fungal cells being surrounded
by platelets.
Phenotypic switching
- allows the yeast to exploit microniches in the
body
- alters a variety of factors (e.g. antifungal drug
resistance, adherence, extracellular enzyme
production
- dominant and controlling virulence factor
5.2 Aspergillus fumigatus
- saprophytic fungus
- growing on decaying vegetation and damp
surfaces
- opportunistic pathogen
- most common etiological agent of pulmonary
aspergillosis
- Aspergillosis results in greater mortality rates
- Produces two elastinases:
o Serine protease – function as allegens;
important in the conduction and
persistence of allergic aspergillosis
o Metaloproteinase
- Proteases induce the release of the
proinflammatory IL-6 and IL-8 cytokines, which
may induce mucosal inflammatory response and
subsequent damage to the surrounding tissue
Phospholipase
- plays a critical role in tissue degradation
- facilitate exit of the fungus from the lung into the
bloodstream
Fungal Protease
- responsible for tissue degradation; and
- neutralization of the immune system
Elastin – constitutes almost 30% of lung tissue and many A.
fumigatus isolates display elastinolytic activity.
Production and secretion of toxins into surrounding tissues
- important virulence attribute and may facilitate
fungal growth in the lungs.
Gliotoxin – main toxin produced by A. fumigatus;
facilitating the colonization of the lung, by retarding
elements of the local immunity
5.3 Histoplasma capsulatum
- dimorphic fungus
- cause of histoplasmosis
o Histoplasmosis – most common fungal
pulmonary infection in the USA;
common amongst AIDS patients
- Grows in the mycelial form in soil
- Round, budding cells in tissues
- Releases large number of airborne spores that
establish pulmonary infection upon inhalation
o Pulmonary macrophages engulf the
yeast cells and provide a protected
environment for the yeat to multiply
and disseminate from the lungs to
other tissues
- Ability to survive within the hostile environment –
key to the success of H. capsulatum
40-50% - mortality rate among infants exposed to a large
dose of H. capsulatum
Chronic pulmonary histoplasmosis
- seen in men between the age of 45-55
- exposed to low levels of spores for a long period
of time
- progressive; leading to a loss of lung function and
death, if untreated
5.4 Cryptococcus neoformans
- encapsulated yeast
- with AIDS patients, meningitis is the most
common clinical manifestation
- facultative intracellular pathogen
- capable of surviving and replicating within
macrophages and withstanding lytic activity
- produces melanin
o ability to bind and protect against
microbicidal peptides
o melanization – allow the cell to
withstand the effects of harmful
hydroxyl radicals
- Capsule of C. neoformans – virulence factor that
protects the cell from the immune response
- Capsular material (Glucuronoxylomannans) –
induces the shedding of host cell adherence
 molecules required for mitigation of host
inflammatory cells to the site of function
SACCHAROMYCES CEREVISIAE
Cryptococcal meningitis
- associated with AIDS patients
- may be controlled by antifungal therapy
- there is a danger of relapse unless antifungal
therapy is constantly maintained
5.5 Dermatophytes
- keratinophilic fungi (can metabolize keratin)
Tinea capitis
- infection of the hair and scalp
- Microsporum canis or Trichophyton violaceum
- Characteruzed by a mild scaling and loss of hair
Tinea corporis
- infection of the trunk, leg, and arms
- Trichophyton spp., Microsporum sp., or
Epidermophyton floccosum
- Lesions are itchy, dry and show scaling
- Lesions retain a citucular morphology (Ringworm)
Tinea cruris
- infection of the skin of the groin
- Epidermophyton floccosum or Trichophyton
rubrum
- Highly cintagious via fomites (towels, sheets, etc.)
