MEDICAL MICROBIOLOGY

Course for medical students

Associate Professor L. Yocheva, Ph.D.

Department of Biology, Medical Genetics and

Microbiology
Medical Faculty
SU “St. Kliment Ohridski”

Email: [email protected]
 Lecture 5
Fungi and Viruses –
General Characteristics

1. Fungi with medical importance - structure,

classification, basic morphology,
cultivation, significance.

2. Viruses - structure and replication. Viral

classification. Bacteriophages. Prions.
 Key words

• Ergosterol
• Yeasts
• Molds
• Fungal spores
• Virion
• Viral genome
• Prions
• Bacteriophages
 FUNGI WITH
MEDICAL IMPORTANCE
 Fungi in the Nature

• Approximately 80 000 fungal species exist in

the nature

• Mostly grow as saprophytes in the soil or on

dead plant material. Some grow on living plants
and animals (parasites or symbionts)

• They are decomposers, can cause decay and

play an important role in the cycling of carbon
and other elements in the nature.

• Some are used in food industry for the

production of wine, beer, bread, etc.
Fungi in the Nature
 Medical Mycology
• The study of fungi with medical
importance that cause human diseases
(mycoses)
• Most of them are commensals, but
can cause opportunistic infections in
special conditions
• Some are pathogens
• Nearly 50 species cause about 90%
of mycotic diseases in humans
 Human Fungal Diseases
• Allergic reactions
• Mycotoxicosis - ingestion of food products,
contaminated by afla toxin. It is product of some fungi
with carcinogenic properties
• Mycetism (mycetismus) - mushroom poisoning
• Infections
– surface (skin, nails, hair)
– subcutaneous (after trauma)
– systemic or visceral - massive invasions in the
bloodstream and lungs, sepsis; lethal outcome
• fungemia - the presence of fungi or yeast in the
blood
• candidemia or invasive candidiasis - caused by
Candida spp.; most common
Increased Number of Fungal Diseases in the
Past Two Decades

• Incidence of mycotic diseases is

increasing rapidly because of:
˗ More invasive procedures in medicine
˗ More immunocompromised patients
˗ More immunosuppressed patients
(autoimmune diseases; transplanted;
stress; starvation; radiation)
˗ Better diagnostic capabilities in
medicine
 Risk Factors for Invasive Fungal Diseases

Others - diabetes,
autoimmune
disorders

SOT - solid organ

transplantation

Active Surveillance Program to Increase Awareness on Invasive Fungal

Diseases: the French RESSIF Network (2012 to 2018); Bretagne S. et al. 2022
https://doi.org/10.1128/mbio.00920-22
Rapid Incidence Increase of Mycotic Infectious
Diseases (Invasive Mycoses)
(CDC, 1998)

Disease Cases /per million/ per year

1980-1982 1992-1993 1996
Candidiasis 2.6 72.8 228.2
Histoplasmosis 13.9 7.1 13.6
Aspergillosis 8.4 12.4 34.3
Cryptococcosis 4.0 65.5 29.6
 Fungal Diseases Restriction

• Many fungal diseases are endemic

to specific geographical regions

• The causal agents are restricted in

their distribution by soil or climatic
conditions
 Determinants of Pathogenicity

• Thermotolerans

• Live well in tissues and in environment

• Resist to the defense mechanisms of the

body
Kingdom Fungi in Domain Eukaryotes
 Structure of Fungal Cell
• Eukaryotic organisms with typical organelles
˗ Nucleus, mitochondria, endoplasmic reticulum, Golgi
apparatus, vacuole, inclusions
• Cell wall, that provides rigidity, strength, shape, protection
• Cytoplasmic membrane
˗ Double phospholipid layer
˗ Ergosterol (no sterols, typical of eukaryotic cells)
˗ Ergosterol - a main target for the corresponding
antimycotics (azoles and allylamines)
• Capsules
˗ Small number of fungi have capsules e.g.
Cryptococcus neoformans (encapsulated
yeast)
˗ Capsules are polysaccharides
˗ Have antiphagocytic function
Structure of Fungal Cell
 Chemical Composition of the Cell Wall in Fungi

• Multi-layered structure
˗ Mainly polysaccharides – 90%
˗ Proteins and glycoproteins – around 10%

