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Bacterial Pathogenesis
Bacterial infection and antibiotics
--------------Bacterial pathogenesis
(细菌致病机制)
• References:
1. Murray, P. et al., Medical Microbiology (5th edition)
2. Samuel Baron, Medical Microbiology (4th edition)
3. Wilson, B.A. et al., Bacterial Pathogenesis: A
Molecular Approach (3rd edition)
4. Falkow, S. and Miller, V.L., Molecular Genetics of
Bacterial Pathogenesis: A Tribute to Stanley Falkow
Outline
Bacterial Pathogenesis
• Introduction
• Host Susceptibility
• Pathogenic Mechanisms
• Virulence Factors
• Steps in Successful Infection
pathogen
Opportunistic pathogens
The significance of normal flora
•
constitute a protective host defense
mechanism: Competition of nutrients and receptors
Metabolic substances by normal flora: e.g.,
bacteriocins, antibiotics, etc.
•
•
serve a nutritional function: several B
vitamins and vitamin K
keep our immune systems in tune
normal flora share many antigenic
determinants with pathogenic organisms
• Definition: normally nonpathogenic
microorganisms capable of causing an infection in
an immunosuppressed host.
• Conditions of causing diseases by opportunistic
pathogens:
o Alteration of colonization sites
o Declination of host immune system function
Introduction of Bacterial Pathogenesis
What a real pathogen will do?
1.
Infection: growth and multiplication of a microbe in or
on the body with or without the production of disease.
2.
The capacity of a bacterium to cause disease reflects
its relative “Pathogenicity （致病性）.”
3.
Virulence（毒力） is the measure of the
pathogenicity of a microorganism.
4.
Pathogenesis refers both to the mechanism of
infection and to the mechanism by which disease
develops.
Host Susceptibility
Obligate pathogens
are more virulent and can
cause diseases in a normal
person.
1.
Susceptibility to bacterial infections
=> Host Defenses vs Bacterial Virulence
2.
Host Defenses:
- Barriers (skin & mucus) – first line
- Innate Immune Responses (complement, macrophages &
cytokines) – the early stage
- Adaptive Immune Responses (Ag-specific B & T cells) – the
later stage
3.
Host defenses can be comprised by destructing barriers or
defective immune response.
e.x. Cystic Fibrosis => poor ciliary function => NOT clear mucus
efficiently from the respiratory tract => Pseudomonas aeruginosa
=> serious respiratory distress.
Entry into the human body
Opportunistic pathogens
are typically members of
normal flora and cause
diseases when they are
introduced into
unprotected sites, usually
occur in people with
underlying conditions.
Pathogen ↔ Disease
The most frequent portals
of entry- Mucus
- Skin
• Koch‘s postulates (科赫法则)
– the pathogen must be present in every case of the
disease
– the pathogen must be isolated from the diseased host
& grown in pure culture
– the specific disease must be reproduced when a
pure culture of the pathogen is inoculated into a
healthy susceptible host
– the pathogen must be recoverable from the
experimentally infected host
Routes:
Ingestion, inhalation,
trauma, needles, catheters,
arthropod bite, sexual
transmission
: infection
: shedding
Stanley Falkow, Ph.D
Molecular Koch's postulates
•
Microbiologist and a professor of microbiology
and immunology at Stanford University School of
Medicine
•
•
The father of molecular microbial pathogenesis,
which is the study of how infectious microbes and
host cells interact to cause disease at the
molecular level.
•
•
He formulated molecular Koch's postulates,
which have guided the study of the microbial
determinants of infectious diseases since the late
1980s.
•
•
President of the ASM (1997- 1998); elected
member of the Institute of Medicine, the National
Academy of Sciences, and the National Academy
of Arts and Sciences. Fellow of AAAS, UK-FRS
(Foreign member)
•
The prestigious Lasker Award for medical
research (2008)
•
•
•
The iceberg concept of infectious disease
Poliomyelitis（脊髓灰质炎） in a child
0.1-1% of infections are
clinically apparent
"The phenotype or property under investigation should be associated with
pathogenic members of a genus or pathogenic strains of a species."
Additionally, the gene in question should be found in all pathogenic strains
of the genus or species but be absent from nonpathogenic strains.
"Specific inactivation of the gene(s) associated with the suspected
virulence trait should lead to a measurable loss in pathogenicity or
virulence." Virulence of the microorganism with the inactivated gene must
be less than that of the unaltered microorganism in an appropriate animal
model.
