Virulence Factors, Host Factors and Genetics Flashcards
Do most mutations confer an advantage?
No
When do mutations occur and what is the error rate of E.coli?
During DNA replication. 10 -7, 10 -8
What are SNPs?
- Single nucleotide polymorphisms
- Single changes might not change protein sequence (it will if in 1st position)
- Protein expression efficiency may be altered
- If advantageous, kept as SNP
How can repeat sequences lead to mutations which alter gene expression?
(Bordetella pertussis)
o Repeats = error can occur and lose or ad base
o Changes reading frame, changes downstream protein sequence
o Can change phenotype (+, -)
o Subsequent replication can add/remove base and change back to original phenotype
What’s an example of internal DNA recombination?
(Neisseria)
o Recombination used to vary pili (adhesion)
o Exchange from non-expressed gene and make mosaic pilus
Why is the location of a gene on a chromosome important?
o Some parts of chromosome duplicated for longer (increased expression of some genes)
o Gene placement takes into account redundant genetic code, operons
What are the slow mechanisms for genetic change and diversification?
- Point mutation (nucleotide change/insertion/deletion)
- Gene duplication
- Gene Deletion
- Chromosomal rearrangement (inversion, intragenic recombination)
What are the fast mechanisms for genetic change and diversification?
- Phase variation (promoter inversion, slipped-strand synthesis)
- Antigenic variation (gene shuffling/conversion)
- HGT (intergenic recombination, transformation, conjugation, transduction)
What are the features of HGT?
- Resistance plasmids
- Transposons
- Pathogenicity island
- DNA from different bacteria
- ssDNA more efficient because DNA sensitive to DNAases
- Restriction enzymes and methylases for protection
What are the main HGT mechanisms?
Transformation
Conjugation
Transduction
What is transformation?
• DNA from donor cell released to environment and taken up by competent recipient cell (progeny = transformants)
How did Griffith show about transformation?
Pneumococcus
o Rough (un-encapsulated) and Smooth (encapsulated)
o Inject S = dead mice
o Inject R = live mice
o Inject denatured S = live mice
o Inject denatured S and live R = dead mice
o R takes up the pathogenicity features of S
How come E.coli cannot be transformed?
It is not competent
What are transformasomes?
Membranous structures that protect DNA during transformation
What is conjugation? What does the donor require?
- Bacterial sex
* Donor has sex pilus (F)
What is transduction?
- DNA transferred by virus/bacteriophage
- Phage replicate, fail to include viral DNA, package bacterial DNA
- Chromosomal DNA injected into cell
- Recombination possible
- Phage have specific receptor recognition
What are pathogenicity islands?
- Often remnant of bacteriophage
- Insert to phage like insertion sites
- Different G, C content %
- Include phage making toxins
- Phage genes integrated to genome
Where are most pathogenicity islands found?
tRNA genes most common
What is a virulence determinant? What factors are commonly counted as virulence determinants?
- Specific trait/factor that increases the virulence of a microbe
- Factors include growth, adhesion, invasion, resistance, damage, dissemination
How can virulence determinants be identified?
- Aim to measure virulence
- Ethical considerations
- Need model reflecting human disease (route, dose, dependencies, infection kinetics, outcomes)
What are Koch’s postulates?
- Microbe associated with symptoms of disease and present at site of infection
- Microbe isolated from disease lesions and grown as pure culture
- Pure culture of microbe reproduces disease when inoculated into new host
- Microbe reisolated in pure culture from experimentally infected host
What are the problems with Koch’s postulates?
o Dissemination o Toxins o Systemic infection o Growth restrictions (syphilis) o Hosts can be selective/restrictive o Require pure cultures o Multiple infections (poly-microbial) common
What are molecular Koch’s postulates?
- Virulent organism
- Isolate gene
- Mutate gene
- See reduction in virulence
- Return gene/revert mutation to WT
- See increase in virulence
How do mice infected with S. pneumoniae demonstrate the application of molecular Koch’s postulates?
