Download Microbiology 1: Bacterial Properties

Microbiology 1: Bacterial Properties
Main difference between Gram + and Gram – bacteria
The vast majority of bacteria are harmless or beneficial
Some bacteria are pathogenic including:

Gram negative bacteria

Gram positive bacteria

Mycobacteria (neither Gram negative nor Gram positive)
Gram negative bacteria
Diseases caused
EP: EP Escherichia coli
Diarrhoea
EHEC: EH Escherichia coli
Dysentery and kidney failure
Salmonella typhimurium
Self-limiting diseases: food poisoning and gastroenteritis
Salmonella typhi
Systemic diseases: typhoid
Shigella
Dysentery
Vibrio cholerae
Cholera
Neisseria meningitidis
Meningitis
Neisseria gonorrhoeae
Gonorrhoea
Gram positive bacteria
Diseases caused
Staphylococcus aureus
Skin diseases, endocarditis, bacteraemia, joint diseases and pneumonia
Streptococcus pneumoniae
Pneumonia, meningitis, otitis media
Streptococcus pyogenes
Tonsillitis, necrotising fasciitis, bacteraemia, scarlet fever
Mycobacteria
Diseases caused
Mycobacterium tuberculosis
Tuberculosis
Mycobacterium leprae
Leprosy
General characteristics of bacteria:

Prokaryotic

Small (typical dimensions of a Gram negative bacterium: 2 μm × 0.5 μm)

Unicellular

Lack internal membrane-bound organelles (chloroplasts, nucleus, ER, mitochondria)

Possess a bacterial chromosome: DNA is organised into a continuous circular loop

May have plasmids: self-replicating circular loops of DNA

Haploid: there is 1 copy of gene

May have flagella: long filamentous structures assembled on the surface of a bacterial cell
Bacterial form
Examples
Cocci (spherical)
Streptococci and Staphylococcus
Bacilli (rod-shaped)
Most Gram negative bacteria: E. coli, Salmonella, Shigella
Spirilli (spiral-shaped)
n/a
Photosynthetic
Cyanobacteria
Gram stain: classifies bacteria according to differences in the structure of the cell wall

Used to differentiate between Gram positive and Gram negative bacteria

Method:
o
Stain with a violet dye (crystal violet) and iodine
o
Rinse in alcohol
o
Counterstain with a red dye (safranin)

The stain binds to peptidoglycan (a peptide sugar polymer) in the bacterial cell wall

Both Gram positive and Gram negative bacteria have peptidoglycan in their cell wall
Gram positive bacteria
1 cell membrane
Structure
Thick peptidoglycan cell wall
Gram negative bacteria
2 cell membranes
Thinner peptidoglycan cell wall
Peptidoglycan is accessible to the dye
Peptidoglycan is not accessible to the dye: it
is present in the periplasmic space
Wash with alcohol
The crystal violet stain is retained
The crystal violet stain is removed
Counterstain
No effect on the stain
Cells are stained pink
Final result
Appear deep violet
Appear pink
Explanation
Peptidoglycan in the cell wall retains the dye
The outer membrane resists the dye
Examples
Lactobacillus, Bacillus, most cocci
E. coli, Salmonella, some cocci
Gram positive cell wall structure:
Gram negative cell wall structure:
Examples of intracellular and extracellular bacteria
Extracellular pathogens: infect the host without entering host cells
Examples of extracellular pathogenic bacteria:

Staphylococcus

Streptococcus

Yersinia

Neisseria
Intracellular pathogens:
2 methods of entry into the host cell:
1. Engulfed by a phagocytic host cell
2. Directly invade a non-phagocytic host cell
Inside the host cell: all intracellular pathogenic bacteria are enclosed in a membrane-bound vacuole which
is derived from the plasma membrane of the host cell
3 modes of action inside the host cell:
1. Escape from the vacuole and into the cytoplasm
E.g. Listeria and Shigella
2. Modify the vacuole and prevent it from interacting with lysosomes; therefore the host cell does not
degrade the vacuole
E.g. Salmonella and Mycobacteria
3. Remain within the vacuole which fuses with a lysosome to form a phagolysosome; the bacteria are
able to resist the harsh conditions and replicate
E.g. Coxiella
Flagella and type III secretion – 2 related bacterial multi-protein machines
Salmonella: an example of motility and invasion
1. The bacteria are motile outside the epithelial cell
2. A bacterium comes into contact with the surface of epithelial cell
3. At point of contact: the bacteria send a signal to the host cell
4. This causes monomeric actin to polymerise into filaments in the vicinity of the bacterium
5. Polymerisation of actin causes ruffling of the plasma membrane of the host cell
6. This caps the bacterium and seals it into a vacuole
7. The bacterium enters the cell
Motility and invasion require 2 related multi-protein machines:
1. Flagella: allow motility
2. Type III secretion systems: allow invasion
Flagella and type III secretion systems are structurally similar but functionally distinct
Flagella: long protein filaments which are assembled on the surface of a bacterial cell
1. Flagella extend out from 1 pole of the cell
2. Rotations of the flagella propel the bacterial cell
3. Rotations are driven by a motor in the bacterial cell
Flagella assembly: flagella are self-assembled from the motor embedded in the inner membrane to the
long filament
1. Protein monomers are introduced into the inner membrane of the bacterial cell
2. Protein monomers oligomerise: they bind to other protein subunits to form a multi-subunit ring-like
structure
3. A channel-like structure is assembled within the ring
Protein monomers are secreted through the channel, including:
o
Filament proteins: make up the flagellum
o
Capping structures: regulate the assembly of filament proteins
4. The channel is assembled towards the outer membrane
5. Enzymatic activity breaks down the peptidoglycans cell wall and the channel pushes through the outer
membrane
6. Another ring-like structure is formed in the outer membrane
7. A hook-like structure is assembled by capping proteins
8. The capping proteins fall off after completion of the hook
Type III secretion systems: deliver virulence proteins into host cells

Type III secretion systems have a similar structure to flagella present in some Gram negative bacterial
pathogens
Structural similarities include the presence of rings and channels

The type III secretion system has a short needle-like structure at its tip

The needle forms a pore in the plasma membrane of the eukaryotic host cell

The bacterial cell injects bacterial proteins into the host cell via the needle
Bacterial effectors can mimic host cell proteins
Bacteria have evolved mechanisms to mimic various host cell pathways in order to evade host cell defence
mechanisms:

Some bacterial proteins mimic the activity of eukaryotic proteins
E.g. GEF and GAP: eukaryotic proteins which turn rho-dependent GTPases on and off respectively

When Salmonella comes into contact with the host cell, it expresses a protein which mimics GEF: this
activates actin polymerisation

Actin polymerisation is only a transient process: it stops once Salmonella has entered the host cell

Once Salmonella has entered the host cell, it expresses a protein which mimics GAP: this deactivates
actin polymerisation
Genome diversity, horizontal transfer and evolution
Genome diversity:

Genomes of bacterial pathogens encode between 500 and 4500 proteins

Genome sequencing of bacterial strains (e.g. strains of E. coli) shows that some genes are present in all
strains; however other genes are unique to a particular strain

As more strains are sequenced, more unique genes are found

The gene repertoire of bacterial pathogens consists of core genes and accessory genes
o
Core genes: always present in all strains of a bacterial pathogen
o
Accessory genes: unique to a particular strain of a bacterial pathogen
Horizontal transfer:
Vertical transmission of DNA: daughter cells inherit DNA from the parent cell during replication

Replication is asexual: it involves clonal, rapid, simple division
Horizontal transmission of DNA: bacterial cells acquire foreign DNA

Variation arises through mutation and acquisition of foreign DNA by horizontal transfer

The vast majority of accessory genes are acquired by horizontal transfer from unknown sources
3 mechanisms of horizontal gene transfer:
1. Transformation
2. Transduction
3. Conjugation
Transformation: DNA uptake

The bacterial cell takes up double-stranded DNA from the extracellular environment

DNA is integrated into the bacterial chromosome by homologous recombination

Neisseria and Streptococcus are subject to transformation
Transduction:

The bacterial cell is infected by bacteriophages (viruses which infect bacteria)

Bacteriophages replicate within the bacterial cell and cut up bacterial DNA

Some bacterial DNA is incorporated into the viral genome in bacteriophage heads

Newly assembled bacteriophages infect other bacterial cells and inject bacterial DNA into them

Many Gram negative and Gram positive bacteria are subject to transduction since they are susceptible
to bacteriophage infections
Conjugation:

A hollow tube is assembled between 2 bacterial cells

DNA can be transferred via the tube

Many Gram negative and Gram positive bacteria are subject to conjugation

It is a common method of transfer of antibiotic resistance since genes encoding antibiotic resistance
are frequently found on plasmids
Conclusion: genetic diversity in the bacterial genome is huge since bacteria can acquire accessory genes
from an unlimited gene pool in the biosphere by horizontal transfer
Evolution:
The old world view of biodiversity:

Most biodiversity is present within Plantae, Fungi and Animalia

Some biodiversity is also present within Protista and Monera
Carl Woese established a method of sequencing small subunit rRNA (ssrRNA):

Sequence alignments of ssrRNA allow quantitative comparisons among all extant organisms

ssrRNA is conserved throughout the bacterial kingdom: all bacteria require ssrRNA

The ssrRNA sequence varies between different bacteria
The new world view of biodiversity:

Very little biodiversity is present within Plantae, Fungi and Animalia

Most biodiversity is present within Bacteria, Archaea and Eukarya
In summary:

There are many bacterial genomes

All genomes are mixable by horizontal transfer and susceptible to mutation

They grow with a very rapid generation time: e.g. Streptococcus pyogenes
Evolution of bacteria is caused by a combination of the following factors:

There is an incredible source of genetic variation within the bacterial genome

Bacteria have a very rapid generation time

Selective pressures act on bacteria

Time: approximately 1,000,000,000 years
Flagella and type III secretion systems are complex structures which have developed by evolution
Listeria: another example of manipulation of actin by a bacterium

It does not possess either a type III secretion system or a flagellum; therefore it exploits the host cell
cytoskeleton for intracellular movement

It causes food poisoning and more serious diseases in the immunocompromised, the elderly and in
pregnant women

It is an intracellular bacterial pathogen which has the ability to invade host
cells
Mechanism of Listeria movement within and between host cells:
1.
The bacterium is present inside a vacuole in the host cell
2.
It breaks down the vacuolar membrane and escapes into the cytoplasm of
the host cell
3.
It reorganises and causes polymerisation of host cell actin filaments at 1
pole of the bacterial cell
4.
Assembly of F-actin propels the bacterium through the cytoplasm
5.
Once the bacterium comes into contact with the plasma membrane, it
breaks through it and invades a neighbouring cell
Microbiology 2: Properties of Bacterial Pathogens
Explain the concepts of infectivity and virulence and define the term infective dose
Introduction and definitions:
Only a minority of bacterial species are pathogenic:

Obligate pathogens: can only replicate inside host cells

Facultative pathogens: replicate in an environmental reservoir; only cause disease in a susceptible host

Opportunistic pathogens: usually benign; may cause disease in an injured/immunocompromised host
Virulent pathogenic bacteria differ from closely related non-pathogenic bacteria by a very small number of
genes

Virulence genes: genes which contribute to the ability of an organism to cause disease

Virulence genes encode virulence factors
o
Pathogenicity islands: sections of the bacterial chromosome which contain virulence genes
o
Virulence plasmids: sections of plasmids which contain virulence genes
o
Bacteriophages: may carry virulence genes
Complement:
The complement cascade is activated by different pathways
Complement activation kills pathogens by the following mechanisms:

MAC complex: inserted into the plasma membrane causing lysis

C3b: opsonises pathogens and facilitates phagocytosis by neutrophils and macrophages
Meningococcal infection is associated with deficiencies or polymorphisms in complement factors
Molecular basis of virulence:

Commensals: harmless organisms which are part of our normal flora

Pathogens: organisms which cause disease

o
Primary pathogens: cause disease in non-immunocompromised hosts
o
Opportunistic pathogens: cause disease in immunocompromised hosts
Virulence: the degree of damage that a pathogen can induce in its host; a quantitative measure
A highly virulent strain causes a greater extent of damage to its host

Infective dose: the number of infectious organisms which are necessary to cause disease in a host
Context:

1013 human cells in the human body

1014 bacterial cells in the human body

There are about 3000 bacterial species in the human body

Only about 50 bacterial species cause disease in humans

Some pathogens have an extremely low infective dose: e.g. 1-10 Shigella cause disease

Inside the gut there is a large diversity of bacteria

Pathogenic bacteria belong to the same part of the bacterial spectrum; they are closely related
Special attributes of pathogens which contribute to virulence:

Tropism: pathogens must find a unique niche to settle in (either inside or outside cells)

Replication: pathogens replicate by acquisition of nutrients

Immune evasion: e.g. Streptococcus and Meningococcus are host-specific bacteria and must be able to
evade the host immune system

Toxic: pathogens elicit damage to the host by producing toxins, which are either secreted or are part
of the outer membrane

Transmission: pathogens are able to spread between hosts
The genetic constitution of bacteria makes them unique: they can acquire DNA from the environment by
horizontal transfer
The genome of pathogens often has extra regions of DNA which make them toxic
Pathogenicity islands: clusters of genes which are required for pathogenesis; encode virulence factors
List the potential sources and possible routes of infection by bacteria
Potential routes of infection:
Respiratory (aerosol spread): e.g. Meningococcus; Streptococcus
Direct contact: primarily STDs and mucosal pathogens (e.g. Meningococcus)
Faecal-oral spread
Vector borne: e.g. malaria; Leishmania
Give examples of extracellular bacterial pathogens transmitted by different routes and outline the
ways in which they cause disease
Vibrio cholerae: causes cholera
John Snow investigated an outbreak of cholera: showed the relationship of the location of cases to a single
water supply

Gram negative bacillus

Symptoms: sudden onset of profuse, watery diarrhoea (rice water stool)

Rice water stool is produced by the secretion of intracellular contents of epithelial cells
Pathogenesis of cholera:

Faecal-oral transmission (e.g. from contaminated water supplies)

Colonisation of the small intestine: the bacteria adhere to the epithelium of the small intestine via the
toxin co-regulated pilus

Replication

Production of toxins
Cholera structures:
Toxin co-regulated pilus: required for the bacteria to bind to epithelial cells
Flagella: required for extracellular movement of the bacteria to reach enterocytes
Cholera toxin: a toxic protein which is secreted by Vibrio cholerae

1A 5B toxin: composed of 1 A subunit coupled with 5 B subunits

B subunits: bind to glycolipids in the plasma membrane of epithelial cells of the gut

A subunit: an enzymatically active component which catalyses ADP-ribosylation of G proteins

ADP-ribosylation: the transfer of an ADP-ribose moiety from NAD to a G protein
Mechanism of action of the 1A 5B cholera toxin:
1. The B subunits transfer the A subunit into the epithelial cell across the plasma membrane
2. A1 catalyses ADP-ribosylation of G proteins: this activates adenylate cyclase
3. Activated adenylate cyclase converts ATP  cyclic AMP
4. Therefore the cholera toxin causes an increase in the intracellular concentration of cyclic AMP
5. Cyclic AMP causes ion channels to open: this creates an ion imbalance, which leads to massive watery
diarrhoea
Normally sodium is absorbed from the lumen of the GI tract and it is retained within cells
Presence of the cholera toxin causes chloride channels to open: chloride leaves epithelial cells; sodium
is not absorbed; water follows chloride into the GI tract
The cholera toxin is acquired by Vibrio cholerae via transduction:

Virulence genes encoding the cholera toxin are carried by a bacteriophage

The bacteriophage infects non-pathogenic strains of Vibrio cholerae

This produces pathogenic strains of Vibrio cholerae which produce toxins
Similar toxins which cause an increase in the intracellular concentration of cyclic AMP are produced by
in Shigella, E. coli and Bordetella pertussis
Shigella spp.: cause shigellosis (dysentery)

Faecal-oral transmission

Causes inflammation and bleeding of the mucosa of the distal colon

Symptoms: severe diarrhoea containing blood and mucous
Clostridium difficile: causes mild to severe infections in the GI tract
Features:

Gram positive bacillus

Resistant: forms terminal spores which are difficult to eradicate

Spore formation enhances transmission of C. difficile

Pathogenic strains are acquired exogenously from the hospital environment and from health care
workers

Opportunistic pathogen: causes C. difficile associated diarrhoea (CDAD):
o
Diagnosis: observation of pseudomembranes at endoscopy or positive stool test
o
No other aetiology for diarrhoea
o
Antibiotic exposure in the last 6-8 weeks
Symptoms:

Contact bleeding

Hyperaemia

Pus formation in pseudomembranes on the mucosa of the colon
Toxins:

C. difficile produces 2 toxins: toxin A and toxin B

Toxin A and toxin B are single-protein toxins (i.e. they act independently)

Each toxin is composed of 3 functionally distinct domains: an enzyme domain (toxic), a translocation
domain and a binding domain

Toxin A (enterotoxin): enters cells at the apical region; damages the GI tract

Toxin B (cytotoxin): enters cells at the basolateral region; damages cells
Mechanism of action of C. difficile toxins:
1. The toxin enters the cell into an endosome (a membrane-bound vacuole) via endocytosis
2. The enzyme domain is cleaved from the rest of the toxin molecule
3. The vacuole is acidified: this causes the enzyme domain to translocate into the cytoplasm
4. The toxin catalyses glycosylation of Rho GTPases in the cytoplasm: this inactivates Rho GTPases
5. This inhibits effector actions of Rho GTPases: e.g. cytokine production; ROS production
Neisseria meningitidis: causes meningitis

Gram negative diplococcus

Important cause of childhood mortality

There are vaccines against N. meningitidis except for serogroup B

Serogroup C vaccine: virtually eradicated meningitis C

Meningitis B is the most prevalent form of meningitis in the UK
Progression of meningitis:
1.
Colonisation: asymptomatic; involves the sub-epithelial layer
2.
Septicaemia: symptoms include fever, rash, cardiac depression and coagulation; may cause fatality
3.
Spread to cerebrospinal fluid: symptoms include neck stiffness, photophobia and vomiting
Type four pili: required for adhesion to epithelial cells

Bind to CD46 (complement regulatory protein)

Host-specific: type four pili only bind to human cells
Complement is important in innate immunity; complement deficiency leads to an increase in disease
Symptoms of meningococcal septicaemia are caused by:

The bacteria avoid complement-mediated killing: this leads to high levels of bacteraemia