• Polymers
˗ Chitin - polymer of N-acetylglucosamine, peptidoglycan
analogue; rigidity
˗ Glucans α and β – polysaccharides of D-glucose
monomers; strength; many β-glucans are drug target for
antifungal medications (echinocandins)
˗ Mannan - polymer of mannose, bind to proteins -
mannoproteins; specific antigens – significance in
diagnostics; are proven by immunological reactions
˗ Cellulose
Chemical Composition of the Cell Wall in Fungi
 Metabolism

• Heterotrophic metabolism
˗ Energy and carbon from organic
sources

• Most are strict aerobes, some are facultative

anaerobes

• The generation time is about 90 min

(colonies after 2-3 days)
 Classification According to
Morphological Features

• Yeasts

• Filamentous (molds)

• Dimorphic forms - can exist as

yeasts or molds depending on the
conditions
 Yeasts
• Unicellular, oval to round (3-15 μm)

• Can multiply by budding and binary fission

• Budding - distribution of cytoplasm is unequal

between the mother and daughter cell

• Can form pseudohyphae – elongation of

daughter cells

Binary fission
Yeasts - Budding

Budding cell Pseudohypha

 Candiada albicans

SEM Cream-colored colonies on

Sabouraud agar used for
cultivation of different fungi
 Molds

• Multicellular, with branching, tubular cells

• Hyphae (2-10 μm long)
• Mycelium (many branched hyphae)
˗ Vegetative hyphae – that part that grow on the surface
of the culture medium; for substrate utilization
˗ Aerial hyphae – structures that project above the
surface of the medium
˗ The aerial hyphae may produce structures known as
spores - a specialized structures of fungal
reproduction
˗ Colonies formed by molds are described as
filamentous or woolly
Structure oh Hyphae
Morphology of Fungi
Molds - Mycelium

Aerial mycelium of molds;

SEM
 Molds - Colony Morphology

Colonies of molds
isolated from soil

Aerial mycelium – differently

Epidermophyton floccosum, pigmented
dermatophyte fungus Surface of the colonies - granular,
cotton, wooly, velvety
 Dimorphic Fungi

• Able to develop as mold or yeast

according to the surrounding conditions
like CO2 , temperature or nutrients

• Temperature dimorphism (typical of the

group of pathogenic fungi)
 Temperature dimorphism

• Yeast forms - parasitic forms in the

tissue, develop at 37 ℃

• Mycelial forms (saprophytic forms)

develop et 25 ℃
 Fungal Reproduction

• Budding
• Binary Fission
• Hyphae fragmentation
• Sporulation
 Sporulation

• The principle means by which fungi

reproduce and spread through the
environment is sporulation

• They can be borne by air or water to new

sites, where they germinate

• They are generated asexually or sexually

 Fungal spores

• N.B.
• Fungal spores are different than the
endospores of bacteria

• Bacterial endospores are not for

reproduction, but an environmentally
resistant life stage

• Fungal spores are for reproduction, do not

provide resistance to environmental
conditions
 Specialized Structures for Fungal Reproduction

• Observation of specialized structures for fungal

reproduction is of great importance for accurate genus
and species identification.
 Asexual Spores
• Asexual spores - are formed by mitosis
• Major types
˗ Chlamydospores
• formed within psedohyphae in yeasts
• thick-walled spores
• example - Candida albicans
˗ Conidospores
• fungal spore formed on a specialized hyphae called
conidiophore
• examples – Aspergillus spp., Penicillium spp.
˗ Sporangiospores
• hundreds formed within a sac called sporangium at
the end of an aerial hypha
• example – Mucor spp.
Chlamydospores of Candida albicans
Conidospores of Aspergillus spp.
Conidospores of Penicillium spp.
Sporangiospores of Mucorales

Bursting stage
 Sexual Spores

• Sexual spores
˗ Formation – after fusion of two cells (plasmogamy) and
their nuclei (karyogamy) diploid zygote is formed; it
undergoes meiosis – meiospores (haploid nucleus)
˗ Small number – 4, 8, 16 spores
˗ In/on specialized structures
˗ More resistant; for surviving of adverse conditions
• Typеs
˗ Zygospores - Mucor spp., Rhizopus spp.
˗ Ascospores – in sacs (ascs) – most of the fungi of
medical importance
˗ Basidiospores – on basidium; yeasts and molds -
Cryptococcus spp., Trichosporon spp., Malassezia spp.
 Laboratory Identification

