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Frontiers in infectious diseases > 3. Bacterial recombineering, Virulence factor identification and charaterisation > Flashcards

3. Bacterial recombineering, Virulence factor identification and charaterisation Flashcards

1
Q

What is recombineering?

A

Using recombination as an engineering tool to generate tools to study bacterial pathenogenesis

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2
Q

What is the purpose of recombineering?

A

To investigate the function of genes in a research setting.

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2
Q

What is the definition of recombineering?

A
  1. Recombination-mediated genetic engineering
  2. Recombineering is a genetic and molecular biology technique based on homologous recombination systems.
  3. Unlike older methods of using restriction enzymes and ligases to combine DNA sequences.
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2
Q

What natural bacterial mechanism does recombineering take advantage of?

A

Homologous recombination during DNA repair.

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3
Q

What is recombineering a form of?

A

Ligation independent cloning

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4
Q

What is the process of homologous recombination?

A
  1. It uses DNA pol and its exonuclease activity.
  2. At a dsDNA break DNA pol chews back the ends.
  3. The 2 strands separate and a D-loop forms.
  4. there is switch over between the broken DNA and the complementary DNA.
  5. The gap is repaired by DNA pol.
  6. The repair is finished, and the two strands separate.
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5
Q

What are the 3 key steps in homologous recombination?

A
  1. Cross over
  2. Replacement
  3. Repair
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6
Q

What is often used as the template for repair for recombineering?

A

Plasmid vectors

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7
Q

How are plasmids engineered to contain DNA of interest?

A
  1. The DNA of interest can be inserted with restriction enzymes or ligand independent methods.
  2. Regions of complementarity can be joined with polymerases or recombinases.
  3. The plasmid now contains the gene of interest and you can put this DNA into cells.
  4. This is used to test pathogenesis in bacteria
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8
Q

What are knockouts?

A
  1. The use of homologous recombination to delete a gene or region of a gene to disrupt the function of that gene.
  2. They are important experiments in understanding the function of genes.
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9
Q

What are knock out experiments used to do?

A

To test a gene to see if it has the function you think it has.

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10
Q

What does making gene knock out require?

A
  1. A knowledge of the sequence of the gene and flanking sequences either side of the gene.
  2. The gene of interest needs to be specifically disrupted.
  3. The surrounding DNA needs to be left undisturbed and uninfluenced.
  4. You need to know the sequence to do this
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11
Q

What is commonly used to replace the gene of interest in knock out experiments?

A
  1. Antibiotic resistance cassettes
  2. this allows successful transformation to be selected for.
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12
Q

What is a common cloning plasmid used to transform bacteria?

A
  1. pBR322
  2. It contains lots of different restriction sites to insert DNA.
  3. Contains an antibiotic resistance gene
  4. Small to optimise cloning efficiency
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13
Q

What is the process for putting the replacement gene cassette into a cloning vector?

A
  1. Identify the flanking regions of the gene of interest and PCR them up.
  2. Incorporate RE sites in the flanking regions in the PCR primers.
  3. Take an antibiotic resistance cassette and cut it out using restriction enzymes. It’s often kanamycin resistance from PUC4K.
  4. Ligate the flanking regions and resistance cassette into the vector. The flanking regions need matching restriction sites with the vector and resistance matching with the resistance cassettes.
  5. The flanking regions and the resistance gene join into one plasmid that can freely replicate but is not expressed.
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14
Q

How is the cloning vector used to insert the knock out into the gene of interest?

A
  1. The plasmid is linearised using a different restriction site that outside the cloning region.
  2. The plasmid is transformed into the bacteria
  3. The natural HR DNA repair mechanism replaces the gene of interest with the gene from the plasmid.
  4. Some of the bacteria will be successfully transformed and have the knockout.
  5. These can be selected for using the antibiotic you inserted the resistance for.
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15
Q

What methods can be used to transform bacterial cells?

A
  1. Electroporation
  2. Chemical transformation
  3. Natural competence
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16
Q

What factors can influence how many cells take up the replacement DNA?

A
  1. How accessible the secondary structure is
  2. The rate of replication
  3. How much DNA is being inserted
  4. How much DNA is available
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17
Q

What is the transformation efficiency?

A
  1. The proportion of cells that successfully take up the DNA.
  2. about 1 in 10,000 to 1 in 1,000,000
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18
Q

What is used to select for successful transformation?

A
  1. The antibiotic resistance cassette
  2. Use of selective media containing the antibiotic.
  3. This ensures the growth of only successfully transformed colonies.
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19
Q

Why do you need lots of controls in knock out experiments?