Blebbing: bacteria shed components of the outer membrane, including lipopolysaccharides (LPS)
LPS is an endotoxin: i.e. it is an integral component of the cell wall of Gram-negative bacteria
Mechanisms of avoiding complement-mediated killing:
Capsule:

Meningococcus coats itself in a polysaccharide capsule composed of sialic acid

Sialic acid expressed on human cells protects them from complement recognition by recruitment of
factor H

Vaccines against Meningococcus target the capsule

Meningococcus serogroup C: coated by a capsule composed of α2-9 linked sialic acid

Meningococcus serogroup B: coated by a capsule composed of α2-8 linked sialic acid
α2-8 linked sialic acid is identical to a self-antigen which is expressed on cells of the developing CNS
LPS: an integral component of the outer membrane of Meningococcus

Part of the LPS molecule is identical to a self-antigen which is expressed on red blood cells

LPS coats Meningococcus with α2-8 linked sialic acid which recruits factor H
Factor H: a complement factor which down-regulates complement

Factor H is recruited by a protein expressed on the surface of Meningococcus

The bacterial factor H binding protein mimics the factor H binding protein expressed on the surface of
human cells

Recruitment of factor H down-regulates complement and prevents recognition of Meningococcus
Replication strategies: involve the acquisition of iron from the host
Levels of free iron in the body are low: free iron is highly toxic and can damage proteins and lipids
Most iron is transported in storage proteins (e.g. transferrin, lactoferrin and myoglobin)
Meningococcus removes iron from transferrin:
1.
Meningococcus expresses 2 transferrin binding proteins on its surface which engage transferrin
2.
Iron is removed from transferrin
3.
Iron is taken up by meningococcal cells by specialised transport mechanisms
Lecture summary:
Bacterial pathogens have evolved specific mechanisms to interact with human hosts:

Often encode extra DNA fragments

Can subvert the immune system

Secrete toxins (cholera toxin and clostridia toxin)
Examples of extracellular bacterial pathogens:

Vibrio cholerae: cholera toxin causes ADP-ribosylation of G proteins

Clostridium difficile: single-protein toxins cause glycosylation of Rho GTPases

Neisseria meningitidis: releases exotoxins (LPS)
Microbiology 3: Defence and Vaccination against Bacteria
List the major non-immune host defence mechanisms and describe how they protect against infection
Non-immune host defence mechanisms:

Skin: a natural physical barrier to bacterial infection

Low pH and antibacterial secretions: make the skin a difficult surface to colonise

Routine application of soap and water: strengthen the defence provided by the skin

Mucosal surfaces: protected by mucous and ciliary clearance
Competition with commensal organisms:

Bacteria constitute 60% of the Earth’s biomass

Most bacteria are harmless and often beneficial (commensals)

Commensal bacteria constitute ~1% of the body mass
List the main components of the innate immune response and the major antimicrobial mechanisms
Innate immune response:

Bacteria that penetrate the skin and mucosal barriers are met by the innate immune response

Complement system: punches holes into bacteria

Neutrophils and macrophages: phagocytose bacteria and expose them to toxic radicals and hydrolytic
enzymes
Adaptive immune response:

Antibodies (produced by B cells): prevent adhesion; neutralise toxins; enhance complement-mediated
killing and phagocytosis

T cells: enhance B cell responses; activate macrophages

Memory response: works better on re-exposure to the same pathogen
Vaccines stimulate both aspects of the immune response; they are primarily aimed at eliciting acquired
immunity
Innate immunity
Acquired immunity
Does not require exposure to the infectious agent
Requires exposure to the infectious agent/its antigens
Immediate
Takes time to develop
Mediated by monocytes and neutrophils
Mediated mainly by T and B cells
Initially an inflammatory reaction
Other cell types are involved: e.g. monocytes and
dendritic cells
Humoral immunity
Cell mediated immunity
Directly mediated by antibodies
Mediated by T lymphocytes and NK cells
Antibodies are produced by B cells
Not primarily mediated by antibodies
Plasma B cells are the primary source of secreted
Other cell types indirectly play a role: e.g. macrophages
immunoglobulins
Explain how an adaptive immune response is involved in defence against major bacterial pathogens
See above
Describe how infectious agents avoid host defences
Pathogenic bacteria have evolved strategies to avoid host defences:
Strategy
Mechanism
Example
Thick cell wall
Mycobacterium tuberculosis
Capsule
Neisseria meningitidis
IgA protease
Streptococcus pneumoniae
Antigenic variation
Neisseria gonorrhoeae
Polysaccharide capsule
Neisseria meningitidis
Debilitate phagocytes
Yersinia pestis
Hide inside other cells
Chlamydia
Escape from phagosome
Listeria monocytogenes
Block phagosome maturation
Mycobacterium tuberculosis
Resist complement
Resist antibodies
Resist phagocytosis
Inhibit intracellular killing
Explain the difference between active and passive immunisation
Active immunity: elicited in the host in response to an antigen
Passive immunity: the acquisition of protection from another immune individual through the transfer of
antibody or activated T cells
Vaccines induce active immunity
Give examples of the different types of vaccine presently available and how they are used
Current routine bacterial vaccination in the UK
Time of immunisation
Immunisation(s)
Method of administration
Diphtheria
Tetanus
2, 3 and 4 months old
Pertussis (whooping cough)
1 injection
Polio
Hib (meningitis and pneumonia)
13 months old
Meningitis C
1 injection
Measles, mumps, rubella: MMR
1 injection
Diphtheria
3 years and 4 months to 5 years old
Tetanus
Pertussis
1 injection
Polio
10 to 14 years old (sometimes
neonatal)
MMR
1 injection
BCG
Skin test and 1 injection (if needed)
Diphtheria
13 to 18 years old
Tetanus
1 injection
Polio
DTP: diphtheria, tetanus, pertussis (whooping cough)

Subunit vaccines

Antibodies neutralise toxins and block adhesion
Conjugate vaccines (carbohydrate):

T cell recognition of protein carriers enhances B cell activation

Promotes efficient antibody response to the polysaccharide capsule
Live attenuated vaccines:

BCG gives some protection against tuberculosis in children; it is ineffective against adult pulmonary
disease

New vaccine strategies are based on genome information

Very effective live attenuated vaccines are being developed for enteric pathogens (e.g. Salmonella
typhi)
Microbiology 4: Fungal Infections
Outline the main differences between fungi (eukaryotes) and bacteria (prokaryotes)
Fungi:

~200 species are human pathogens

Eukaryotic organisms: genetic material is organised into a membrane-bound nucleus

Perform extracellular digestion: secrete hydrolytic enzymes which break down biopolymers

Disperse over large distances because of spores; humans are constantly exposed to spores

Commensal organisms and skin colonisers are transmitted by contact
Summarise briefly the ecology and epidemiology of infectious fungi
Fungal allergies:

Inhalation of/contact with fungal spores may induce a wide range of allergic diseases
E.g. rhinitis; dermatitis; asthma; allergic broncho-pulmonary aspergillosis (ABPA)

Allergic responses differ according to (a) the individual and (b) the species
E.g. upper respiratory disease (URD):

Affects 115 million Americans

Symptoms: congestion; sneezing; itching; watery eyes; coughing; headache

Diagnosis: immunoCAP; specific IgE blood test (this in vitro quantitative assay measures allergenspecific IgE in human serum/plasma)
List the major groups of disease causing fungi and specify their growth forms
3 types of fungal infections:
1.
Allergies
2.
Mycotoxicoses
3.
Mycoses: superficial; cutaneous; systemic
Mycotoxicosis: a toxic reaction caused by ingestion/inhalation of a mycotoxin
Mycotoxins: secondary metabolites of moulds which exert toxic effects on animals and humans

Symptoms: breathing problems; dizziness; severe vomiting; diarrhoea; dehydration; hepatic and renal
failure (6 days later)

Therapy: gastric lavage and charcoal; liver transplant
Mushrooms which have psychedelic properties include: Liberty Cap and Fly Agaric

Symptoms: vomiting; nausea; stomach pains

Therapy: supportive; correction of hypoglycaemia and electrolyte imbalance
Mycoses: classified by the level of tissue affected

Superficial

Cutaneous

Subcutaneous

Systemic (deep)
Define the terms superficial mycoses and deep mycoses, giving appropriate examples of each type of
infection
Superficial mycoses: superficial cosmetic fungal infections of the skin or hair shaft
No living tissue is invaded and no cellular response is initiated from the host
E.g. black piedra: caused by Piedraia hortae
Cutaneous mycoses: caused by dermatophytes (aka keratinophilic fungi)
Dermatophytes: produce extracellular enzymes (keratinases) which hydrolyse keratin
Inflammation is caused by the host response to metabolic by-products
Dermatophytoces/dermatomycoses are characterised by the part of the body affected:

Tinea capitis: affects the head/neck

Tinea pedis: affects the feet (Athlete’s foot)
Tinea capitis: a superficial fungal infection of the skin of the scalp, eyebrows and eyelashes with a
propensity for attacking hair shafts and follicles
Subcutaneous mycoses: chronic localised infections of the skin and subcutaneous tissue following
traumatic implantation of the aetiological agent
Rare compared to superficial and cutaneous mycoses
E.g. Sporotrichosis
Systemic mycoses:
Primary systemic mycoses: establish infection in a normal immunocompetent host (i.e. no breach of the
host defence is required and an immunocompromised host is not required)

