Upload
mark-pallen
View
620
Download
0
Tags:
Embed Size (px)
DESCRIPTION
Citation preview
Bio305 Genetics of Bacterial Virulence
Professor Mark Pallen
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
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
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)
A Bacterial Genome: WYSIWYG
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
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
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
Genetic Terminology
Genotypic designation uses 3 letters, lowercase, underlined or italicized e.g. ara represents the ara locus involved in arabinose utilization
ara+ indicates all genes in locus are wild-type, not mutant araA represents a gene that 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 in araB
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
Genetic Designations
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. l) has it listed in
genotype zde1, zde2, etc. = mutations in unknown genes
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)
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
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?
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
Interactions with amoebae, insects, nematodes, annelids, fungi
Interactions with predatory bacteria and bacteriophages
Interactions with humans as commensals
An ecological perspective
Non-mammalian systems are exploited experimentally as models of infection
Yeast as a model of human infection
Case Study: STEC and Shiga toxin STEC is one of several
“pathotypes” of E. coli to cause diarrhoea
Classically E. coli O157:H7 More recently other
serotypes, e.g. O104:H4 in Germany
Those that have a type-III secretion system called enterohaemorrhagic E. coli or EHEC
Shiga Toxin
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...
A twist in the tale: bacteriophages Many
bacteriophages encode “virulence factors” that help bacteria in their interactions with eukaryotes
Lambda
Virulence effectors dominate the passenger compartments of lambdoid prophages in EHEC
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
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??
Why do bacteriophages encode virulence factors?
Phage and protozoa both eat E. coliScrapping 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...?
Another use of genetics… Genetic approaches to the study of virulence Using genetic modification to understand
pathogenesis
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!
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
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
Tn-Seq
Nat Methods. 2009 Oct;6(10):767-72
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
TraDIS Genome Res. 2009 Dec;19(12):2308-16.
Profile changed after serial passage through bile
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