A
  1. To ensure what you are seeing is due to the reason you think it is.
  2. The same outcomes could be due to different reasons.
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20
Q

How do you confirm knock out experiments are successful?

A
  1. Using PCR and measuring gene expression (ELISA/ Western blot)
  2. Functional assays to see if the gene of interests has the function you thought it had
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21
Q

What are things that need to be considered in knock-out experiments?

A
  1. Multiple gene presence
  2. Off target integration
  3. Undesirable polar effects
  4. Undesirable global effects
  5. Is the gene essential to the bacteria
  6. What effect does introducing the antibiotic have
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22
Q

How does multiple gene presence effect knock out experiments?

A
  1. Lots of different copies of the same gene in the same genome eg Opa
  2. This redundancy is important for the bacteria but means when you knock out 1 gene the function is still preserved
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23
Q

How does off target integration effect knock out experiments?

A
  1. These often happen due to incomplete knowledge of the genome sequence.
  2. The flanking regions could be present elsewhere so it could knock out a different gene or no gene at all.
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24
Q

How can undesirable polar effects effect knock out experiments?

A
  1. Polycistronic mRNA contains lots of gene transcripts on 1 DNA.
  2. knocking out one gene that is on these polycistronic mRNA can affect the function of the other genes.
  3. This can cause different effects or you could mistake the function of 1 gene as the function of the knockout.
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25
Q

How can undesirable global effects effect knock out experiments?

A
  1. Proteins don’t work in isolation so knocking 1 out can have knock on effects.
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26
Q

How does knocking out an essential gene effect the results?

A
  1. Sometimes, we don’t know what genes are essential to bacterial life.
  2. When you know these genes, you can tell what their function is as all the transformed cells die.
  3. You get no colonies to select from, so you don’t know if the transformation has failed or if the gene is essential
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27
Q

How can introducing antibiotic resistance effect knock out experiments?

A
  1. Antibiotic production can carry a fitness cost or cause a stress response.
  2. Often, it gives a bacteria that doesn’t normally produce antibiotics something else to do.
  3. It could change the function or expression of other proteins
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28
Q

What do you need to use to validate your knock out experiment?

A

Knock ins or recomplementation

29
Q

What is complementation or recomplementation?

A
  1. Complementation is knocking in a function
  2. Recomplementation is knocking in a gene that has been knocked out
30
Q

What are knock in experiments used for?

A
  1. Studying essential gene function
  2. Checking for undesirable effects on expression of other genes.
  3. Knocking in a function in another bacteria can allow the study of function that could be masked in its native bacteria
31
Q

What are the 3 main ways to knock in genes?

A
  1. Putting the gene back in where it was knocked out
  2. Putting the gene back elsewhere in the genome
  3. Putting the gene into another bacteria.
32
Q

Knock ins: Putting the gene back in the same place

A
  1. Rereplacing the antibiotic resistance gene
  2. See if the function is restored
  3. Needs to have a good functional selection assay for this gain of function to prove successful transformation. No resistance to select from.
33
Q

Knock ins: Putting the gene elsewhere in the genome

A
  1. More common method of knock ins
  2. Put the gene back in with another antibiotic resistance cassette downstream of the gene for selection.
  3. The transformed bacteria has both function and selection method.
  4. Use recombination to replace a gene or put it in an area of non functioning in the genome.
  5. The area needs to be carefully selected to not disrupt something else.
34
Q

Knock ins: Putting the genome into another host bacteria

A
  1. Most common method of knock ins
  2. Does another host gain that function
  3. This is called a heterologous host
  4. Just put the gene on an expression plasmids with an antibiotic resistance cassette.
  5. Make sure it has the correct structure and can attach signal peptides.
  6. Can be used to put into E.coli to make tonnes of the protein to purify and test the function
35
Q

What needs to be considered in knock in experiments?

A
  1. Epigenetics
  2. Post translational modification if being expressed in a different host
  3. Codon bias
  4. Levels of gene expression
  5. Expression tropism
  6. Effect of antibiotic resistance
  7. Ethical considerations
36
Q

Why do epigenetic changes need to be considered in knock in experiments?

A

Epigenetic differences in other areas in the genome can lead to silencing and incorrect results.

37
Q

Why does codon bias need to be considered in knock in experiments?

A
  1. As codons appear at different frequencies some organisms with lower GC content might not have all the tRNAs for those codons.
  2. This means some organisms won’t be able to make knocked-in genes from different organisms due to lack of tRNAs.
  3. CG content needs to be considered when choosing a heterologous host.
  4. You can supplement other hosts with extra tRNAs or by changing the gene sequence so it has the right codons for that organism.
38
Q

Why does matching expression need to be considered in knock in experiments?