"Reversion or allelic replacement of the mutated gene should lead to
restoration of pathogenicity." In other words, reintroduction of the gene into
the microbe should restore virulence in the animal model.
"The gene, which causes virulence, must be expressed during infection."
"Immunity must be protective.“
For many pathogenic microorganisms, it is not currently possible to apply
molecular Koch's postulates to a gene in question. Testing a candidate
virulence gene requires a relevant animal model of the disease being
examined and the ability to genetically manipulate the microorganism that
causes the disease. Suitable animal models are lacking for many important
human diseases. Additionally, many pathogens cannot be manipulated
genetically.
Characteristics of Pathogenic
Bacteria
1. Transmissibility
classical
clinical disease
2. Adherence to host cells
less severe
disease
3. Invasion of host cells and tissue
4. Evasion of the host immune system
Rubella（风疹）
50% of infections are
clinically apparent
5. Toxigenicity
asymptomatic infection
Spectrum
of virulence
A bacterium may cause diseases by
1. Destroying tissue (invasiveness)
2. Producing toxins (toxigenicity)
Rabies（狂犬病）
100% of infections
are clinically apparent
3. Stimulating overwhelming host immune
responses
Pathological Mechanisms of Bacterial
Infections
1.
Bacteria-mediated
Pathogenesis
2.
Host-mediated
Pathogenesis
3.
Bacterial virulence
factors
Bacterial Virulence Mechanisms
=> bacterial factors
causing diseases
Adopted from Samuel Baron “Medical Microbiology”
Bacterial virulence factors
Adhesins
Pili (fimbriae)
Nonfimbrial adhesins
Invasion of host cells
Tissue damage
Growth byproducts
Tissue-degrading enzymes
Immunopathogenesis
Toxins
Exotoxins (cytolytic enzymes
and A-B toxins); enterotoxins;
superantigens;
endotoxin and other cell wall
components
Antiphagocytic factors
Intracellular survival
Antigenic heterogeneity
Antigenic variation
Phase variation
Iron acquisition
Siderophores
Receptors for
iron-containing molecules
Resistance to antibiotics
Microbial defenses against host immunologic clearance
Encapsulation (Inhibition of phagocytosis and serum
bactericidal effect)
Antigenic mimicry
Antigenic masking
Antigenic or phase variation
Intracellular multiplication
Escape phagosome
Inhibition of phagolysosome fusion
Resistance to lysosomal enzymes
Production of anti-immunoglobulin protease
Inhibition of chemotaxis
Destruction of phagocytes
Mechanisms for escaping
phagocytic clearance and
intracellular survival
Mechanisms for escaping
phagocytic clearance and
intracellular survival
Mechanisms for escaping
phagocytic clearance and
intracellular survival
Steps in successful infection
• Sex comes before disease
– acquire virulence genes
• Sense environment
– and Switch virulence genes on
and off
• Swim to site of infection
• Stick to site of infection
• Scavenge nutrients
– especially iron
• Survive stress
• Stealth
– avoid immune system
• Strike-back
– damage host tissues
• Subvert
– host cell cytoskeletal and
signalling pathways
• Spread
– through cells and organs
• Scatter
Bacterial Sex
Bacterial Sex
acquiring virulence genes
Mobile genetic elements
• Bacteria have three
ways of exchanging
DNA
– Transformation
• cells take up naked DNA
– Transduction
• phages carry DNA
– Conjugation
• cells mate through
specialised appendages
• Transposons
– ST enterotoxin genes
• Virulence Plasmids
– e.g. TTSSs in Shigella,
Yersinia; toxins in
Salmonella, E. coli, anthrax
• Phage-encoded virulence
– e.g. botulinum toxins,
diphtheria toxin, shiga-like
toxin (linked to lysis),
staphylococcal toxins,
TTSS substrates in
Salmonella.
Bacterial Sex
Bacterial Sex
Pathogenicity Islands
Pathogenicity Islands
• Concept originated from study of uropathogenic
E. coli strains
• Defining Features
– Carriage of (many) virulence genes
– Presence in pathogenic versus non-pathogenic
strains
– Different G+C content from host chromosome
– Occupy large chromosomal regions (10-100 Kb)
– Compact distinct genetic units, often flanked by DRs,
tRNAs, ISs
– Presence of (cryptic) mobility genes
– Unstable, prone to deletion
• often encode secretion systems
– LEE region in EPEC
– Spi1, Spi2 in Salmonella
– Cag in H. pylori
• can also encode adhesins, siderophores, toxins
– Uropathogenic E. coli (Pai I, II, IV, V)
– Yersinia spp. (HPI)
– V. cholerae (VPI or TCP-ACF element)
Sense environment
• Bacteria can sense changes in environment
– e.g. in temperature, nutrient availability, osmolarity, cell density
(“quorum sensing”).