E.g. Histoplasma capsulatum
Secondary systemic mycoses: require an immunocompromised host in order to establish infection

E.g. Candida; Aspergillus
Pathogen: a microorganism capable of causing disease
True pathogen: a microorganism capable of infecting and causing disease in apparently
immunocompetent individuals
Opportunistic pathogen: a usually harmless microorganism which becomes pathogenic under favourable
conditions
Endemic mycoses (true systemic mycoses):

Caused by thermally dimorphic fungi which exist in natural surroundings, soil, bird and bat droppings

Geographically limited to Central and Southern America

Infection occurs by inhalation, pulmonary involvement and dissemination
Candida infections:

Candida albicans is an opportunistic commensal: i.e. it colonises and invades the host tissues of
immunocompromised individuals

In healthy individuals: it is found in the GI tract, the genitourinary tract and the skin

In immunocompromised individuals: it causes superficial, mucosal and systemic infections
The incidence of hospital-acquired fungal infections has risen in recent decades due to the development of
more aggressive and invasive surgical techniques
Pathogenic Candida species:

6/100 Candida species predominate as pathogens

They colonise humans: oral colonisation
Superficial Candida infections:

Regions affected: mouth; throat; skin; scalp; vagina; fingers; nails; bronchi; lungs; GI tract

Usually occur due to impaired epithelial barrier functions

Occur in all age groups, most commonly in the newborn and elderly

Respond readily to treatment
Mucosal Candida infections:

Infections are symptomatic

Occur in neonates and the elderly

In HIV patients: oropharyngeal, oesophageal and vulvovaginal regions are affected

Frequently seen
Systemic Candida infections:

Not seen in normal healthy individuals

Risk factors include: chemotherapy; gut-related surgery; catheters
Mechanism of Candida virulence:
1.
Colonisation
2.
Superficial infection
3.
Deep-seated infection
4.
Disseminated infection
Aspergillus infections:

Infections are macroscopic

There are mitotic spores at the end of each hypha: these can spread easily

More difficult to diagnose and treat compared to Candida infections

Higher mortality than Candida infections
Diagnosis of fungal infections:
1.
Sample acquisition
Skin; sputum; bronchoalveolar lavage; blood; vaginal swab/smear; spinal fluid; tissue biopsy
2.
Microscopy: rapid and cheap
Allows detection of distinct morphological features associated with a particular fungal species
3.
Culture and identification: slow and prone to contamination
Requires skilled sample collection
Positive identification allows susceptibility testing
Non-culture methods: antibody- and antigen-based detection assays detect: glucan; mannan; enolase;
proteinase
Disadvantage: none of these techniques has been validated for widespread use in laboratories
Describe briefly the main classes of antifungal agents
3 targets for antifungal therapy:
1.
Cell membrane: fungi primarily use ergosterol instead of cholesterol
2.
DNA synthesis: some compounds may be selectively activated by fungi, arresting DNA synthesis
3.
Cell wall: fungi have a cell wall, unlike mammalian cells
Cell membrane active antifungals:
Mode of action:

Inhibit synthesis of ergosterol in the fungal membrane

Act upon fungal cytochrome P450 enzymes
Classes:

Polyene antibiotics: amphotericin B; nystatin)

Azole antifungals: ketoconazole; itraconazole; fluconazole; voriconazole; miconazole; clotrimazole
DNA/RNA synthesis:
Pyrimidine analogues: flucytosine
Cell wall active antifungals:
The fungal cell wall is the primary target of antifungal therapy
Major components of the fungal cell wall: glucans and chitin
Mode of action: non-specific inhibition of ß-1,3 glucan synthase
Echinocandins: caspofungin acetate (Cancidas)
Case study:
Sprotrichosis: a subcutaneous mycosis which occurs due to trauma

Response time to fungal therapy is relatively slow
Microbiology 5: Antibiotic Resistance and Hospital Acquired
Infection
Definitions
Antimicrobial: any substance which interferes with growth and reproduction of a microbe (bacteria,
viruses and fungi)

A broad category of substances which includes detergents, soaps and disinfectants
Antibacterial: any substance which can reduce or eliminate bacteria
Antibiotic: a type of antimicrobial which is used as a medicine for humans and animals

Originally: referred to naturally occurring compounds

Now: refers to either synthetic derivatives of naturally occurring compounds or to purely synthetic
compounds
Nosocomial infections: hospital acquired infections which occur after 48 hours of admission
The scale/impact of the problem and its underlying causes
1940-1970s: the golden era of antibiotic discovery

Penicillin: the first widely used antibiotic

Cephalosporins: synthetic analogues of penicillin (i.e. they have the same structure as penicillin but
possess different side chains)
Misconceptions regarding bacteria:

Bacteria are unlikely to become resistant to more than 1 class of antibiotics

Bacteria cannot exchange genetic material (i.e. horizontal gene transfer is not possible)

Resistant bacteria are less fit
Evolution of resistance in bacteria:

Antimicrobial-resistant strains of Staphylococcus aureus have evolved including:
o
Penicillinase-producing Staphylococcus aureus: resistant to penicillin
o
Methicillin-resistant Staphylococcus aureus (MRSA): a significant cause of bacteraemia

Resistance in Staphylococcus aureus is due to the spread of 2 virulent strains which are resistant:
EMRSA-15 and EMRSA-16

E. coli is developing increasing resistance to ciprofloxacin
Infection with resistant organisms is associated with:

Increased mortality

Increased duration of hospital stays

Increased costs
Reasons for resistance
Resistance is emerging in bacteria due to the following factors:

Normal flora: composed of many bacterial cells

Organic biomass: there is significantly more bacterial biomass than plant organic biomass
There are many environmental bacterial reservoirs, including oceans and deep sea vents

Mutation rates are very high because generation times are low

There is a huge amount of genetic diversity which may contain resistance elements

Horizontal transfer of genetic material: occurs by conjugation, transformation and transduction

There is significant production and release of antibiotics into the environment:
90-180 million kg of antibiotics are used per year
The predominant use of antibiotics is in the agricultural industry to increase meat yield
Reasons for the high rate of hospital acquired infections
Hospitals are a potent source of infection as a result of:

Intervention methods including:
o
Lines: intravenous; central; arterial; CVP/pulmonary artery
o
Catheterisation
o
Intubation

Chemotherapy: this may reduce the innate immune response by causing reduced neutrophil and
macrophage function

Prophylactic usage of antibiotics: this may eliminate normal flora (i.e. commensals)

Inappropriate prescription of antibiotics

Prosthetic material in patients: e.g. joints and pacemakers
Bacteria present on abiotic surfaces are highly resistant to antimicrobial treatment

Dissemination due to vectors: e.g. hospital care workers, stethoscopes and computer terminals

Concentration of bacteria: e.g. in multi-bed wards and in ICUs
Mechanism of action of some important classes of antibiotics
Selective toxicity: antibiotics must be selectively toxic to bacteria in order to limit damage done to
eukaryotic host cells
Targets and mechanisms of antibiotic action:
1.
Cell wall components: antibiotics inhibit cell wall synthesis
Presence of a peptidoglycan cell wall in bacteria differentiates them from eukaryotic cells
E.g. penicillins (cephalosporins) and glycopeptides (vancomycin)
Bactericidal: kill bacteria
2.
Protein synthesis: antibiotics target ribosomes and inhibit protein synthesis
Bacteria have 70S ribosomes (30S + 40S); eukaryotic cells have 80S ribosomes (40S + 60S)
E.g. aminoglycosides (streptomycin) and tetracyclines
Bacteriostatic: inhibit replication of bacteria
3.
Metabolic targets: antibiotics inhibit nucleotide synthesis
There are sufficient differences in nucleotide synthesis between bacteria and eukaryotic cells
E.g. sulphonamides and trimethoprim
4.
Nucleic acid synthesis: antibiotics inhibit nucleic acid synthesis
Rifampicin: inhibits RNA synthesis (anti-tuberculosis)
Quinolones and fluoroquinolones (e.g. ciprofloxacin): inhibit DNA gyrase (urinary tract infections)
5.
Other targets: metronidazole treats infections caused by anaerobic bacteria
The nitro group of metronidazole is chemically reduced by ferredoxin
Ferredoxin is only present in anaerobic bacteria; therefore metronidazole is non-toxic to eukaryotic
cells
Mechanism of antibiotic action
Inhibition of cell wall synthesis
Inhibition of protein synthesis
Metabolic targets (inhibition of nucleotide synthesis)
Antibiotic families
Penicillins (cephalosporins)
Glycopeptides (vancomycin)
Aminoglycosides (streptomycin)
Tetracyclines
Sulphonamides
Trimethoprim
Inhibition of RNA synthesis
Rifampicin
Inhibition of DNA synthesis
Quinolones and fluoroquinolones (ciprofloxacin)
Other
Metronidazole
Mechanisms of antibiotic resistance
General mechanism of antibiotic action:
1.
The antibiotic is taken up into the bacterial cell
2.
It binds to its target
3.
It inhibits its target
4.
The bacterial cell dies
Bacteria have evolved mechanisms of antibiotic resistance:
1.
Decreased influx: the antibiotic is not taken up into bacteria
2.
Increased efflux: bacteria have specific membrane pumps which pump out the antibiotic
E.g. tetracycline resistance
3.
Drug inactivation: bacteria produce enzymes which cleave the antibiotic, inactivating it
E.g. penicillin resistance (penicillinases are produced which cleave penicillin)
4.
Target modification: the target site is modified so that the antibiotic can no longer bind to it
E.g. quinolone resistance and penicillin resistance (the structure of the penicillin binding protein is
modified)
5.
Target amplification: target site production is increased so that it outcompetes the antibiotic
E.g. sulphonamide resistance
Mechanisms of passing on antibiotic resistance:
Integrons: genetic structures which act as shopping trolleys for AbR cassettes