A
  1. Different Expression vectors or systems can express proteins at different levels.
  2. The expression level needs to be similar to the native level otherwise it can inhibit function
  3. You can drive protein expression if you want to purify the protein or do crystallography.
39
Q

Why does matching tropism need to be considered in knock-in experiments?

A
  1. Tropism is where in the cell the protein is expressed.
  2. This needs to be correct to show correct function.
  3. If too much protein is made, it can affect folding and processing, which disrupts function and trafficking to the right location.
40
Q

Why do ethical reasons need to be considered in knock in experiments?

A
  1. Knock ins can give pathogens more functions and potentially dangerous functions.
  2. You need to be mindful that the gain of function is not worse than what is in nature.
  3. The containment needs to be considered very carefully.
41
Q

What is codon bias?

A
  1. Due to the redundant nature of the genetic code there are about 60 codons for the 20 amino acids.
  2. Each tRNA is specific for the codon.
  3. Some codons appear more frequently than others. These higher frequency codons usually are more GC based
  4. Some organisms have different or lower GC content so might not have all the tRNAs for those codons
42
Q

Why do post-translational modifications need to be considered in knock-in experiments?

A
  1. Different bacteria can have different systems to modify proteins
  2. It is less of a concern for prokaryotes but still the correct modifications need to happen.
43
Q

What is recombinant protein expression?

A
  1. Making a protein and studying it outside of the bacterial system.
  2. Normally studying just 2 proteins like a receptor and ligand.
  3. Safer as it’s just protein and no chance of infection.
44
Q

What considerations need to be made when doing recombinant protein expression?

A
  1. Amplification of cDNA for eukaryotic proteins
  2. Cloning method
  3. Sequence tags
  4. Using the right expression system
  5. GC content
45
Q

Why does cDNA amplification need to be considered when doing recombinant protein expression?

A
  1. Eukaryotic genes contain introns.
  2. Bacterial expression systems cannot deal with introns.
  3. So you need to make cDNA from mRNA so the bacteria can use it.
  4. The correct exons need to present and in the right order.
46
Q

Why does the cloning method need to be considered when doing recombinant protein expression?

A
  1. Ligation independent methods or restriction enzyme based.
  2. Choose for maximum efficiency
47
Q

Why do sequence tags need to be considered when doing recombinant protein expression?

A
  1. Protein purification without tags is painstaking and based on lots of assumptions.
  2. You can add poly His tags to separate proteins using columns.
  3. For eukaryotic proteins you can use Fc tags
48
Q

What are poly His tags?

A
  1. 6 histidines
  2. They have high affinity for divalent metal cations like nickel.
  3. These catch the desired protein and wash the other away.
  4. You can obtain highly purified protein.
49
Q

What are Fc tags?

A
  1. Add an Fc antibody fragment to the protein.
  2. These are then secreted and you can purify them from the supernatant.
  3. This often replaces transmembrane domains in receptors.
50
Q

Why does expression system need to be considered when doing recombinant protein expression?

A

You need to consider a variety of factors:
1. Right plasmid
2. Right promoter (eukaryotic vs prokaryotic)
3. Efficient expression
4. Codon bias
5. GC content

51
Q

What are the typical characteristics of prokaryotic expression vectors?

A
  1. Start codons
  2. 6 his tag
  3. Multiple cloning site with restriction enzyme sites to insert the gene of interest.
  4. Stop codons
52
Q

What are the typical characteristics of eukaryotic expression vectors?

A
  1. Clone gene of interest between the signal peptide and Fc tag.
  2. The protein gets secreted.
  3. Use protein A or protein G to purify.
53
Q

What is protein A/G?

A
  1. They originate from organisms like S.aureus.
  2. They bind the Fc regions of antibodies.
  3. This is used for immune evasion to make the bacteria look like self by being coated in antibodies.
  4. You can use a resin coated in protein A or G to extract protein from a supernatent.
54
Q

What are the common factors between N. meningitidis, H. influenzae and Moraxella catarrhalis?

A
  1. They are all human restricted pathogens.
  2. They live in the back of the throat.
  3. They are opportunistic pathogens.
  4. They bind to CEACAMS
55
Q

What are CEACAMs?

A
  1. Carcinoembryonic antigen related cell adhesion molecule.
  2. First discovered in embryonic cancers
  3. They are a superfamily of immunoglobulin proteins.
  4. Most pathogens bind to CEA, CEACAM1, CEACAM3 and CEACAM6.
  5. Highly evolved human pathogens don’t bind CEACAM3 to avoid activating neutrophils.
56
Q

Where is CEACAM1 expressed?