Tinea pedis
- infection of the feet
- Epidermophyton floccosum or Trichophyton
rubrum
Tinea manuum
- fungal infection of the hands
- Trichophyton mentagrophytes
- Seen in association with eczema
*both modulate immune system response sa infection;
Tinea unguium
allow degradation ng cell membrane*
- infection of the fingernails or toenails
Brewing strains of S. cerevisiae
- often descibed as onychomycosis
- associated with psoriasis
6 EMERGING FUNGAL PATHOGEN
- Widely distributed in nature
- Cause systemic/ mild systemic infection
- Impairment of immune system
- Causes vulvovaginitis in women (5% case)
- Sole pathogen
o Responsible for:
 Pneumonia
 Septicaemia
 Induction of fever & cough in transplant
patient
 Widespread dissemination in AIDS patient
 Postoperative peritonitis
o Used as:
 production of bread “Brewer’s
yeast//Baker’s Yeast
 alcoholic beverages
 dietary supplements
*NO longer in GRAS (generally regarded as safe) and
now classified as “Biosafety Level 1 Pathogen”*
ISOLATES
- Ability to grow Pseudohyphal form (penetration
of tissue)
- Facilitate their persistence & dissemination in
the host
*42C (greater upper temp range for brewing yeast)---
febrile patients( may fever )*
EXTRACELLULAR ENZYME
1. Acid proteinase
2. phospholipases
- Flocculate at the end of fermentation
- Also seen in isolates (obstructing capillaries
mostly sa brain)
Pathogenic Isolates
- Adhere to epithelial tissue via proteinaceous
adhesin
- Critical to yeast para di mabilis mawwash away
when swallowing
Virulence: ability to alter phenotype
Azole & Polyenes
- Conventional therapy for superficial/systemic
infection
*clinical isolates high level of resistance sa
fluconazole*
NON-ALBICANS CANDIDA SPECIES
 Candida dubliniensis
- Oral candidosis
- Normally green  Oropharyngeal candidosis;
but now diff shade in carbohydrate assimilation
& DNA restriction patterns
- Previously unrecognized pathogen to emerge
out-compete the perceived dominant pathogen
 C. krusei
- Before: harmless, transient eomensal found in
the environment or in human body
- Now: both solid tumor & leukemia; can
colonize gastrointestinal respiratory & urinary
tract
Virulence: reduced ability to adhere to epithelial cell
compared to C. albicans (even tho high cell surface
hydrophobicity to stick).
- High level to colonize catheters & inherent
resistance implants to fluconazole
 C. glabrata
- Serious cause of disease neutropenic cancer
patients (Mortality rate: 5-38%)
- Late stages of haematological malignancies
(Mortality rate: 70-100%)
- 4TH most commonly isolated C. spp
 - Common in leukemia patients & bone marrow  Spleen inflammation high intracellular levels and retards
recipients *fatal if not treated* production or kills the cell
*yeast relatively low virulence but emergence a  to reverse, amphotericin B can be introduced
*Tuberculosis & Pneumonia seen along P. marneffei*
serious pathogen* in low levels which increases membrane
7 ANTIOBIOTIC PRODUCTION BY FUNGI permeability, leading to decrease in
*resistance in fluconazole*
 Penicillium notatum intracellular antibiotic levels and a
Azole prophylaxis  discovered by Fleming in 1929 concomitant increase in production
 produced substance capable of killing Gram-  antibiotic production
- Eliminates C. albicans but not C. krusei  maximized by optimizing production as result
positive bacteria
& C. glabrata of random mutagenesis selection
 first member of B-lactam class of antibiotics
Risk factor:  another approach: to fuse or mate high-
 function by inhibiting peptidoglycan
synthesis in bacteria producing strains with good secretors
1. Prolonged hospitalization
 Penicillium chrysogenum  rational selection
2. Use cytotoxic drug
 superior producer process where a chelating agent is introduced into the
3. Catherization
 following a series of mutagenic and selection fermentation to complex all the metal ions present (has
PENICILIUM MARNEFFEI
beneficial effect on antibiotic production)
procedures the strain used in conventional
- Most frequent cause of fungal disease in AIDS fermentation: can produce penicillin at rate
patients that visited or live in Southeast Asia of 7000mg/L compared to 3mg/L of P.
- 1st: China & Thailand notatum
- 2nd: Taiwan, Hong Kong, Malaysia, Japan  typical penicillin fermentation: yields 3 types:
- Soil/decaying vegetation F, G, and V
- Asexual, dimorphic fungus growing as mycelium at  G is modified by action of penicillin acylase to
37C(tissue) or 28C(single cell) give variety of penicillins which shows
- Reproduction: fission resistance to action of bacterial penicillases
o Monoclonal antibody based assays  majority of antibiotics from fungi
-detection of mannoproteins associated  produced via fermentation
w/ P. marneffei  most are 20 metabolites
- hard to distinguish between histoplasma  occurs in stationary phase
or Cryptococcus  linked to sporulation
o PCR (polymerase chain rxn) fingerprinting  catabolite repression
-identity & distinguish infecting solution  can inhibit antibiotic production (avoided by
using low levels of glucose, either in the
Infection in humans: Inhalation / Aids
fermentation medium or to obtain mutant
*pulmonary problems can be seen in AIDS patients which is not catabolite repressed)
but more affected with people with the ff  chemical content of medium
conditions:  must be monitored since high levels of
nitrogen or phosphate retard antibiotic
 Anemia
production
 Skin lesion
 feedback inhibition
 Fever
 affects productivity of antibiotic
 Liver
fermentation, where the antibiotic builds to
 Weight loss