Integrons have 3 elements: a promoter, a single AbR cassette and a recombination site

AbR cassette 2 from another integron is integrated into the bacterial genome at the recombination
site of the original integron
Plasmids: circular, extrachromosomal DNA which are capable of independent replication

Plasmids often carry multiple AbR cassettes

They are transferred between different bacterial strains by transformation or conjugation

Bacterial cells contain many copies of a plasmid
Development of antibiotic resistance in Neisseria gonorrhoeae:
Neisseria gonorrhoeae has developed multiple antibiotic resistance mechanisms to various antibiotics
Antibiotic
Resistance mechanism
Mechanism of transfer
Penicillinase production (drug inactivation)
Plasmid transformation
Target modification
Point mutation
Tetracycline
Efflux pump (increased efflux)
Plasmid conjugation
Quinolone
Target modification
Point mutation
Penicillin
Some of the approaches to prevent the emergence of drug-resistant bacteria and nosocomial
infections
Strategies for the preventing the spread of resistance:

Hygiene

Monitor lines and change lines regularly

Single rooms

Screen high risk patients

Discharge patients as soon as possible

Avoid unnecessary prescription of antibiotics

Know local sensitivities

Always prescribe narrow spectrum antibiotics rather than broad spectrum antibiotics

Directly observed therapy (DOTS): completion of the antibiotic course

Use drug combinations

Antibiotic policies

Develop new drugs
Lecture summary:

Antibiotic resistance is increasing: this is a major problem as it costs lives and money

Hospitals are an important focus for spread of infection

New antibiotics are needed
Microbiology 6: Viral Properties
Describe the nature of viruses: their small size, mode of replication and diversity
Properties of viruses:

Size: 20-450 nm

Obligate intracellular parasites: i.e. viruses are dependent upon a host cell for replication; viruses are
inert outside a host cell

Structure: nucleic acid and protein; lipid and carbohydrate may be present

Unique mode of replication (different to all other organisms)

Diversity:
o
Viruses infect all species and contribute to evolution
o
Viruses may cause great plagues or be asymptomatic
Virus classification: the poxvirus family
Family
Poxviridae
Subfamily
Chordopoxvirinae
Genus
Orthopoxvirus
Species
Vaccinia virus
Strain
Lister
Other parameters used for classification include:

Type of disease caused: e.g. pox diseases, such as chickenpox and smallpox

Mode of transmission: e.g. arthropod born (arbo) viruses are transmitted by biting insects

Structure: viruses with common structures are likely to belong to the same family

Immunological relatedness: viruses which are immunologically related are likely to belong to the same
family

Nucleic acid sequence: viruses which have a common nucleic acid sequence are likely to belong to the
same family

Mode of replication (Baltimore classification): viruses with a common mode of replication are likely to
belong to the same family
Virus structure:
Nucleic acid: constitutes the viral genome

DNA or RNA

Linear or circular

Monopartite (i.e. a single segment) or segmented (i.e. multiple segments)

Double-stranded (ds) or single-stranded (ss)

Polarity of single-stranded nucleic acids: either + or −
o
+ polarity: the nucleic acid has the same sense as mRNA
o

− polarity: the nucleic acid has the complementary sense to mRNA
Size range: 3-1200 kb
Nucleocapsid: composed of viral proteins

Helical or icosahedral (an icosahedron is a 20-faced polygon)

Surrounds and protects the viral nucleic acid, especially while the virus is in transit
Lipid envelope: may be present

Derived from the host cell

Fragile: destroyed by organic solvents

May contain embedded proteins which allow the virus to bind to the host cell
Examples of virus structure:
Virus
Helical/icosahedral
Envelope
Tobacco mosaic virus (TMV)
Helical
−
Polio
Icosahedral
−
Influenza
Helical
+
Herpes simplex virus (HSV)
Icosahedral
+
Examples of viral nucleic acid:
Virus
Size (kb)
DNA/RNA
ss or ds
Segments
Polio
7.5
RNA
ss +
1
Measles
15.9
RNA
ss −
1
Influenza
13.6
RNA
ss −
8
HIV
10
RNA*
ss +
1 (diploid)
HSV
152
DNA
ds
1
HBV
3.2
DNA*
ds
Circular
*HIV and HBV are retroviruses and carry reverse transcriptase
HIV: packages RNA

Reverse transcriptase converts RNA  DNA which is integrated into the host cell genome
HBV: packages DNA

Reverse transcriptase converts DNA  RNA  DNA
Methods of measuring viruses:

Observation of disease symptoms in the host

Plaque assay: measures the number of infectious virus particles (i.e. the viral titre)

Electron microscopy: measures the total number of virus particles; analyses viral structure

Polymerase chain reaction: amplifies the viral genome and measure the total number of viral genomes
present

Immunological evidence of infection: serological evidence of infection is provided by a blood sample
Plaque assay: measures the viral titre
1.
Make a series of dilutions of virus particles
2.
Place the dilution on a culture of host cells
3.
The virus infects host cells and replicates
4.
Virus particles are released by host cell lysis and infect new host cells
5.
The virus destroys an area of host cells and forms a plaque (i.e. an area of dead cells)
6.
The area of the plaque and the dilution factor can be used to calculate the viral titre
Viral titres are expressed as plaque forming units per mℓ
Not all viruses can be titrated by plaque assay since they cannot be cultured: e.g. HBV
Virus replication:
One step growth curve:

Latent period: time taken from the point of infection until the first virus particles are produced

Eclipse phase: no virus is present; components of new virus particles are being synthesised

Mean burst size: the average increase in viral titre from infection to burst
A mean burst size of 50 implies that 50 new virus particles are produced on average per host cell
T4 bacteriophage
HSV
Host cell
E. coli
Human fibroblasts
Latent period
20 minutes
10 hours
Mean burst size
50
50
Mechanism of virus replication:
1.
Binding to host cell: this is often highly specific since host cells express specific surface receptors
2.
Penetration: e.g. enveloped viruses fuse with the host cell membrane
3.
Eclipse phase: expression of virus proteins and replication of viral nucleic acid; highly regulated
Temporal control: enzymes are synthesised before structural proteins
Quantitative control: enzymes are produced in lower concentrations than structural proteins
4.
Assembly: production of new infectious virus particles; spontaneous
5.
Release: virus particles are released either by cell lysis or by budding
Cell lysis: all virus particles are released from the cell
Budding: virus particles continuously bud out of the cell over a long period causing a persistent
infection
Binding: occurs via a specific interaction between viral surface proteins and receptors expressed on the
surface of the host cell
Virus
Viral surface protein
Receptor
HIV
gp120
CD4
Epstein-Barr virus (EBV)
gp340
CD21
Influenza
Haemagglutinin (HA)
Sialic acid
Penetration:
Enveloped viruses: enter the host cell by fusion between the virus envelope and host cell membrane
Examples:

HIV and measles: fuse at the cell surface with the plasma membrane (able to fuse at neutral pH)

Influenza: fuses with acidified intracellular vesicles (requires a low pH for fusion)
Influenza entry:
1.
Binding to sialic acid
2.
Endocytosis of the virus into an endosome
3.
Acidification of the endosome: this causes conformational changes in HA
4.
Fusion between the virus envelope and the cell membrane (of the endosome)
5.
Release of RNA into the cytosol
Non-enveloped viruses: cause disruption of the integrity of the host cell membrane; the viral genome or
core enters the cytosol
Examples:

Polio virus

T4 in E. coli
Eclipse phase:
1.
Disassembly of virus particles: no infectious virus particles are present
2.
Expression of viral proteins: this is highly regulated- temporal and quantitative control
3.
Replication of viral nucleic acid
Assembly: viral proteins and nucleic acids are assembled to form new infectious virus particles
Release:
Release may be…

Spontaneous: e.g. tobacco mosaic virus (TMV)

Over a long period

Complex with multiple stages: e.g. vaccinia virus (the smallpox vaccine)
Release may occur at the cell surface or internally
Budding: lytic infections e.g. HIV

Viral envelope glycoproteins are inserted into specific areas of the host cell plasma membrane

Newly synthesised virus particles are attracted to these areas

The plasma membrane forms buds around the virus particles and the buds pinch off the cell
Latency: latent infections e.g. HSV; varicella zoster virus (VZV)

After lytic infection viruses enter host cells (neurones) and may remain dormant

The viral genome is maintained as circular DNA (episomes) in the nucleus of host cells