• In simplest cases, change in intracellular concentration
of ion linked directly to gene expression
– e.g. fall in intra-cellular iron levels triggers de-repression of
diphtheria toxin gene
• In more complex cases, sophisticated signal
transduction cascades allow bacteria to regulate gene
expression in response to environmental cues
– the pathogen as an information processor
Switch virulence factors on and off
A multi-layered hierarchy
• Changes in DNA
sequence
– Gene amplification
– Genetic rearrangements
– Enterics, Campylobacter,
Helicobacter, spirochaetes
• Motility crucial for
virulence in some cases
• Usual organelle of
motility=flagellum
• Variants
– Twitching motility
– Swarming
– Stability of protein,
controlled cleavage
– Covalent modifications
• e.g. Hin flip-flop control of
flagellar phase variation
• Transcriptional
Regulation
• e.g. phosphorylation in
two-component sensorregulator systems
– Activators and Repressors
(helix-turn-helix motif)
– mRNA folding and stability
Swim
• Many bacterial pathogens
are motile
• Translational Regulation
• Post-translational
Regulation
Stick
• To avoid physical and
immunological removal,
bacteria must adhere to
– cell surfaces and
extracellular matrix
e.g. in respiratory,
gastrointestinal and
genitourinary tracts
– solid surfaces
e.g. teeth, heart valves,
prosthetic material
– other bacteria
• Direct interaction
• Molecular bridging via
e.g. fibronectin
• Adherence often
combined with
manipulation of host cell
signalling and
cytoskeleton
– Invasion
– Intimate adherence
Stick
Stick
• Common adherence mechanisms
– Capsules and slime
– Biofilm formation
• Gram-positive adhesins
– MSCRAMMs (microbial surface components
recognizing adhesive matrix molecules), e.g. protein
A
– Fimbriae
• Gram-negative adhesins (CHO and protein
receptors)
– Fimbriae, Afimbrial adhesins (FHA, Pertactin etc.)
– Outer Membrane Proteins
– Types III-IV secretion
Scavenge nutrients
e.g. iron
• Free iron levels very low in body fluids
– Acute phase response causes further drop
– Iron overload increases susceptibility to infection
• Many different bacterial systems for scavenging iron
– Siderophores chelate available iron & transport it into bacteria
– Iron can be scavenged direct from host iron-binding proteins, e.g by
lactoferrin-binding proteins
– Often co-ordinately regulated e.g. by fur locus in E. coli
– Some pathogens avoid the problem by cutting out need for iron, e.g.
Treponema pallidum
• Iron used to regulate aggressive virulence factors
– Diphtheria toxin (DtxR repressor)
– Shiga-like toxin
– Pseudomonas aeruginosa exotoxin A
Survive Stress
• In addition to nutrient-limitation stress, pathogens face
many other stresses
– Acid stress within stomach
– Heat shock during fever
– Oxidative stress within phagocytes
• Stress response proteins, such as chaperonins, feature
as immunodominant antigens
• Detoxification proteins play a role in virulence, e.g.
