2. Introductory Lectures
1: Pathogen Biology
2: Genetics of Bacterial Virulence
3: Regulation of Bacterial Virulence
Later lecture blocks from me on
Bacterial Genomics
Bacterial Protein Secretion
3. Learning Objectives
At the end of this lecture, the student will be able to
provide a definition of terms and jargon related to
bacterial pathogenesis
describe the multifactorial nature of bacterial virulence
outline the steps in a successful infection
describe the varied macromolecules implicated in
virulence, including endotoxin and exotoxins
4. Bacterial Genetics is Different
Single circular DNA chromosome (usually)
often also contain plasmids
No histones
so no nucleosomes
No nuclear membrane
coupled transcription and translation
No mitosis or meiosis
Rarely any introns
Genes often in clusters of related function
controlled as a unit (operon)
6. Genetic Terminology
Gene
smallest region of DNA (RNA) that encodes a polypeptide
OR is transcribed (tRNA) OR is a "regulatory element"
Locus (pl. loci)
location of a gene on the chromosome, often referring to
group of related genes, e.g., trp locus contains several
genes involved in tryptophan biosynthesis
Allele
alternative form of a gene
7. Genetic Terminology
Wild-type organism
carries standard/reference gene which is usually but not
always functional.
Mutant organism
carries altered form.
Genotype
genetic or allelic composition of strain
Phenotype
observable properties of strain
8. Genetic Terminology
Mutation
• permanent, heritable change in the DNA
Mutant
• organism/cell carrying a mutation.
Forward mutation
• results in change from wildtype phenotype to mutant
phenotype
Backward mutation (reversion)
• mutant phenotype reverts to wild-type (=revertant)
Genome
• entire genetic complement: chromosomes + plasmids
9. Genetic Designations
Genotypic designation uses 3 letters, lowercase, underlined or
italicized
e.g. ararepresents the ara locus involved in arabinose utilization
ara+ indicates all genes in locus are wild-type, not mutant
araA represents a genethat is part of the ara locus
araA1 indicates araA contains mutation #1 creating a distinct allele
araA2 represents another mutation that results in another distinct allele
araB235 indicates a mutation inaraB
ara-25 indicates mutation in the ara locus but not known which gene
∆araC43 indicates a deletion (∆) in araC
araB::Tn5 indicates an insertion (::) in araB of Tn5, a transposon
10. Genetic Designations
Phenotypic designation
not underlined/italicized, first letter capitalized
wild type = Ara+
mutant = Ara-, regardless of which gene carries mutation
antibiotic resistance/sensitivity
Strr or Str-r = streptomycin resistant
Strs or Str-s = streptomycin sensitivity
Genotype of organism
list only mutations
trpE38 araD139 lamB::Tn10
a lysogen containing a phage (e.g. ) has it listed in
genotype
zde1, zde2, etc. = mutations in unknown genes
11. Genetics of virulence
Many virulence genes
acquired via horizontal
gene transfer
On plasmids or
chromosome via
conjugation
As naked DNA via
transformation
On bacteriophage via
transduction
(generalised or
specialised)
12. Mobile genetic elements and virulence
Transposons
e.g ST enterotoxin genes
Virulence Plasmids
e.g type III secretion systems in Shigella, Yersinia; toxins
in Salmonella, E. coli, B. anthracis
Phage-encoded virulence
e.g. botulinum toxins, diphtheria toxin, Shiga-like toxin
(linked to lysis), staphylococcal toxins, T3SS effectors
Pathogenicity islands
e.g. Locus for enterocyte effacement, Spi1, Spi2
13. But where do virulence genes originate?
How can genes from a non-pathogen become
virulence genes in a pathogen?
How do pathogens originate in the first place?
Why do we see “virulence factors” in non-
pathogens?
