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MIIM30011 Bacteriology > Virulence Factors, Host Factors and Genetics > Flashcards

Virulence Factors, Host Factors and Genetics Flashcards

1
Q

Do most mutations confer an advantage?

A

No

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2
Q

When do mutations occur and what is the error rate of E.coli?

A

During DNA replication. 10 -7, 10 -8

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3
Q

What are SNPs?

A
  • 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
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4
Q

How can repeat sequences lead to mutations which alter gene expression?

A

(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

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5
Q

What’s an example of internal DNA recombination?

A

(Neisseria)
o Recombination used to vary pili (adhesion)
o Exchange from non-expressed gene and make mosaic pilus

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6
Q

Why is the location of a gene on a chromosome important?

A

o Some parts of chromosome duplicated for longer (increased expression of some genes)
o Gene placement takes into account redundant genetic code, operons

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7
Q

What are the slow mechanisms for genetic change and diversification?

A
  • Point mutation (nucleotide change/insertion/deletion)
  • Gene duplication
  • Gene Deletion
  • Chromosomal rearrangement (inversion, intragenic recombination)
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8
Q

What are the fast mechanisms for genetic change and diversification?

A
  • Phase variation (promoter inversion, slipped-strand synthesis)
  • Antigenic variation (gene shuffling/conversion)
  • HGT (intergenic recombination, transformation, conjugation, transduction)
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9
Q

What are the features of HGT?

A
  • Resistance plasmids
  • Transposons
  • Pathogenicity island
  • DNA from different bacteria
  • ssDNA more efficient because DNA sensitive to DNAases
  • Restriction enzymes and methylases for protection
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10
Q

What are the main HGT mechanisms?

A

Transformation
Conjugation
Transduction

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11
Q

What is transformation?

A

• DNA from donor cell released to environment and taken up by competent recipient cell (progeny = transformants)

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12
Q

How did Griffith show about transformation?

A

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

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13
Q

How come E.coli cannot be transformed?

A

It is not competent

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14
Q

What are transformasomes?

A

Membranous structures that protect DNA during transformation

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15
Q

What is conjugation? What does the donor require?

A
  • Bacterial sex

* Donor has sex pilus (F)

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16
Q

What is transduction?

A
  • 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
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17
Q

What are pathogenicity islands?

A
  • Often remnant of bacteriophage
  • Insert to phage like insertion sites
  • Different G, C content %
  • Include phage making toxins
  • Phage genes integrated to genome
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18
Q

Where are most pathogenicity islands found?

A

tRNA genes most common

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19
Q

What is a virulence determinant? What factors are commonly counted as virulence determinants?

A
  • Specific trait/factor that increases the virulence of a microbe
  • Factors include growth, adhesion, invasion, resistance, damage, dissemination
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20
Q

How can virulence determinants be identified?

A
  • Aim to measure virulence
  • Ethical considerations
  • Need model reflecting human disease (route, dose, dependencies, infection kinetics, outcomes)
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21
Q

What are Koch’s postulates?

A
  • 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
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22
Q

What are the problems with Koch’s postulates?

A 
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
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23
Q

What are molecular Koch’s postulates?

A
  • Virulent organism
  • Isolate gene
  • Mutate gene
  • See reduction in virulence
  • Return gene/revert mutation to WT
  • See increase in virulence
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24
Q

How do mice infected with S. pneumoniae demonstrate the application of molecular Koch’s postulates?

A
  • Respiratory disease in mice
  • Mutant lives longer than WT
  • Mutant with restored gene wore survival than mutant, slightly better than WT
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25
Q

What are the requirements for an animal model to be considered valid when studying human disease?

A

• Need model reflecting human disease (route, dose, dependencies, infection kinetics, outcomes)

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26
Q

What’s an example of using a transposon to create mutants?

A

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)

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27
Q

What is a transposon?

A

DNA with self-encoding insertion capability, transposase, inverted repeats and selectable marker (e.g. TetR)

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28
Q

What is an insertion sequence?

A

2 inverted repeats flanking transposase gene

29
Q

What is a compound transposon?

A

has two IS elements flanking resistance gene

30
Q

What is a simple transposon?

A

has resistance gene within two inverted repeats

31
Q

What is the aim of using transposition for mutagenesis?

A

to make transposon jump from delivery plasmid into bacterial chromosome, occasionally will jump into toxin gene resulting in loss of function

32
Q

What is ID50?