- Respiratory disease in mice
- Mutant lives longer than WT
- Mutant with restored gene wore survival than mutant, slightly better than WT
What are the requirements for an animal model to be considered valid when studying human disease?
• Need model reflecting human disease (route, dose, dependencies, infection kinetics, outcomes)
What’s an example of using a transposon to create mutants?
Gene = toxA
o Introduce transposon
o Select transposon mutants using resistance
o Test each in high through assay
o Identify nontoxic mutants (transposon is in toxA gene)
What is a transposon?
DNA with self-encoding insertion capability, transposase, inverted repeats and selectable marker (e.g. TetR)
What is an insertion sequence?
2 inverted repeats flanking transposase gene
What is a compound transposon?
has two IS elements flanking resistance gene
What is a simple transposon?
has resistance gene within two inverted repeats
What is the aim of using transposition for mutagenesis?
to make transposon jump from delivery plasmid into bacterial chromosome, occasionally will jump into toxin gene resulting in loss of function
What is ID50?
ID50 = dose required to infect 50% of animals
What is LD50?
LD50 = dose required to kill 50% of animals (les subjective)
What is the process of competition assays?
- WT vs mutant
- Inject both into organism (mouse in vivo, cultured phagocytes in vitro)
- See surviving bacteria, has one time (WT/mutant) been selected for?
- More important the gene that has been mutated, the stronger the selection
What is signature tagged mutagenesis?
- Competition assay between transposon mutants
- Different groups in pool of transposon mutants
- Subject pool to competition
- Assay and see which group is missing (suggests transposon probably hit and interrupted a key virulence gene)
- PCR can show specific tags
How can host factors be added?
- Antibodies
- Cells
- Transgenic/genetically modified
How can host factors be removed?
- Depletion with antibodies
- Genetic depletion
- Transgenic depletion
How can host determinants be identified?
- Compare infection in normal vs those with factors removed/added
- LD50, ID50
- Bacterium growth
- Pathology
- Transmission
What are the features of S. Typhimurium?
- Multi-host pathogen
- Poultry
- Gastroenteritis (self-limiting unless issues with Cd4 T cells or IL-12)
- Very common cause of bacteraemia in HIV+ patients
- Invasive non-typhoidal salmonellosis (iNTS)
Why would an iNTS vaccine be ineffective in the immunocompromised?
- Would need antibody response (adaptive immunity)
- Antibodies poor action against intracellular bacteria
- Need helper Tcells
How can iNTS be stopped in the immunocompromised?
- Could control intracellular bacteria with IFNy
* Use model for iNTS (mouse)
What did Murine Model iNTS show?
- Some things make mice more sensitive (e.g. lack of CD4)
- Mice lacking lymphocytes or IFNy had trouble controlling infection
- CD4+ deficient (HIV like) mice had chronic infection
- CD4+ and CD8+ lacking mice had very bad chronic infection
What other methods can be used to analyse host and bacteria interactions?
- IVUS camera (reporter strain) e.g. GFP , non invasive
- Knock down host gene function
- 2D gel electrophoresis and western blotting
- GWAS
How might host gene function be knocked down?
o siRNA, works against specific genes
o study contribution of specific host genes in response to infection (tissue culture)
How can GWAS be used to study host gene function?
o Need lots of info, expensive, data
o Groups of different people (sick, not sick, really sick etc)
o Look for SNPs that correlate
o Study gene, look for functional importance
What was the process used to construct the Murine Model iNTS?
- C57BL6 mice (S. Typhimurium lethal)
- Normal course usually takes a while, need to mimic in mice (chronic) therefore used attenuated S. Typhimurium BRD509
- Inject with S. Typhimurium, measure growth in spleen at various times
- Use animals with various immunedeficiencies (B cells, T cells, no lymphocytes etc)
- Compare growth kinetics in the different animals
What is the basis of transposon mutagenesis?
