Pharmaceutical Microbiology

Chapter 4
Antifungal agents, Groups,
Mechanisms of action and resistance
 Structure of the Fungal Cell…
• Fungal cell wall:
• The cell wall which contains a number of structural
polysaccharides and may account for up to 25% of the dry
weight of the cell wall.

• Glucan accounts for 50–60%, mannan for 15–23% and chitin

for 1–9% of the dry weight of the wall, respectively, with
protein and lipids also present in smaller amounts.

• Glucan, the main structural component of the fungal cell wall,

is a branched polymer of glucose which exists in three forms
in the cell, that is, β-1,6- glucan, β-1,3-glucan and β-1,3–β-1,6-
glucan complexed with chitin.
 …Structure of the Fungal Cell…
• Mannan is a polymer of the sugar mannose and is found in
the outer layers of the cell wall.

• The third principal structural component, chitin, is

concentrated in bud scars which are areas of the cell from
which a bud has detached.

• Proteins and lipids are also present in the cell wall and under
some conditions may represent up to 30% of the cell wall
contents.
 …Structure of the Fungal Cell…
• Mannoproteins form a fibrillar layer that radiates from an
internal skeletal layer which is in turn formed from the
polysaccharide component of the cell wall.

• The innermost layer is rich in glucan and chitin, which

provides rigidity to the wall and is important in regulating cell
division.

• The periplasmic space is a thin region that lies directly below

the cell wall. It contains secreted proteins that cannot pass
out through the wall and is thus the location for a number of
enzymes required for processing nutrients prior to their entry
into the cell core.
 …Structure of the Fungal Cell
• Plasma membrane:
• It is located directly underneath the periplasmic space and is a
phospholipid bilayer which contains phospholipids, lipids,
protein and sterols.

• The plasmalemma is approximately 10 nm thick and, in

addition to being composed of phospholipids, also contains
globular proteins.

• The dominant sterol in fungal cell membranes is ergosterol

which is the target of the antifungal agent amphotericin B.
Sterols are important components of the plasmalemma and
create regions of rigidity within the fluidity provided by the
phospholipid bilayer.
 Antifungal agents…
• Finding suitable fungal targets for chemotherapy is difficult
because fungi, like humans, are eukaryotes. Many of the
cellular and molecular processes are similar, and there is often
extensive homology among the genes and proteins of fungi
and their mammalian hosts.

• The increasing number of antifungal drugs are becoming

available, and additional compounds are currently under
evaluation in clinical trials. Most have limitations, such as
significant side effects, a narrow antifungal spectrum, poor
penetration of certain tissues, and the development of
resistance.
 …Antifungal agents
• Antifungal agents classes:
1. Polyene Macrolide.
2. Azole Drugs.
3. Echinocandins.
4. Allylamines.
 Polyene Macrolide…
• They represent a class of biologically active fungal metabolites
isolated from the genus Streptomyces.

• More than 100 polyene antibiotics have been described,

amphotericin B and nystatin are the two agents most
commonly used to treat fungal and some protozoal infections
in humans.

• Other polyene macrolide agents: Griseofulvin, 5-Fluorocytosine

(Flucytosine).
 …Polyene Macrolide…
Agent Nystatin Griseofulvin Flucytosine
Source Produced by Produced by a Fluorinated
Streptomyces species of derivative of
noursei Penicillium cytosine
Mechanism of Bind ergosterol Binding Antimetabolite
action in fungal cell microtubular converted
membrane, alters proteins, which are to fluorouracil,
the selective required for perturbing
permeability of this mitosis synthesis of
membrane pyrimidines and
RNA
Usage Used locally (topical Oral agent used to Used in
agent )to treat oral treat combination
or vulvovaginal dermatophytoses, therapy for treating
candidiasis. which are not infections
responsive to azole by Candida spp.
antifungal therapy. and Cryptococcus
spp.
 …Polyene Macrolide…
• Amphotericin B:
• Amphotericin B is produced by the actinomycete
Streptomyces nodosus.

