Transcript Principles of Pathogenesis Bacterial Infection

Principles of Pathogenesis
Bacterial Infection
Professor Mark Pallen
University of Birmingham
Microbes and humans
Very few microbes are
always pathogenic
Many microbes are
potentially pathogenic
Most microbes are
never pathogenic
Microbes and humans
Disease can come about in several overlapping ways
1. Some bacteria are entirely adapted to the pathogenic way of
life in humans. They are never part of the normal flora but may
cause subclinical infection, e.g. M . tuberculosis
2. Some bacteria which are part of the normal flora acquire extra
virulence factors making them pathogenic, e.g. E. coli
3. Some bacteria from the normal flora can cause disease if they
gain access to deep tissues by trauma, surgery, lines, especially
if associated with a foreign body, e.g. S. epidermidis
4. In immunocompromised patients many free-living bacteria and
components of the normal flora can cause disease, especially if
introduced into deep tissues, e.g. Acinetobacter
How do we know that a given
pathogen causes a specific disease?
• Koch's postulates
– 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
The iceberg concept of infectious disease
poliomyelitis in a child
0.1-1% of infections are
clinically apparent
classical
clinical disease
less severe
disease
rubella
50% of infections are
clinically apparent
asymptomatic infection
Spectrum
of virulence
rabies
100% of infections
are clinically apparent
How do we know that a given pathogen
causes a specific disease?
Diagnosis and effective treatment of
infection depends not just on isolating an
organism, but in establishing a plausible
link between the laboratory findings,
recognised syndromes and the patient's
clinical condition
potential pathogen
isolated from or
detected in clinical
samples
Recognised syndromes
e.g.
septicaemia, endocarditis,
osteomyelitis meningitis,
UTI, pneumonia
pharyngitis
patient's clinical
condition
Microbes and humans
• Evidence for a potential pathogen being clinical
significant (particularly for bacteria)
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Isolated in abundance
Isolated in pure culture
Isolated on more than one occasion
Isolated from deep tissues
Evidence of local inflammation
Evidence of immune response to pathogen
Fits with clinical picture
Normal flora
• All body surfaces possess a rich normal bacterial flora,
especially the mouth, nose, gingival crevice, large bowel, skin
– This can be a nuisance in that
• it can contaminate specimens
• it can cause disease
– This is beneficial in that
• it can protect against infection by preventing pathogens colonising
epithelial surfaces (colonisation resistance)
• removal of the normal flora with antibiotics can cause
superinfection, usually with resistant microbes
• Endogenous viruses reside in the human genome
– worries about similar pig viruses in xenografts
Bacterial Virulence
A simplistic view
• Some bacterial proteins (“exotoxins”) can elicit
the features of a bacterial infection when
injected as pure proteins, e.g.
– tetanus toxin, botulinum toxin
– diphtheria toxin, anthrax toxin
• Vaccination with inactivated toxins (“toxoids”) led
to a spectacular decline in the incidence of many
bacterial infections.
• Leading to the simplistic idea that all bacteria
need to cause disease is a single toxin…
Bacterial Virulence
A more sophisticated view
• There are many different ways to define a “virulence
factor”…
• needed to colonise and/or damage tissues
– “Molecular Koch’s postulates”
• Delete gene, show loss of virulence in model system, add gene
back (e.g. on plasmid), show restoration of virulence
– Biochemical evidence of damaging potential
• distinguishes pathogen from commensal
• Comparative genomics
• expressed or essential in vivo…
…but not in the lab?
Bacterial Virulence
A more sophisticated view
• Virulence as a process is
– MULTIFACTORIAL
• A bacterial army, like a human army, needs more than just
its firearms to enter and secure enemy territory…
“An army marches on its stomach” Napoleon
– MULTIDIMENSIONAL
• A programme of events organised in time and space
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
acquiring virulence genes
• Bacteria have three
ways of exchanging DNA
– Transformation
• cells take up naked DNA
– Transduction
• phages carry DNA
– Conjugation
• cells mate through
specialised appendages
Bacterial Sex
Mobile genetic elements
• 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
Pathogenicity Islands
• Concept originated from study of uropathogenic E.
coli strains
• Defining Features
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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
Bacterial Sex
Pathogenicity Islands
• 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
• e.g. Hin flip-flop control
of flagellar phase
variation
• Transcriptional Regulation
– Activators and Repressors
(helix-turn-helix motif)
– mRNA folding and stability
• Translational Regulation
• Post-translational
Regulation
– Stability of protein,
controlled cleavage
– Covalent modifications
• e.g. phosphorylation in
two-component sensorregulator systems
Swim
• Many bacterial pathogens
are motile
– Enterics, Campylobacter,
Helicobacter, spirochaetes
• Motility crucial for
virulence in some cases
• Usual organelle of
motility=flagellum
• Variants
– Twitching motility
– Swarming
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
• 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
Stick
Scavenge nutrients
e.g. iron
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Free iron levels very low in body fluids
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Many different bacterial systems for scavenging iron
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Iron used to regulate aggressive virulence factors
– Acute phase response causes further drop
– Iron overload increases susceptibility to infection
– 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
– 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
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
– e.g. sialic acid capsule of group B meningococcus
Stealth
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
67700
67710
67720
GAAGTGCATTTAACTT**GGGGGGGGGGGTAAT
GAAGTGCATTTAACTT*GGGGGGGGGGGGTAAT
GAAGTGCATTTAACTTGGGGGGGGGGGGGTAAT
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GAAGTGCATTTAACTT****GGGGGGGGGTAAT
GAAGTGCATTTAACTT*GGGGGGGGGGGGTAAT
GAAGTGCATTTAACTT**GGGGGGGGGGGTAAT
GAAGTGCATTTAACTT***GGGGGGGGGGTAAT
GAAGTGCATTTAACTT***GGGGGGGGGGTAAT
GAAGTGCATTTAACTT***GGGGGGGGGGTAAT
GAAGTGCATTTAACTT**GGGGGGGGGGGTAAT
GAAGTGCATTTAACTT*GGGGGGGGGGGGTAAT
Homopolymeric tract in Campylobacter jejuni
Strike-back
Damage host tissues
• Endotoxin
• Exotoxins
– Toxins acting on cell membranes
– Toxins active inside cells
– Superantigens
Endotoxin of
Gram-negatives
Gramnegativ e
cell
cytopl.
mem.
peptidoglycan
outer mem.
Lipopolysaccharide
(LPS)
Lipid A
The toxic part
Core
polysaccharide
Helps solubilise Lipid A
O sidechain
Somatic antigen
Strike-back
Endotoxin
• Actions of Endotoxin
– Pyrogenicity
– Leucopenia then leucocytosis
– Hypotension
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“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
Membrane-Damaging Exotoxins
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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
LYSIS!
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
AB5 Toxins
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
Spread
…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
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
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Counter-arguments
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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
Summary
• Spectrum of virulence
– Commensals
– Potential pathogens
– Obligate pathogens
• Difficulties in linking
pathogen to disease
– 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
Further Reading
Bacterial Pathogenesis: A
Molecular approach,
Salyers and Whitt
(2nd Ed if possible)
Cellular Microbiology
Cossart, Boquet, Normark,
Rappuoli