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