• It is commonly infused intravenously to treat deep-seated

fungal infections (e.g., invasive aspergillosis) and those caused
by Candida spp., Cryptococcus spp.

• Resistance to amphotericin B is uncommon but it is increasing

in the context of emerging pathogens, such as Candida
lusitaniae, and species of Aspergillus, Fusarium,
Scedosporium, and Trichosporon.
 …Polyene Macrolide…
• Mechanism of action: acts mainly at the plasma membrane,
and impairs membrane barrier function.

• Susceptibility to polyenes depends on membrane structure,

Sterols are essential components of eukaryotic cells, and
ergosterol is the principal sterol in the fungal cell membrane.

• Role of ergosterol in fungal cell membrane: it serves as a

bioregulator of membrane fluidity, and of membrane
integrity and permeability. Ergosterol also has a role in active
growth phases of fungal cells.
Amphotericin B structure
 …Polyene Macrolide…
• The antifungal effects of amphotericin B are believed to be by
two primary mechanisms:
1. Binding sterols in cellular membranes and the formation of
membrane pores causes altered membrane permeability and
leakage of potassium ions, and of other vital cytoplasmic
components, leading to membrane disruption, and possible
fungal cell death.

2. Pro-oxidant effect: cell membrane damage is due to the

formation of reactive oxygen species (ROS), such as superoxide,
hydrogen peroxide, and hydroxyl radicals, that results in
membrane disruption and cell death through membrane lipid
peroxidation.
Amphotericin B: mechanism of action
 …Polyene Macrolide…
• Mechanisms of Resistance:
• Resistance can be categorized into three main categories:
1. Primary or Intrinsic: occurs without exposure to antifungal
agents, rare among pathogenic fungi infecting humans.

2. Acquired: develops during treatment, and often occurs as a

result of one or several genetic mutations.

3. Clinical resistance: less common.

 …Polyene Macrolide…
• Mechanisms of resistance to polyenes include alterations in
membrane sterols, defense mechanisms against oxidative
damage, defects in ergosterol biosynthetic genes, factors such
as fatty acid composition of the cell membrane, and
alterations in sterol-to-phospholipid ratio.

1. Alterations in the sterol content: occur in different ways: the

total ergosterol content of the fungal cell can be decreased
without concomitant changes in the sterol composition.
Some or all of the polyene-binding sterols may be replaced
by sterol intermediates, such as fecosterol or episterol, which
bind polyenes less well.
 …Polyene Macrolide…
2. Genetic alterations in the ergosterol biosynthetic pathway or
ERG genes have been shown to decrease sensitivity to
polyenes and azoles.

3. Resistance to oxidation: it is involved in the resistance of

C. albicans cells to the lethal effects of amphotericin B.
Increased levels of intracellular or extracellular catalase, as
well as incubation under hypoxic conditions, have been
shown to reduce the lethal effects of amphotericin B.
 …Polyene Macrolide
4. Biofilm formation: Candida spp. produce biofilms on
biological and inert surfaces. The resistance of Candida
biofilms to antifungal drugs has been previously documente.
One possible resistance mechanism is related to the slow
growth rate of biofilm cells.

5. Cell wall alterations: Cell wall components may affect the

interaction of polyenes with the cytoplasmic membrane e.g.
low chitin content is associated with increased resistance to
amphotericin B in C. albicans.
 Azole group…
• Azole derivatives represent one of the major groups of
antifungal drugs used in clinical practice to treat fungal
infections in humans, including skin and vaginal infections in
the general population and more serious life-threatening
invasive mycoses in severely immunocompromised patients.

