1. Principles of Pathogenesis
Bacterial Infection

2. Microbes and humans
Very few microbes are
always pathogenic
Many microbes are
potentially pathogenic
Most microbes are
never pathogenic

3. Microbes and humans
Disease can come about in several overlapping
ways
1. PATHOGENS: Not normal flora
subclinical infection (M tuberculosis)
2. Normal flora acquire extra virulence factors
to be pathogenic, e.g. E. coli
3. Normal flora: Disease if they gain access to
deep tissues by trauma, surgery, lines,
especially if a foreign body, e.g. S. epidermidis
4. In immunocompromised patients free-living
bacteria & normal flora can cause disease,
especially if introduced into deep tissues, e.g.
Acinetobacter

4. 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

5. Spectrum
of
virulence
poliomyelitis in a child
0.1-1% of infections are
clinically apparent
rubella
50% of infections are
clinically apparent
rabies
100% of infections
are clinically apparent
The iceberg concept of infectious disease
asymptomatic infection
classical
clinical disease
less severe
disease

6. potential pathogen
isolated from or
detected in clinical
samples
Recognised syndromes
patient's clinical
condition
e.g.
septicaemia, endocarditis,
osteomyelitis meningitis,
UTI, pneumonia
pharyngitis
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

7. Microbes and humans
• Evidence for a potential pathogen being clinical
significant (particularly for bacteria)
– 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

8. 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

9. 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…

10. 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?

11. 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

12. 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

13. 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

14. Bacterial
Mobile genetic elements
• Transposons
– ST enterotoxin genes
• Virulence Plasmids
– e.g. TTSSs in Shigella,
Yersinia; toxins in
Salmonella, E. coli,
anthrax
• Phage-encoded virulence
- botulinum toxin
-diphtheria toxin,
- shiga-like toxin(lysis),
-staphylococcal toxins,
TTSS substrates in
Salmonella.

15. Bacterial Sex
Pathogenicity Islands
• Concept from study of uropathogenic Ecoli 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

16. 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)

17. 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

18. 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
sensor-regulator
systems

19. Swim
• MOTILE bacterial
pathogens
– Enterics,
Campylobacter,
Helicobacter,
spirochaetes
• Motility crucial for
virulence in some cases
• Usual organelle of
motility=flagellum
• VariantTwitching
motility
– Swarming

20. 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

21. Stick
• COMMON ADHERENCE MECHANISMS:
- CAPSULES/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

22. Stick

23. Scavenge nutrients
e.g. iron
• FREE IRON LEVELS: very low in body fluids
– Acute phase response causes further drop
– Iron overload increases susceptibility to infect
• SCAVANGING IRON:
– Siderophores chelate available iron & transport
it into bacteria
– Iron can be scavenged direct from host iron-
binding proteins, e.g by lactoferrin-binding prot
– Often co-ordinately regulated fur locus in Ecol
cutting out need for iron, e.g. T pallidum
• Iron used to regulate aggressive virulence factors
– Diphtheria toxin (DtxR repressor)

24. Survive Stress
• nutrient-limitation stress, 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)

25. 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

26. Stealth
avoid immune system
.Antigenic or phase varia
– Involves surface
structures as LPS
proteins, capsules
– Variety of mechani .
– slip-strand mispair
• flip-flop
• cassettes
.Adopt cryptic niche in
phagocytes
in biofilm
67700 67710 67720
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

27. Strike-back
Damage host tissues
• Endotoxin
• Exotoxins
– Toxins acting on cell membranes
– Toxins active inside cells
– Superantigens

28. Endotoxin of
Gram-negatives
cytopl.
mem.
peptidoglycan
outer mem.
Gram-
negative
cell
Lipid A Core
polysaccharide
O sidechain
The toxic part
Helps solubilise Lipid A Somatic antigen
Lipopolysaccharide
(LPS)

29. Strike-back
Endotoxin
• ENDOTOXIN EFFECTS:
– PYROGENICITY
– LEUCOPENIA,THEN LEUCOCYTOSIS
– HYPOTENSION
• “Gram-negative Shock”
• Life-threatening complication of septicaemia
• e.g. in meningococcal infection, in ITU or oncology pts
• Endotoxic shock seen with dirty intravenous equipmen
Most effects of endotoxin mediated by tumour necrosis factor
– THERAPY ATTEMPTS: ANTI-ENDOTOXIN
– or ANTI TNF ANTIBODIES

30. Strike-back
Membrane-Damaging Exotoxins
TOXINS:
1.FORM PORES in eukaryotic
cell membranes, producing
oligomeric rings,
-streptolysin O of S pyogenes
-listeriolysin of L
monocytogenes
-alpha-toxin of S aureus
2. DEGRADE CM as
phospholipases
– e.g. Clostridium
perfringens alpha toxin
LYSIS!LYSIS!

31. Strike-back
Toxins active inside cells
• Toxins consist of:
• 1 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

32. AB5 Toxins

33. 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 Phagocytes e.g.
Yersinia & Pseudomonas
-remaining within a vacuole by manipulating
host cell vesicular transport & endocytosis

34. Spread
…through CELLS &
organs:
• In Macrophages, e.g. in
typhoid
• Via BLOOD (need to
be complement-
resistant)
• within cells
– actin-based motility
of L monocyogenes,
depends on ActA
protein.

35. 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
• 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 incidenta
disease (e.g. Legionella) no
selective pressure towards
low virulence
– Virulence as a local
adaptation (why
meningitis?)
– Bad vaccines and effect

36. Summary
• Spectrum of virulence
– Commensals
– Potential pathogens
– Obligate pathogens
• Difficulties in linking
pathogen to disease
– Koch’s postulates
• Multi-dimensional view of
virulence
• Sex
• Sense
• Switch
• Swim
• Stick
• Scavenge
• Survive stress
• Stealth
• Strike-back
• Subvert
• Spread
• Scatter

37. Further Reading
Bacterial Pathogenesis: A
Molecular approach,
Salyers and Whitt
(2nd Ed if possible)
Cellular Microbiology
Cossart, Boquet, Normark,
Rappuoli