A

It is highly expressed on most human cells.

57
Q

Where is CEACAM3 expressed?

A

Neutrophils.

58
Q

How does N. meningitidis bind to CEACAM?

A
  1. It binds CEACAM via Opa.
  2. These are ß-barrels in the outer membrane of the bacterial cell.
  3. They bind through the hyper variable loop 2 and 3.
59
Q

How does H. influenzae bind to CEACAM?

A
  1. It binds CEACAM through the P5 protein.
  2. These are ß-barrels in the outer membrane of the cell.
  3. They bind through the hyper variable loops 1 and 3.
60
Q

How does Moraxella catarrhalis bind to CEACAM?

A
  1. It binds with a trimeric autotransporter adhesin.
  2. It is ubiquitous surface protein A1 or UspA1.
  3. ß-barrel anchor in the outer membrane with a coiled stalk and a ß-barrel head.
  4. The functional region is the stalk and the head.
  5. It sticks up from the bacterial cell surface.
61
Q

How was H. influenzae shown to bind to CEACAM?

A
  1. Knockout of P5 to removed P5 expression.
  2. Attach Fc tag to CEACAM by replacing the transmembrane region.
  3. Antibodies specific for the Fc region are used to detect CEACAM binding.
  4. The control showed good CEACAM binding.
  5. There was no CEACAM binding in the P5 knockouts.
  6. However later experiments show only binding to soluble CEACAM was effected.
  7. Further experiments showed a second ligand P1 that bound to CEACAM on other cells.
  8. This redundancy is essential for function.
62
Q

How was M. catarrhalis’s UspA1 shown to bind to CEACAM?

A

Use of different knockouts.
1. Started with the whole protein and it bound to CEACAM1.
2. A knockout of the back half showed the front half couldn’t bind to CEACAM.
3. A knockout of the front half showed the Back half could still bind CEACAM.
4. The Back half of UspA1 contains the binding motif.
5. Making smaller and smaller fragments of UspA1 located the binding motif.
6. This motif was found to be rD-7.

63
Q

What is rD-7?

A
  1. A small fragment of UspA1 that is responsible for binding to CEACAM.
  2. It showed monomeric, dimeric and trimeric binding to the his tag and CEACAM1.
  3. It is located in the coiled stem of the protein.
  4. Different from other autotransporters that bind through the head.
64
Q

What did site directed mutagenesis of CEACAM show about its binding?

A
  1. This was used to change single nucleotides in rD7 and in the N domain of CEACAM1.
  2. Showed All 3 species target CEACAM and the same face and binding site of CEACAM.
  3. This is an example of convergent evolution.
  4. Show I91 is an essential residue in CEACAM for pathogen binding.
65
Q

How was the sequence of rD-7 discovered?

A
  1. It was mapped by using mutations.
  2. Mutations in most areas didn’t affect binding.
  3. Found an isolated binding pocket of about 15 residues that are essential for CEACAM1
66
Q

How can we use this knowledge of rD-7?

A
  1. It could be used as a vaccine antigen if conserved enough.
  2. It could be turned into an anti-adhesin peptide as a treatment for infection.
67
Q

What needs to be considered when turning rD-7 into a potential treatment?

A
  1. Ensure rD-7 is present in all strains of M. catarrhalis.
  2. Some key strains don’t contain it including O35E which is the worldwide benchmark for M. catarrhalis.
  3. This shows it is important to use different strains when studying a species
68
Q

Can M. catarrhalis alter the UspA1 phenotype by acquiring DNA from heterogeneous strains?

A
  1. They are naturally competent so can take up DNA.
  2. DNA encoding UspA1 from Mx2 was provided to O35E.
  3. These bacteria were grown up without selective pressure.
  4. Colony blotting was used to detect O35E binding to CEACAM.
  5. O35E gained the ability to bind CEACAM1.
69
Q

What does M. catarrhalis O35E gaining the ability to bind CEACAM demonstration?

A
  1. They are highly mutable.
  2. They can take up DNA from other strains or species to change their phenotype.
70
Q

How could rD-7 be used as an anti-adhesin?

A
  1. When soluble rD-7 is added to cultures of all 3 species, CEACAM1 binding is inhibited.
  2. This can be used as a treatment to prevent bacterial colonisation.
  3. This can be used in lots of infections
71
Q

Could rD-7 be used as a vaccine antigen?

A

Yes but only for M. catarrhalis infections.

Frontiers in infectious diseases (4 decks)

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