- Conjugation of transposon in to recipient
- Jumps, plasmid to genome (random insertion)
- Each colony = different insertion
- Transferred plasmid: conditional replicon
- Recipient loses plasmid
- Retain transposon (resistance marker e.g.), it must have jumped to genome
- Select for resistant recipient (has transposon on chromosome)
- Transposon insertion inactivates gene, test mutant for phenotype
What is the importance of virulence genes? At what levels can virulence gene expression be regulated?
- Show if pathogenic
- Can be linked together (PAIs)
- May not be required if bacterium external to host or in niche not causing disease
- Regulation needed for efficiency
- Multiple environments, stresses, opportunities
- Regulation levels = DNA, transcription, translation, DNA structure, Quorum sensing
What is phase variation? What’s an example?
o Gene on, gene off
o N. gonorrhoeae Protein II (OM protein, Opa, adherence and invasion)
o Can occur through slip strand mis-repair
What is antigenic variation? What’s an example?
o Expression locus, homologous recombination into locus of alternate alleles
o N. gonorrhoeae PilE (on all the time but alternates so antibodies may not recognise)
What are operons and regulons?
- Operon: gene/genes on single transcript
* Regulon: multiple transcripts, multiple promoters controlled by same regulator
How does transcriptional regulation work?
• Increase/decree RNA pol activity (RNA pol binds promoter, block or encourage binding)
What makes up the Prf regulon and where is it found?
o Prf (Listeria monocytogenes) o prfA as master switch o 3 gene operons, 1 gene operons
What is the basis of the lac operon? What kind of regulation does it show?
o LacZ = Bgal
o No lactose, LacI bound, gene off
o High glucose, no cAMP activator protein, gene off
o High lactose, allolactose binds LacI, removes it from operator
o Low glucose, CAP site bound by cAMP activator protein, RNA pol present and activator protein can activate transcription
Transcriptional regulation
What does +1 correspond to?
The transcriptional start point
Where does LacI bind?
The operator
How is diphtheria toxin produced?
o Diphtheria (toxin essential, from phage)
o Iron limiting in host (binding proteins, e.g. transferrin)
o Low iron → DtxR repressor dimer disassembles → toxin produced
o DtxR/Fe2+ binds -10 region of promoter and blocks RNA pol
What happens in two component systems?
o Common for g-, respond to environment
o Membrane sensor kinase protein (membrane) (S)
o Regulator protein (cytoplasm) (R)
o Signal in periplasm →sensor dimerises → kinase autophosphorylates histidine residue→ transferred to aspartic acid residue on regulator → activated as transcription factor
What happens in the Bvg regulon of Bordetella pertussis?
Many virulence determinants
In vivo expression controlled by BvgS and BvgA (not R)
BvgS= 3 intracellular domains
Phosphate transferred from his to asp to his then BvgA for activation
Bvg+ = toxins, adhesins produced
What are sigma factors required for?
o RNA pol needs them to bind promoter
o Recycled
o Different factors = different promoters
o Control regulons
What changes in DNA topology can control genes and how?
o Supercoiling
o Histone like-proteins
Alter DNa shape
E.g. curve DNA, RNA pol can’t bind
What are examples of genetic rearrangements as regulation mechanisms?
Phase variation, antigenic variaiton, gene duplication and amplification
What are examples of transcriptional regulation as regulation mechanisms?
activators, repressors, complex networks, two component systems, cic-acting thermosensors, DNA topology
What are examples of translational regulation as regulation mechanisms?
mRNA efficiency
What are examples of quorum sensing as regulation mechanisms?
Vibrio fischeri in squid
What is quorum sensing?
• Communicating between species (autoinducers) • Certain community size = quorum • Al-1: acyl homoserine lactones (g-) o Alter transcription with regulators • G+ use peptide signals
What is Al-1?
• Al-1: acyl homoserine lactones (g-)
o Alter transcription with regulators
What are Vibrio fischeri?
o Colonise light organs of squid
o Help squid, attract prey
o Bacteria reach quorum → AI-1, AI-2 reach threshold → light emitted due to luciferase, GFP