periplasmic Cu,Zn-superoxide dismutases
• Infectious dose for enteric pathogens much lower in
achlorhydria (no need to overcome acid stress)
Stealth
Stealth
avoid immune system
• IgA proteases
– metalloproteases active against IgA
• Immunoglobulin-binding proteins
– e.g. protein A of S. aureus
• Resist complement, opsonisation
– Capsule (usually polysaccharide)
– Lipopolysaccharide
– Surface proteins and OMPs
• Antigenic mimicry
avoid immune system
• Antigenic or phase
variation
– Involves surface structures
such as proteins, LPS,
capsules
– Variety of mechanisms
• slip-strand mispairing
• flip-flop
• cassettes
• Adopt cryptic niche
– inside phagocytes
– in biofilm
– e.g. sialic acid capsule of group B meningococcus
Strike-back
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GAAGTGCATTTAACTT**GGGGGGGGGGGTAAT
GAAGTGCATTTAACTT*GGGGGGGGGGGGTAAT
GAAGTGCATTTAACTTGGGGGGGGGGGGGTAAT
GAAGTGCATTTAACTT***GGGGGGGGGGTAAT
GAAGTGCATTTAACTT**GGGGGGGGGGGTAAT
GAAGTGCATTTAACTT****GGGGGGGGGTAAT
GAAGTGCATTTAACTT*GGGGGGGGGGGGTAAT
GAAGTGCATTTAACTT**GGGGGGGGGGGTAAT
GAAGTGCATTTAACTT***GGGGGGGGGGTAAT
GAAGTGCATTTAACTT***GGGGGGGGGGTAAT
GAAGTGCATTTAACTT***GGGGGGGGGGTAAT
GAAGTGCATTTAACTT**GGGGGGGGGGGTAAT
GAAGTGCATTTAACTT*GGGGGGGGGGGGTAAT
Homopolymeric tract in Campylobacter jejuni
Endotoxin of
Gram-negatives
Damage host tissues
• Endotoxin
• Exotoxins
Gramnegative
cell
cytopl.
mem.
peptidoglycan
outer mem.
– Toxins acting on cell membranes
– Toxins active inside cells
– Superantigens
Lipopolysaccharide
(LPS)
Lipid A
Core
polysaccharide
O sidechain
The toxic part
Helps solubilise Lipid A
Somatic antigen
Strike-back
Endotoxin
• Actions of Endotoxin
– Pyrogenicity
– Leucopenia then leucocytosis
– Hypotension
•
•
•
•
“Gram-negative Shock”
Life-threatening complication of septicaemia
e.g. in meningococcal infection, in ITU or oncology patients
Endotoxic shock seen with dirty intravenous equipment
• Most of the effects of endotoxin are mediated by
tumour necrosis factor
– Attempts at therapy using anti-endotoxin or anti-TNF
antibodies
Strike-back
Toxins active inside cells
• Toxins often consist of
translocation and binding
B subunit that delivers
the active A subunit into
the host cell cytoplasm
• Example of AB toxin:
diphtheria toxin
an ADP-ribosyltransferase
Strike-back
Membrane-Damaging Exotoxins
• Many bacterial toxins form
pores in eukaryotic cell
membranes, producing
oligomeric rings, e.g.
– streptolysin O of
Streptococcus pyogenes
– listeriolysin of Listeria
monocytogenes
– alpha-toxin of S. aureus
• Other toxins, such as
phospholipases, degrade
components of the membrane
– e.g. Clostridium perfringens
alpha toxin
AB5 Toxins
LYSIS
Spread
Subvert
• inject proteins into host cells to subvert the
cytoskeleton and signal-transduction pathways:
– manipulating (e.g. Rho GTPases and the
cytoskeleton) to induce membrane ruffling and
bacterial invasion
– preventing uptake by phagocytic cells, e.g. Yersinia
spp. and Pseudomonas spp.
– remaining within a vacuole by manipulating host cell
vesicular transport and endocytosis
…through cells and organs:
• within macrophages, e.g.
in typhoid
• through blood (need to be
complement-resistant)
• within cells
– actin-based motility of
Listeria monocyogenes,
depends on ActA protein.
Scatter
Summary
Transmission, virulence and evolution
• Established dogmas
– balanced
pathogenicity
– being too virulent is no
good
– high virulence is a sign
of recent emergence
of a pathogen
– pathogens evolve
towards symbiosis
•
Counter-arguments
– Where pathogens rely on spread
through biting arthopods, high
bacteraemias advantageous
– Where pathogens rely on
shedding into water, highest
possible shedding rates good for
pathogen
– Where pathogens cause
incidental disease (e.g.
Legionella) no selective pressure
towards low virulence
– Virulence as a local adaptation
(why meningitis?)
– Bad vaccines and effect on
virulence
• Spectrum of virulence
– Commensals
– Opportunistic pathogens
– Obligate pathogens
• Difficulties in linking
pathogen to disease
– Koch’s postulates
– Molecular Koch’s
postulates
• Multi-dimensional view of
virulence
•
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Sex
Sense
Switch
Swim
Stick
Scavenge
Survive stress
Stealth
Strike-back
Subvert
Spread
Scatter
Home Work
1. Name at least 3 ways bacterial pathogens can evade
host defence.
2. What is endotoxin? What is exotoxin?
3. Why both escaping and overly stimulating immune
system are bad for the host being infected?