The viral genome may reactivate many years later to cause recurrent infections:
o
HSV causes cold sores
o
VZV causes shingles
Examples of virus replication HIV, polio, influenza, herpes simplex virus
Baltimore classification of viruses: viruses are grouped according to their replication strategy
Virus
Replication strategy
Examples
ss − RNA
Encode a virus RNA-dependent RNA polymerase: RNA  mRNA
Orthomyxo; paramyxo; rhabdo
ds RNA
Encode a virus RNA-dependent RNA polymerase: RNA  mRNA
Reo; rota
RNA is directly translated since it is the same sense as mRNA
Picorna; alpha; flavi; corona
Encode reverse transcriptase: RNA  ds DNA  mRNA
Retro
ss DNA
DNA  ds DNA  mRNA
Parvo
ds DNA
ds DNA  mRNA
Pox; adeno; herpes; papilloma
ss + RNA
Polio virus:
Properties:

Size: 20 nm

ss + RNA genome

Icosahedral capsid

Very stable to acid pH and proteases
Replication: ss + RNA  ss − RNA  mRNA
Genome replication occurs via a complementary RNA intermediate
1.
mRNA is translated into a single giant polyprotein
2.
The polyprotein is cleaved into mature individual proteins by proteolytic enzymes
Influenza virus:
Properties:

Segmented ss − RNA genome (8 segments)

Helical capsid

Enveloped
Replication: ss − RNA  mRNA and cRNA
1.
The genome is transcribed into mRNA and cRNA by virus RNA-dependent RNA polymerases
2.
mRNA is translated into virus proteins
3.
cRNA is converted into virus RNA (vRNA): this is viral nucleic acid
HIV:
Properties:

Retrovirus: HIV replicates via reverse transcription

Size: 110 nm

Diploid ss + RNA genome

Enveloped

The genome encodes:
o
Functional proteins: gag, pol and env
o
Regulatory proteins: tat and rev
Replication: ss + RNA  ds DNA  mRNA
1.
The RNA genome is transcribed into ds DNA by reverse transcriptase
2.
DNA integrates into the host cell DNA to form a provirus and may remain dormant
Provirus: a virus genome in the form of DNA which has integrated itself into the DNA of a host cell
This allows vertical transmission of the virus when host cells replicate
3.
DNA is expressed by host RNA polymerase II to produce HIV mRNA; this is regulated by splicing
Herpes simplex virus:
Properties:

Size: 130 nm

Linear ds DNA

Icosahedral capsid

Enveloped

HSV infection occurs via skin abrasions

It may replicate via a lytic cycle or it may become latent

HSV-1 causes cold sores

HSV-2 causes genital herpes
Lytic replication: HSV replicates in the nucleus and undergoes a cascade of gene expression
1.
IE (initial early)/α genes are expressed
2.
DE (delayed early)/ß genes are expressed
3.
Late/γ proteins genes are expressed
N.B. Expression of each class requires prior expression of genes of the previous class
Functions of proteins:

IE proteins: regulatory function

DE proteins: replicative function (e.g. DNA polymerase)

Late proteins: structural function
Define the following terms as used in the description and classification of viruses: DNA virus, RNA
virus, capsid type, enveloped, non-enveloped
Microbiology 7: Viral Disease
How are viruses transmitted?
Virus transmission occurs by: gaining entry, replication and dissemination to new hosts
The host is hostile:



Non-specific defences:
o
Skin: forms an impervious barrier; viruses can only enter via breaks in the skin
o
Mucous membranes: swept by cilia and mucous secretions
o
Stomach: acidic pH and proteases form a hostile environment
Innate immunity:
o
Complement
o
NK cells
o
Phagocytes
o
Interferons
o
Fever
o
Inflammation
o
Apoptosis: may not allow the virus to complete its replication cycle
Adaptive immunity: specific for viral proteins and virus infected cells
o
Antibodies
o
Cytotoxic T cells
Factors affecting virus transmission:

Particle stability:
o
Presence of a lipid envelope affects particle stability: enveloped viruses are less stable
The lipid envelope is fragile: if it is destroyed, viral proteins are removed and infectivity is
destroyed
o
Viruses are usually spread by close contact: e.g. influenza virus in winter
In winter we are indoors and closer together; therefore this helps influenza transmission
o



Some viruses are able to spread over long distances: e.g. foot and mouth disease virus
Duration of virus shedding:
o
Short duration viruses cause acute infections and require higher viral titres: e.g. rhinoviruses and
influenza
o
Long duration viruses cause chronic infections and require lower viral titres: e.g. HSV-1
Virus concentration:
o
Viruses causing acute infections tend to be released in higher concentrations
o
E.g. rotaviruses may be shed at 1010 pfu/ml
Availability of new hosts:
o
Population size: e.g. measles virus may cause epidemics in isolated communities
There must be a sufficient number of hosts to continue transmission of measles
o
Availability of animal reservoirs: e.g. yellow fever virus (YFV)
YFV is transmitted between biting mosquitoes and humans (urban cycle)
YFV is also transmitted between biting mosquitoes and monkeys (jungle cycle)
Entry routes:

Respiratory tract: influenza; mumps; measles; variola; varicella-zoster virus; rhinovirus

Skin: papilloma viruses; HSV-1 and HSV-2; rabies (bites); yellow fever virus (biting mosquitoes)

Blood products: HIV; HBV; HCV

Genital tract: HIV; HSV-2; HPV 16 and HPV 18

Alimentary canal: polio virus; Hepatitis A virus (HAV); rotavirus
Outcome of infection: cell death, persistent infection, cell transformation / cancer
Factors which influence outcome of infection:

Virus dose

Route of entry: e.g. smallpox causes variolation by an unusual route (via the skin)

Age, sex and physiological state of the host:
o
HBV: neonatal infection results in a greater chance of establishing chronic infection
Males have a greater chance of developing Hepatitis B
o
Epstein-Barr virus (EBV): children are asymptomatic; young adults develop glandular fever
o
VZV: symptoms of chickenpox are more severe in adults than in children
Outcomes of infection:

Cell death

Persistent (i.e. latent) infection

Cell transformation and cancer

Local or systemic infection
Cell death
Virus
Cell type affected
Disease caused
Poliovirus
Motor neurones
Paralysis
Rotavirus
Gut epithelium
Diarrhoea
HIV
CD4+ T lymphocytes (T helper cells)
Immunodeficiency
HBV
Hepatocytes
Hepatitis
Rabies virus
Perkinji cells in the cerebellum
Hydrophobia
Virus
Cell type affected
Disease caused
HBV
Hepatocytes
Hepatitis
Measles
Neurones
Subacute schlerosing
Persistent infection
panencephalitis
HSV-1 and HSV-2
Neurones
Cold sores; genital herpes
VZV
Neurones
Chickenpox; shingles
Virus
Cell type affected
Disease caused
HBV
Hepatocytes
Hepatocellular carcinoma
HPV 6 and HPV 11
Epithelial cells
Common warts (benign)
HPV 16 and HPV 18
Epithelial cells
Cervical/penile carcinoma
EBV
B cells
Burkitt’s lymphoma; nasopharyngeal
carcinoma
Cell transformation and cancer
Local infections:

Replication only occurs locally

Shorter incubation period (i.e. the period between infection and appearance of symptoms)

E.g. rhinovirus; rotavirus; influenza; HPV 6 and HPV 11
Systemic infections:

Replication proceeds through multiple phases

Longer incubation period

Infection may disseminate via the bloodstream or the nerves

Infection is more severe

Clearance of infection is more dependent on cellular immunity (esp. CTLs)

E.g. VZV; variola virus; measles
Give examples of different viruses associated with infectious disease in humans and describe the way
in which they cause disease
Virus release
From
Virus
Blood
YFV; HBV; HCV; HIV
Skin
HPV; VZV; measles; variola
Gut
Polio virus; rotavirus; HAV
Respiratory system
Rhinovirus; influenza virus; VZV; measles; variola
Saliva
EBV; rabies virus; mumps
Semen
HIV; HBV
Breast milk
HCMV
Placenta
Rubella; HCMV
Influenza virus antigenic shift and drift
Influenza virus causes new epidemics
It does this by undergoing antigenic variation to produce new strains which evade existing immunity
Existing immunity is of limited value against new strains
Methods of antigenic variation:

Antigenic drift

Antigenic shift
Influenza viruses from avian species infect humans and may acquire human-human transmission
Components of influenza virus:

Lipid envelope: contains embedded glycoproteins
o
Haemagglutinin (HA): important in causing influenza epidemics and pandemics
o
Neuraminidase (NA)

M2: ion channel

Matrix protein

Nucleoprotein

Polymerase: PB1, PB2 and PA

RNA genome
Haemagglutinin (HA):

Structure: trimer

Each monomer consists of 2 polypeptides: HA1 and HA2
o
HA1: globular head
o
HA2: stalk

HA binds to sialic acid (a cell surface receptor) via the sialic acid binding pocket at the top of HA

Antibodies bind to HA and sterically block its ability to bind to cells: this prevents infection

HA2: contains a fusogenic peptide buried internally in HA
Fusogenic peptide: necessary to induce fusion between the virus envelope and the host cell membrane
Influenza entry:
6.
Binding: HA binds to sialic acid
7.
Endocytosis of the virus into an endosome
8.
Acidification of the endosome: this causes conformational changes in HA
9.
Fusion between the viral envelope and the host cell membrane (of the endosome)
10. Release of RNA into the cytosol
Conformational changes: caused by the low pH
a.
The fusogenic peptide is transferred to the top of HA where it can engage with the host cell
membrane and induce fusion
b. Alpha helices at the bottom of HA are rearranged so that the membrane anchor is moved to the top of
HA; therefore the virus envelope and host cell membrane are moved closer together
Antigenic drift:

Involves the H3 HA

Amino acid mutations occur in HA and accumulate with time

There is continual change in HA which is mediated by antibodies: new HA variants which escape
existing antibodies are selected

It is a less dramatic change than antigenic shift
Antigenic shift:

The genome of each influenza particle is segmented: it consists of 8 RNA segments

The segmented genome is crucial for the ability of the virus to cause antigenic shift

If a cell is simultaneously infected by human influenza virus and avian influenza virus: the viral RNAs of
both strains travel to the nucleus and replicate simultaneously

Reassortment of genetic material occurs during the RNA replication of both strains

New virus strains may acquire genetic material from both parental strains

Danger: if the new strain acquires the avian influenza gene encoding HA, this may cause a pandemic
since existing human antibodies are completely ineffective avian HA
E.g. the 1918 H1N1 pandemic killed 40 million people with a mortality rate of 2.5%
Birds (i.e. avian species) are natural hosts for influenza viruses:

16 different HA subtypes: H1-H16

9 different NA subtypes: N1-N9

All HA and NA subtypes are present in avian species

Only 3 HA subtypes (H1-H3) and 3 NA subtypes (N1, N2 and N8) are present in man

Reassortment of HA and NA genes has the potential to produce a large variety of influenza strains

Avian influenza viruses may be transmitted to other species, including humans
H5N1 influenza:

H5N1 influenza viruses are endemic in avian species; they are spread globally by migratory birds

H5N1 is transmitted to other birds: e.g. in the poultry industry, the density of birds is high, which leads
to high virus concentrations

H5N1 has transmitted from birds to man (initially in S and SE Asia; now elsewhere)

H5N1 has a very high mortality rate in man (63%)

Human-human transmission has not developed yet… but there is a serious threat of this if the virus
adapts
Mechanisms by which H5N1 may adapt to man:

Better binding to human cells

Better replication in human cells

Better evasion of human innate immunity

Better transmission between humans
N.B. adaptation may be multi-factorial
Binding to host cells:


Sialic acid is present on the surface of both human and avian cells, but with different linkages:
o
Avian cells have α-2,3 sialic acid
o
Human cells have α-2,6 sialic acid
HA binds to sialic acid on the surface of the host cell:
o
HA from avian influenza viruses binds to α-2,3 linkages
o
HA from human influenza viruses binds to α-2,6 linkages

The sialic acid binding pocket on the surface of HA has a complementary fit to sialic acid

Surrounding amino acid residues may induce this complementary fit
o
Amino acid residues 226 and 228 influence the ability of HA to bind to sialic acid
o
Therefore it these residues are altered, this will alter the HA binding specificity
Replication efficiency in host cells:


Avian influenza and human influenza have different amino acids at amino acid residue 627 of PB2
o
Avian influenza virus: glutamic acid; replicates well in avian cells but poorly in human cells
o
Human influenza virus: lysine; replicates well in both human and avian cells
Experimental change (E627K): glutamic acid is changed to lysine at amino acid residue 627 of PB2
o
This allows better replication of avian influenza in human cells
o
Avian influenza strains that have adapted to man have lysine at amino acid residue 627
Resistance to host innate immunity:

NS1 (non-structural protein 1): confers resistance to interferon-mediated inhibition of influenza
replication

Avian NS1: resistant to avian interferon but less resistant to human interferon

Changes in avian NS1 may increase the resistance of avian influenza virus to human interferon and
hence increase its virulence in man
Anti-influenza virus drugs:
Amantidine and rimantidine:
Mechanism of drug action: inhibition of influenza virus by raising the pH of endosomes

Low pH of endosomes is critical for the conformational changes in HA which induce fusion of the viral
envelope and host cell membrane

Therefore if the pH of endosomes is increased, HA does not undergo conformational changes to
enable fusion
Disadvantage: drug-resistance mutations arise quickly; therefore these drugs are of limited clinical use
Relenza and tamiflu:
Mechanism of drug action: inhibition of the viral neuraminidase (NA)
NA: an enzyme which removes sialic acid from the cell surface

It prevents budding influenza particles from sticking to the host cell surface and aggregating

Therefore if the viral NA is inhibited, influenza dissemination is inhibited
The DOH has purchased stocks of tamiflu
HIV life cycle and epidemic
The HIV epidemic:
Los Angeles: there was an unusual cluster of opportunistic infections in young men, including:
pneumocystis carinii; disseminated cytomegalovirus; mucosal candida; chronic HSV infection

Patients had T lymphocyte dysfunction

Initial patients: homosexuals; intravenous drug users
Disease spread to haemophiliacs, blood transfusion recipients and sexual partners of individuals at risk
Human immunodeficiency virus (HIV) was isolated and identified as the cause of AIDS
More than 95% of AIDS is in LEDCs
There is no vaccine, no cure once infected and without drugs, death is the inevitable outcome
Retrovirus:

Enveloped virus

RNA genome

Replicate via a DNA intermediate: RNA  ds DNA  mRNA

Possess reverse transcriptase and integrase:
o
Reverse transcriptase converts RNA into DNA
o
Integrase: integrates DNA into the host cell genome
HIV structure:

Core: contains 2 copies of RNA

The core is surrounded by a capsid, which is in turn surrounded by a lipid envelope

p24: viral protein which constitutes the capsid

gp120 and gp41: viral envelope glycoproteins which are embedded in the envelope
These proteins are analogous to HA in influenza
HIV genome:

Diploid ss + RNA

Lentivirus: subgroup of retroviruses which have more complex genomes, which contain extra
regulatory genes (in addition to 3 common genes possessed by all retroviruses)

Genes which are common to all retroviruses: encode structural proteins
o
gag (group-specific antigen): encodes viral capsid proteins (i.e. p24)
o
pol (polymerase): encodes viral enzymes (i.e. reverse transcriptase and integrase)
o

env (envelope): encodes viral envelope proteins (i.e. gp120 and gp41)
Regulatory genes: expressed by differential splicing
o
Differential splicing: primary mRNA transcript  smaller mRNAs  small regulatory proteins
HIV infection:
Transmission methods:

Sexual: initially homosexual transmission; now predominantly heterosexual transmission

Intravenous drug use

Mother to baby transmission

Contaminated blood products (particularly initially)
Mode of entry: viral gp120 binds to cells expressing CD4 and a co-receptor (CCR5 or CXCR4)

gp120 embedded in the viral envelope binds to CD4 on the plasma membrane of the host cell

This causes a conformational change

gp41 mediates fusion of the viral envelope and the plasma membrane

Fusion requires the presence of a co-receptor:
o

Co-receptor molecules (CCR5 and CXCR4): 7-transmembrane molecules which normally function
as chemokine receptors
Viruses are tropic (i.e. they bind preferentially) to either CCR5 or CXCR4:
o
CCR5 tropic viruses: macrophage tropic; this is the initial tropism in HIV infection
o
CXCR4 tropic viruses: T cell tropic; this is the subsequent tropism in HIV infection
Human polymorphism in CCR5:

Mutant CCR5 allele: has a 32 nucleotide deletion which causation termination of CCR5 after
transmembrane domain 4

Mutant CCR5 is non-functional: it does not function as a co-receptor for HIV-1

The mutant CCR5 allele is found Caucasians; not in Japanese or Africans

Homozygotes: resistant to HIV infection because transmission is predominantly cause by CCR5 tropic
viruses

Heterozygotes: less resistant to HIV infection than homozygotes

Both homozygotes and heterozygotes for the mutant CCR5 allele may still be infected by CXCR4
tropic viruses

The mutant CCR5 allele may still exist at a high frequency due to natural selection
HIV pathogenesis:
1.
2.
Acute infection: 2-3 months

Active virus replication occurs (burst of viral replication)

Viraemia occurs: i.e. the virus enters the bloodstream

CD4+ cell count declines temporarily
Minor/asymptomatic phase: 15 years

Host immune response starts: HIV-specific CD8+ CTLs are produced

Viral titres decline

CD4+ cell count recovers and plateaus; CTL count increases and plateaus
3.