A

ID50 = dose required to infect 50% of animals

33
Q

What is LD50?

A

LD50 = dose required to kill 50% of animals (les subjective)

34
Q

What is the process of competition assays?

A
  • 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
35
Q

What is signature tagged mutagenesis?

A
  • 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
36
Q

How can host factors be added?

A
  • Antibodies
  • Cells
  • Transgenic/genetically modified
37
Q

How can host factors be removed?

A
  • Depletion with antibodies
  • Genetic depletion
  • Transgenic depletion
38
Q

How can host determinants be identified?

A
  • Compare infection in normal vs those with factors removed/added
  • LD50, ID50
  • Bacterium growth
  • Pathology
  • Transmission
39
Q

What are the features of S. Typhimurium?

A
  • 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)
40
Q

Why would an iNTS vaccine be ineffective in the immunocompromised?

A
  • Would need antibody response (adaptive immunity)
  • Antibodies poor action against intracellular bacteria
  • Need helper Tcells
41
Q

How can iNTS be stopped in the immunocompromised?

A
  • Could control intracellular bacteria with IFNy

* Use model for iNTS (mouse)

42
Q

What did Murine Model iNTS show?

A
  • 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
43
Q

What other methods can be used to analyse host and bacteria interactions?

A
  • IVUS camera (reporter strain) e.g. GFP , non invasive
  • Knock down host gene function
  • 2D gel electrophoresis and western blotting
  • GWAS
44
Q

How might host gene function be knocked down?

A

o siRNA, works against specific genes

o study contribution of specific host genes in response to infection (tissue culture)

45
Q

How can GWAS be used to study host gene function?

A

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

46
Q

What was the process used to construct the Murine Model iNTS?

A
  • 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
47
Q

What is the basis of transposon mutagenesis?

A
  • 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
48
Q

What is the importance of virulence genes? At what levels can virulence gene expression be regulated?

A
  • 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
49
Q

What is phase variation? What’s an example?

A

o Gene on, gene off
o N. gonorrhoeae Protein II (OM protein, Opa, adherence and invasion)
o Can occur through slip strand mis-repair

50
Q

What is antigenic variation? What’s an example?

A

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)

51
Q

What are operons and regulons?

A
  • Operon: gene/genes on single transcript

* Regulon: multiple transcripts, multiple promoters controlled by same regulator

52
Q

How does transcriptional regulation work?

A

• Increase/decree RNA pol activity (RNA pol binds promoter, block or encourage binding)

53
Q

What makes up the Prf regulon and where is it found?

A 
o	Prf (Listeria monocytogenes) 
o	prfA as master switch
o	3 gene operons, 1 gene operons
54
Q

What is the basis of the lac operon? What kind of regulation does it show?

A

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

55
Q

What does +1 correspond to?

A

The transcriptional start point

56
Q

Where does LacI bind?

A

The operator

57
Q

How is diphtheria toxin produced?

A

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

58
Q

What happens in two component systems?

A

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

59
Q

What happens in the Bvg regulon of Bordetella pertussis?

A

 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

60
Q

What are sigma factors required for?

A

o RNA pol needs them to bind promoter
o Recycled
o Different factors = different promoters
o Control regulons

61
Q

What changes in DNA topology can control genes and how?

A

o Supercoiling
o Histone like-proteins
 Alter DNa shape
 E.g. curve DNA, RNA pol can’t bind

62
Q

What are examples of genetic rearrangements as regulation mechanisms?

A

Phase variation, antigenic variaiton, gene duplication and amplification

63
Q

What are examples of transcriptional regulation as regulation mechanisms?

A

activators, repressors, complex networks, two component systems, cic-acting thermosensors, DNA topology

64
Q

What are examples of translational regulation as regulation mechanisms?

A

mRNA efficiency

65
Q

What are examples of quorum sensing as regulation mechanisms?

A

Vibrio fischeri in squid

66
Q

What is quorum sensing?

A 
•	Communicating between species (autoinducers)
•	Certain community size = quorum
•	Al-1: acyl homoserine lactones (g-)
o	Alter transcription with regulators 
•	G+ use peptide signals
67
Q

What is Al-1?

A

• Al-1: acyl homoserine lactones (g-)

o Alter transcription with regulators

68
Q

What are Vibrio fischeri?

A

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

MIIM30011 Bacteriology (5 decks)

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