• Universal nutrient medium - Sabouraud agar

• Microscopical observation of specific
structures for reproduction
• Colony morphology
• Biochemical tests for identification
• Immunological methods for proving specific
antigens or antibodies
 Fungi Causing Opportunistic Mycoses

• Rare cause infections in normal

persons
• Affect immunocompromissed
individuals
• Examples of fungal infections:
– Candidiasis
– Cryptococcosis
– Aspergillosis
– Mucormycosis
 Superficial and Cutaneous Mycoses Caused by
Dermatophytes

Tinea corporis
(ringworm)

Tinea pedis (athlete's foot): the

onychomycosis,
skin between the toes is scaly
and itchy.
Cause: Epidermophyton
floccosum
Fungal nail infection
 Mucocutaneous Candidiasis

Thrush or Oral candidiasis in a child

who has taken antibiotics

Skin infections caused by C. albicans is

found in wet areas

Onychomycosis
 C. аlbicans – Systemic Infections

C. аlbicans present in a urine

sample during UTI

C. аlbicans forms biofilms on indwelling devices and

epithelial cells. A common cause of nosocomial
infections
Mucormycosis
 Aspergillosis

Aspergillus fumigatus

Aspergillus niger
 Cryptococcosis

Cryptococcosis of lung in patient India ink staining of cerebrospinal

with AIDS. fluid.
The capsules are like a shining halo
Alveolar septum containing a few
around the cells.
inflammatory cells and numerous
Cryptococcus neoformans –
yeasts of Cryptococcus
opportunistic yeast; causes
neoformans.
meningitis or pneumonia in AIDS
The inner layer of the yeast
patientsIt; forms a thick
capsule stains red.
polysaccharide capsule
General Characteristics and
Classification of Viruses.
Prions
 General Characteristics of Viruses
• Obligate intracellular parasites
• Viruses are not living; infectious particle
(no cellular organization)
• Contain genetic material either DNA or
RNA, surrounded and protected by
proteins
• Have a wide range of hosts - can infect
bacteria (called bacteriophages), plants,
animals and humans
 Virion

• Virion - the infectious form of a virus that

exists outside the host cell

• Consist of a nucleic acid core, a protein

coat, and in some cases, an external
envelope

• It may contains certain essential or

accessory specific viral enzymes to facilitate
initial replication and final assembly of the
virion
 Size

• Viruses are extremely small

• The units for measurement of virion size

are nanometers (nm)

• The clinically important viruses range from

18 nm (parvoviruses) to 300 nm (poxviruses)

• Viruses can be seen only with an

electron microscope
 Giant Viruses

• Newly discovered in 2003, their number is

rapidly increasing
• Isolated from the amoebas, environment,
clinically healthy individuals
• dsDNA is gigantic in size and capable of
encoding a large number of virus-specific
proteins
• Health impacts (early and long-term) of this
viruses is not fully defined and is subject to
further research
 Giant Viruses
Siberian viruses

◼
Giant Мimivirus Infected with
Virofag
Acanthocystis turfacea chlorella virus 1 (ATCV-1)
ATCV-1 virus is responsible for stupidity???
 Cultivation of the Viruses

• They can reproduce only in living cells

• Cultivation is possible only in experimental

animals, chicken embryos or cell cultures

• They are obligate intracellular parasites that

depend on the host cell’s synthetic machinery for
reproduction

• The cultivation of some viruses in laboratory is

difficult or impossible
 Genome

• Genomes may be RNA or DNA but not both

• Either of which may be single-stranded (ss) or
double-stranded (ds)
• ssRNA viral genomes are divided into two types:
– positive stranded that have a properties of
mRNA and can be used as template for protein
synthesis
– negative stranded which can not be used
directly as a template
• The RNA genome may be single segment, some
RNA genomes are segmented into pieces
• Most of the viruses have a linear genome
 Genome

DNA virus RNA virus

 Viral Proteins according to their Function

• Structural proteins – capsid, matrix

• Enzymes
˗ Viral polymerases
◼ DNA viruses
◼ RNA(+) viruses
◼ RNA(–) viruses
• Proteases
• Endonucleases and ligases
 Viral Capsid