Virus replication continues in lymph nodes and variation occurs

The balance between the T cell response and ongoing viral replication maintains the virus but
confers protection to the host

The patient shows minor symptoms or is asymptomatic
AIDS: 1-3 years

Host immune response is overcome: virus variants are no longer controlled by CTLs

Viral titres increase

CD4+ cell and CTL counts decline to low levels

The patient develops AIDS, suffers opportunistic infection and dies
Outcomes of HIV infection:
Outcome
CTL response
CD4+ cell and CTL counts decline after
Slow progressors
Good
10-15 years
Long term non-progressors
Strong
15+ years
Rapid progressors
Poor
1-5 years
HIV vaccine:
Antibody-based vaccines: target HIV env protein gp120

Some anti-gp120 antibodies neutralise HIV

Problem: HIV undergoes antigenic variation and escapes from the effects of antibody; therefore the
vaccine is ineffective
CTL-based vaccines: target internal conserved epitopes in gag, pol and nef
The vaccines stimulate high and long-lasting levels of CTLs and kill HIV-infected cells
Problem: CTLs are only effective against infected cells; therefore the vaccine cannot prevent infection
Possible solution: an effective vaccine requires a combination of antibodies and CTLs
Anti-HIV drugs:
AZT (zidovudine, retrovir): nucleoside analogue
Mechanism of action: chain terminator
Advantage: specific to HIV-infected cells- AZT is a better substrate for the HIV reverse transcriptase than
the host cell DNA polymerase
Dideoxycytidine (ddC) and dideoxyinosine (ddI) 3TC, (lamivudine): nucleoside analogues
Disadvantage: have some toxicity
Ritonavir, saquinovir, indinavir: protease inhibitors
Mechanism of action: target the HIV aspartate protease by preventing cleavage of HIV polyprotein
precursors into mature proteins
Classes of antiretroviral drugs:

Nucleoside/nucleotide reverse transcriptase inhibitors

Non-nucleoside reverse transcriptase inhibitors

Protease inhibitors

Fusion (gp41) inhibitors
HIV drug resistance:
1.
Mutations arise in HIV due to the error prone nature of reverse transcriptase and RNA pol II
2.
With time the virus becomes resistant to a single drug
3.
If the drug is changed, mutations arise again
Solution:

HAART (highly active antiretroviral therapy): use several drugs simultaneously

Advantage: it is more difficult for the virus to develop multidrug resistance

Disadvantage: expensive
Virus immune evasion
Mechanisms of immune evasion:

Antigenic variation: viruses either evade either the antibody response or the CTL response
E.g. influenza; HIV; HCV

Hiding: viruses establish a latent infection in which virus proteins are not expressed; therefore the
infected cell is not recognised by the immune system
E.g. HSV hides in neurones

Viruses express proteins which inhibit the immune response in the following ways:
o
Proteins block antigen presentation of viral peptides via class I MHC molecules; therefore CTLs do
not recognise virus infected cells
o
Proteins block recognition of virus infected cells by NK cells
o
Secreted proteins capture cytokines, chemokines or interferons (in solution)
o
Proteins block intracellular signalling pathways or apoptosis
Examples of immune evasion:
Vaccinia virus: encodes a protein (B15) which can control the body temperature of the infected host

Mechanism of action: B15 only binds to human IL-1ß (it does not bind to any other cytokines)

IL-1ß: the principal endogenous pyrogen that regulates body temperature
Interferons (IFNs): specific, soluble glycoproteins which induce an antiviral state in cells which express the
appropriate surface receptors

Type I IFNs (IFN-α and IFN-ß): bind to a common receptor (type I IFN-R)

Type II IFNs (IFN-γ): binds to a different receptor (type II IFN-R)

Type III IFNs (IFN-λ): bind to another receptor
All interferons have:

Direct antiviral activity

Immunomodulatory roles: e.g. IFN-γ promotes the Th1 response
Mechanism of interferon action: blocking virus replication
1.
The cell signalling pathway is activated
2.
The IFN gene is transcribed
3.
IFN is expressed
4.
IFN is secreted and binds to IFN receptors
5.
Engaged receptors trigger another signalling pathway
6.
Antiviral proteins are expressed (this is induced by IFN)
7.
Antiviral proteins remain in the cytoplasm and mediate antiviral activity
If virus infection occurs: antiviral proteins block virus replication
Protein kinase (PKR): an antiviral protein which is induced by IFN and activated by ds RNA
1.
PKR activation: inactive PKR + ds RNA  active PKR
2.
Auto-phosphorylation: active PKR + ATP  active PKR + ADP
3.
eIF2-α inactivation: active eIF2-α + ATP + active PKR  inactive eIF2-α + ADP
4.
Inhibition of protein synthesis: viral and cellular protein synthesis stops and the cell dies
Vaccinia virus: gene E3L encodes an intracellular protein (E3) which binds to ds RNA

Viruses lacking gene E3L: avirulent

Mechanism of action of E3: binds to ds RNA and inhibits PKR activation; therefore virus replication
continues
Vaccinia virus: produces soluble interferon receptors which are secreted

Mechanism of action: the virus produces and secretes soluble proteins which mimic cellular proteins
and capture interferons in solution; therefore it blocks the ability of interferon to bind to host cell
interferon receptors

B18 blocks type I IFN: B18 binds to type I IFN in solution and on the cell surface
Interfering with interferon:

Many mammalian viruses interfere with interferon; if they did not they would probably not exist
Virus-host evolution:

Threat of viruses has driven the evolution of interferons and shaped the human genome

Threat of interferons has driven the evolution of viruses and shaped the virus genome
Microbiology 8: Prevention and Treatment of Viral Disease
Prevention is better than cure; give examples of other viral infections for which vaccination is a
successful strategy

Vaccinia virus (the smallpox vaccine) has eradicated variola virus (smallpox)

Vaccines have controlled several diseases including: diphtheria; tetanus; yellow fever; pertussis;
measles; mumps; rubella; poliomyelitis

New vaccines are needed for other unchecked diseases: e.g. AIDS; malaria
The eradication of smallpox and lessons from this for control of other diseases
Smallpox (variola virus): the first and only disease which has been eradicated

1796: Edward Jenner produced a vaccine

1980: the WHO certified the eradication of smallpox
Smallpox eradication was possible due to the following factors:

Smallpox did not have an animal reservoir (i.e. smallpox was a strictly human disease)

Smallpox did not cause latent or persistent infections (i.e. smallpox was an acute disease)

Easily recognisable disease

The vaccine was effective against all strains of smallpox (i.e. all strains of variola virus)

Vaccine properties: effective; potent; low cost; abundant; heat stable; easy to administer

Determination by the WHO to eradicate smallpox
Reason for the effectiveness of smallpox vaccination:

Outer envelope proteins of vaccinia virus and variola virus are highly conserved

Capsid proteins of vaccinia virus and variola virus are highly conserved
These facts were only discovered after the eradication of smallpox, so…
Lesson: to have an effective vaccine, you don’t need an understanding of all the antigens recognised by
the immune system  if the vaccine works, use it!
Considerations for vaccine development:

Which antigens should be used (for antibody-based vaccines)?

What type of immunity is needed? Humoral (antibody-mediated) or cell-mediated?
o


In the case of humoral immunity: are secretory (IgA) or systemic (IgG) antibodies needed?
When is the immunity needed?
o
When does the pathogen induce disease?
o
Consider exposure in endemic areas (i.e. vaccination before travelling)
How should the vaccine be administered?
Types of vaccine:
Passive vaccines:

Immune globulin (e.g. for rabies; infants born to HBV positive mothers)

Maternal antibodies
Advantages: offer immediate protection
Disadvantages: protection is short-lived; cause serum sickness; induce immune responses to foreign
proteins
Live vaccines:

Attenuated forms of virulent organisms (e.g. yellow fever virus)

Organisms which are immunologically related to viruses (e.g. vaccinia for smallpox)

Maternal antibodies
Advantages: induce both T cell and antibody responses; protection is long lasting; low cost; easy to
manufacture and administer
Disadvantages: unsafe (may revert to virulence or cause serious infection in immunocompromised people);
heat labile; may be shed into the environment

Many virus vaccines used in humans are live attenuated vaccines
Dead vaccines: antigen preparations which have been chemically treated to inactivate infectivity and
toxicity

Whole viruses (e.g. rabies)

Specific viral proteins (e.g. surface antigen for HBV; HA for influenza)

Peptides to important epitopes
Advantages: safe (provided that the inactivation is complete)
Disadvantages: require multiple administration (i.e. boosters) to achieve solid immunity; may require
adjuvants; protection is short-lived; high cost; reduce cell-mediated immunity
Sabin (live) polio vaccine
Salk (dead) polio vaccine
Methods of producing new vaccines:
Genetically engineered subunit vaccines:
1.
Identify the gene encoding the desired antigen
2.
Express the gene: clone the gene using vectors
3.
Purify the protein to use as an immunogen
Advantages: safe; specific; cheap; abundant
E.g. Hepatitis B vaccine
Genetically engineered live recombinant viruses:
1.
Identify the gene encoding the desired antigen
2.
Express the gene: insert the gene into the genome of a live virus to produce a recombinant virus
3.
Use the live recombinant virus as the vaccine: this simultaneously expresses the antigen and delivers it
to the immune system
This method enables the development of polyvalent vaccines which target multiple antigens
simultaneously
Explain why it is difficult to develop drugs which selectively act against viral infections
Antiviral chemotherapy:
Must inhibit the virus without toxicity for the host
Viruses have fewer targets than bacteria
Targets in viruses: binding to the host cell; replication (esp. enzymes); assembly; dissemination
Give examples of classes of drugs which have been used successfully in antiviral therapy
Anti-herpes virus drugs:
Acyclovir (ACV) (Zovirax): nucleoside analogue

Active against HSV-1 and HSV-2

Apply topically

Activity: requires HSV thymidine kinase (TK) and DNA polymerase

Mode of action: once incorporated into the DNA, ACV blocks further elongation

Specificity: ACV is active in HSV-infected cells and not in uninfected cells
o
ACV is a better substrate for HSV TK than host cell TK
o
ACV triphosphate is a better substrate for HSV DNA polymerase than host cell DNA polymerase
Ganciclovir and cidofovir: nucleoside analogues

Used for human cytomegalovirus (HCMV) infections

Specificity: due to use by viral DNA polymerase

Cidofovir must be given intravenously and has some renal toxicity
Describe the strategies underlying the search for novel antiviral agents
Viruses: friend or foe?
Viruses are valuable research tools:

For studying cell biology and immunology

For gene therapy

As new vaccines

As tools for molecular biology