14. The Eco-Evo perspective
Studies of bacterial pathogenesis and of bacterial
genomes have forced a re-appraisal of host-microbe
interactions
Bacteria need to be viewed in the light of their
evolutionary history and usual ecological context
15. An ecological perspective
Interactions with predatory
bacteria and bacteriophages
Interactions with amoebae, insects,
nematodes, annelids, fungi
Interactions with humans as
commensals
18. Case Study: STEC and Shiga toxin
STEC is one of several
“pathotypes” of E. coli to
cause diarrhoea Shiga Toxin
Classically E. coli O157:H7
More recently other
serotypes, e.g. O104:H4 in
Germany
Those that have a type-III
secretion system called
enterohaemorrhagicE. coli or
EHEC
19. STEC: why virulence?
Why does STEC possess virulence factors active
in human infection when human-to-human
transmission is unable to sustain STEC in the
human population?
Usual explanation: EHEC is a commensal of cattle,
and uses these factors to colonise the bovine
intestine
But the German outbreak showed that not all STEC
come from cattle
Alternative explanation: STEC has to deal with
micro-predators...
20.
21. A twist in the tale: bacteriophages
Many
bacteriophages
encode “virulence
factors” that help
bacteria in their
interactions with
eukaryotes
23. Why do bacteriophages encode virulence
factors
An obvious answer is that when resident in the
bacterial genome as prophages, the interests of
the phage and of the bacterium coincide, so that
by aiding the bacterium, the virulence factors also
aid the phage...
• probably true for type III secretion effectors
24. Why do bacteriophages encode virulence factors?
Shiga toxin is also
phage-encoded
BUT provides a spanner
in the works for the idea
that phage and
bacterium’s interests
coincide!
Shiga toxin is a suicide
bomber
released from bacterial
cell only when the cell
has been lysed by
bacteriophage
why? how can the
bacterium benefit??
25. Why do bacteriophages encode virulence factors?
Phage and protozoa both eat E. coli
Scrapping over common food source!
But lysis isn’t an all-or-none
phenomenon
Maybe bacteria benefit because
low-level lysis and toxin release is a
form of kin selection for the
bacteria...?
26. Another use of genetics…
Genetic approaches to the study of virulence
Using genetic modification to understand
pathogenesis
27. Candidate gene approach
Molecular Koch’s postulates
A specific gene should be consistently associated with the
virulence phenotype
When the gene is inactivated, the bacterium should
become avirulent
If the wild type gene is reintroduced, the bacterium should
regain virulence
If genetic manipulation is not possible, then induction of
antibodies specific for the gene product should neutralize
pathogenicity
[Falkow, 1988. Rev. Infect. Dis. Vol. 10, suppl 2:S274-276]
BUT slow progress when you have 4,000 genes to
assay!
28. Signature-tagged mutagenesis (STM)
A negative selection method invented by David Holden, used to
determine which genes are essential under a given condition
e.g. survival during infection in animal tissues
Sets of mutants are created by random transposon insertion
All mutants have to be capable of survival on laboratory media
Each transposon within a set contains a different 'tag' sequence that
uniquely identifies it and which can be retrieved easily by PCR with
common primers
29. Signature-tagged mutagenesis (STM)
Mutants within each set are pooled
Input pool is then used to infect an animal
Comparison between input and output pools allows us to
identify genes needed for survival in the host and
therefore necessary for virulence
Hundreds of genes surveyed in each experiment
31. Tn-Seq
First part JUST LIKE STM!
Tn library constructed in vitro
transformed into bacterial population
each bacterium with single Tn insertion
DNA is isolated from input pool
selection applied to pool (e.g. infection)
DNA isolated from output pool
But then:
PCR up160-bp sequence (20 bp insert-specific)
massively parallel amplicon sequencing
20-bp reads mapped to the genome
counted for each insertion
fitness effects of each gene calculated
36. Summary
Bacterial genetics is different
Definition of terms
Role of horizontal gene transfer and mobile genetic
elements
Origins of virulence genes
Genetic methods for analysing virulence