• Protects and provides virus attachment

and penetration
• N.B. Viruses having capsid are resistant to
desiccation, temperature, enzymes in the gut
and denaturation
• Composition: different number of capsomers,
each composed of one or more proteins
• Proteins may associate with the genome to form
a nucleocapsid
 Viral Envelope

• Some viruses have an additional outer

layer (envelope)
• Envelope is obtained from the cell membrane
and contains virus-coded proteins and
glycoproteins
• Provides protection and attachment
• N.B. These viruses are sensitive to drying,
temperature, enzymes and detergents
 Viral Envelope

Naked virus Enveloped virus

 Capsid Symmetry

• The protein shell surrounding the genome

is found in either of two geometric
configurations:
– Helical symmetry like a rod
– Icosahedral symmetry (regular polyhedron
with 20 triangular faces), like a sphere
Capsid Symmetry
 Viruses with Helical Symetry

Virion is rod shaped

Viruse with Icosahedral Symmetry

Poliovirus Herpes simplex virus

Virion looks like a sphere

 Taxonomic Classification and
Nomenclature of Viruses

• Viral classification starts at the level of Order and

continues as follows, with the taxon suffixes
given in italics
• Binominal name for the species
 Means of Classification and Naming of Viruses

• Structure and mode of replication: the current

means of taxonomic classification (size,
morphology, nucleic acid, envelope,
symmetry)

• Disease: e.g. encephalitis and hepatitis

viruses

• Means of transmission: e.g. arboviruses

are spread by insects
 Families of RNA Viruses

• Picornaviridae • Coronaviridae
• Caliciviridae • Retroviridae
• Reoviridae • Bunyaviridae
• Togaviridae • Orthomyxoviridae
• Flaviviridae • Paramyxoviridae
• Arenaviridae • Rhabdoviridae
• Filoviridae
 RNAViruses
RNA Viruses

Rotavirus
Influenza virus

Paramyxovirus

Coronavirus
Enterovirus
 RNA Viruses
• Most RNA viruses are single-stranded (half
are positive (+) stranded and half are
negative(-); enveloped and replicate in the
cytoplasm
• Exceptions:
˗ Reoviridae are double-stranded
˗ Three are nonenveloped:
• Picornaviridae
• Caliciviridae
• Reoviridae
˗ Two undergo replication in the nucleus:
• Retroviridae
• Orthomycoviridae
 Families of DNA Viruses

• Parvoviridae
• Papillomaviridae
• Polyomaviridae
• Adenoviridae
• Herpesviridae
• Poxviridae
• Hepadnaviridae
 DNA Viruses

Papillomavirus
Adenovirus

Herpes Simplex
Virus
Hepatitis B Virus
 DNA Viruses

• Most DNA viruses are double stranded,

show icosahedral symmetry and replicate
in the nucleus

• Two exceptions: Parvoviridae is with ssDNA

and Poxviridae does not have icosahedral
symmetry and replicates in the cytoplasm
Steps in the Replication Cycles of Viruses

1. Attachment
2. Penetration
3. Uncoating
4. Genome replication
5. Assembly of viral
particles
6. Release
 Stage of adsorption
• Attachment sites on the virus surface: some viruses
have specific attachment structures such as the
glycoprotein spikes in viral envelopes

• Host cell receptor molecules: the receptor molecules

on the host cell membrane are specific for each virus
family and are attachment sites for viruses. Examples:
CD4 (HIV); ICAM-1 (Rinovirus); CR-2 (EBV)
 Penetration and Uncoating

• Stage of penetration:
– Receptor-mediated endocytosis: The cell membrane
invaginates, enclosing the virion in vesicle
(endosome)
– Membrane fusion: In some enveloped viruses (e.g.
Retroviruses). Fusion of the viral membrane with
that of the cell membrane. The end result is that
nucleocapsid is free in the cytoplasm, the viral
membrane remains associated with the plasma
membrane