• This class of antifungal agents was originally developed in the

1960s and 1970s, the first available azole derivative for the
oral treatment of systemic fungal infections, ketoconazole, an
imidazole, was released in the early 1980s.
 …Azole group…
• The azole group of antifungal agents consists of the imidazoles
and the triazoles. These compounds contain six carbon ring
structures with conjugated double bonds, chloride residues,
and five carbon ring structures that contain at least two
nitrogen molecules

• Traditionally used agents in this group include clotrimazole,

miconazole, fluconazole, itraconazole, voriconazole, and
ketoconazole. The newer triazoles are voriconazole,
posaconazole, and, most recently, ravuconazole.
 …Azole group…
• Mechanism of action:
• The mode of action of azole derivatives is by binding to and
inhibiting lanosterol 14-α demethylase (Cyp51p or Erg11p), a
cytochrome P₄₅₀ enzyme responsible for the C-14
demethylation of lanosterol, thus blocking ergosterol
biosynthesis and leading to a fungi static effect in the majority
of cases.
 …Azole group…
I. Imidazole agents:
• Ketoconazole: It is useful
in mild cases of
Paracoccidioidomycosis
and is an alternative to
amphotericin B for
infections caused by
Blastomyces or
Histoplasma spp.
 …Azole group…
2. Clotrimazole and Miconazole:
• Synthetic imidazoles covered
together because of their
many similarities.

• These agents are available for

topical or intravaginal
application.

• They are useful in mild cases

of dermatophytosis, including
tinea versicolor.
 …Azole group…
II. Triazoles Agents:
1. Fluconazole:
• It has excellent activity against
most Candida spp. and
Cryptococcus spp.; therapeutic
levels are easily reached in the
central nervous system.

2. Itraconazole: has a spectrum of

activity that is similar to
ketoconazole, effective in cases
of aspergillosis, sporotrichosis,
cryptococcosis, and
onychomycosis.
 …Azole group…
3. Voriconazole: one of the
new triazoles, has an
expanded spectrum of
activity compared with
itraconazole.

• Demonstrates useful
activity against some
Fusarium strains. All strains
of C. glabrata and some
strains of other Candida
spp.
 …Azole group…
• Mechanism of resistance:
1. Alterations in the target enzyme (lanosterol 14-α-demethylase),
including point mutations and over expression, lead to
decreased susceptibilities to azole drugs, which may also lead to
cross-resistance to other azole derivatives.

2. Increased drug efflux: This mechanism is mediated by two types

of multidrug efflux systems, the major facilitator super family
and the ATP-binding cassette super family (ABC transporters).
 …Azole group
3. Biofilm resistance: Mechanisms contributing to azole
resistance in fungal biofilms include:
1) Increased numbers of fungal cells within the biofilm.

2) Subpopulations of “persister” cells that develop tolerance to

azoles.

3) Activation of efflux systems, which may occur physiologically

as a means to facilitate the removal of waste products.

4) Changes in the membrane sterols of cells within the biofilms.

 Echinocandins group…
• The Echinocandins are semisynthetic lipopeptide antifungal
agents, are now recommended as primary therapy for non-
neutropenic patients with invasive candidiasis. It is estimated
that 60 % of candidemia patients now receive an
echinocandin for treatment or prophylaxis.

• There are currently three echinocandins approved for

treatment, including anidulafungin, caspofungin, and
micafungin.
 …Echinocandins group…
• Mechanism of action:
• The echinocandins are glucan synthesis inhibitors. More
specifically, the echinocandins are selective inhibitors of the
fungal enzyme 1,3-beta-D-glucan synthase that is involved in
fungal cell wall synthesis.
 …Echinocandins group…
• Mechanism of resistance:
• Candida species isolates resistant to echinocandin drugs were
first reported in 2005. Their frequency remains relatively low
at less than 2–3 % with C. albicans and most other Candida
species.

1. Target modification: modification of the catalytic subunit of

glucan synthase, which is encoded by genes FKS1and/or FKS2
by restricted mutations in two highly conserved “hot-spot”
regions of the FKS genes.
 …Echinocandins group
2. Biofilm:
• It has been shown for echinocandin drugs that the extensive
production of β-glucan within the extracellular glucan matrix
helps sequester drugs by decreasing their concentration at
the cell membrane surface.
Thank you

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