Bio305 Lecture on Genetics Presentation Transcript
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
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
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
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
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
An ecological perspective Interactions with predatory bacteria and bacteriophages Interactions with amoebae, insects, nematodes, annelids, fungi Interactions with humans as commensals
Non-mammalian systems are exploitedexperimentally 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 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
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
Why do bacteriophages encode virulencefactors 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. 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...?
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