• Stage of uncoating: the nucleic acid is released

from the capsid into the cytoplasm
Penetration
 Transcription, Translation and Replication
of RNA Viruses
• Usually undergo in the cytoplasm
• Positive stranded RNA viruses are read by the
host directly as mRNA
• Negative stranded RNA viruses use viral RNA
polymerase (RNA-dependent RNA polymerase)
to transcribe the negative strand RNA to a
positive
• The positive strand than acts as like mRNA
and undergoes both transcription and
translation
Transcription, Translation and Replication of
Retroviruses

• The RNA of Retroviruses is transcribed into

dsDNA, using unique viral enzyme called RNA-
dependent DNA polymerase (reverse
transcriptase)
• dsDNA is integrated into the host genome by the
action of a viral “Integrase”
• Viral mRNAs and progeny RNA genomes are
transcribed from this integrated DNA by the
host cell RNA polymerase
Transcription, Translation and Replication
of DNA Viruses

• Transcription and replication usually occur in

the nucleus

• The positive stranded DNA is used as a

template for transcription into mRNA
 Stages of Synthesis of Viral Proteins

• Viral proteins are synthesized in the

cytoplasm of the cell
• Early proteins - enzymes necessary for
nucleic acid replication and regulatory
proteins
• Late proteins - necessary for completion
and assembly of the virion
 Virus Assembly

• Viral components (proteins and RNA

or DNA) assemble into the intact virion

• Assembly is based on the principle

of complementarity

• It is performed in the cytoplasm or in

the nucleus of the infected cell
 Release of Progeny Viruses

• Three types for releasing of progeny

viruses exist:
– By budding
– By cell lysis
– By exocytosis
Release of Progeny Viruses by „Budding"
• Enveloped viruses use the cell membrane of the
host
• Specific viral glycoproteins are transported to
the host cell membrane and inserted in it
• The provirus binds to modified membrane and
the virion buds from the membrane
• Membrane does not lyse, but exhausted slowly
in time
Viral Budding
 Release of Progeny Viruses by Cell Lysis
• The virus is released from the infected cell by
bursting of the membrane; this kills the cell
• Most non-enveloped viruses and few enveloped
viruses require cell lysis
• Cell lysis is actively induced by viruses using various
mechanisms e.g. production of viroporins in the late
phase of the infection
 Release of Progeny Viruses by Reverse
Endocytosis (Exocytosis)
• Cell transported the virus out through vesicles
• Vesicles containing the virus are excreted out
of the infected cell
• Mostly non-enveloped viruses use exocytosis
• The cell is not degraded
Exocytosis
 N.B. Viral Infections at the Cellular Level

• Cytolytic viral infection – death of the cell

• Latent infection – the virus exists in an inactive
dormant state. Can be periodically reactivated.
No effect on the cell.
• Chronic slow infection – some viruses can
cause disease only after many years
• Some RNA and DNA viruses are oncogenic.
They can induce transformation of the cell
(activation or introduction of oncogenes)
 Prions

• The most simple infectious agents,

consisting only of proteins, without
nucleic acids
• Can cause chronic slow infections
• Incubation period of many years and
progressive course of the disease which
ends with dementia and death
 Prion Structure

• Aggregates of filamentous form of

abnormal configured self-proteins

• Extremely resistant to heating above

80℃, formaldehyde, proteases, etc.
˗ Destruction by autoclaving
121 ℃ /1 atm for 1 h
Prions Structure
 Pathology

• Infectious prion protein causes the

normal form to fold into the infectious
form

• Infectious prions reach the highest

concentration in the brain
• They form amyloid plaques in the brain and
destroyed gray matter
 Slow Viral Infections

• In humans:
– Creutzfeldt-Jakob disease (CJD)
• In animals:
– Scrapie (sheep and goats)
– Spongiform encephalopathy (mad cow disease)
• All form of diseases are characterized with
transmissible spongiform encephalopathy
• There is no known treatment of CJD and it is
invariably fatal
Pathological Changes in Prion Diseases
 Mode of Transmission of Infection

• The mode of transmission among animals

is not clear, but feed prepared from other
diseased animals is suspected

• In humans, some routes of transmission

are:
– Alimentary route
– Blood
– Contaminated tools
– Transplants
 Epidemiology

• Outbreak of mad cow disease in 1980 in

England

• Unusually frequent accidents in young

people with CJD in 1996

• It is assumed that the infection in humans

occurs mainly after use of food from
infected animals