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Bacterial Pathogenicity > L8. S. pneumoniae Molecular Genetics 2 > Flashcards

L8. S. pneumoniae Molecular Genetics 2 Flashcards

1
Q

What was 1. Synthetic lethal screen (old method) and 2. Next gen (TraDIS/ Tn-seq) synthetic lethal screening used to discover?

A
  1. LpoA and LpoB in E.coli
  2. CosE and MacP in S. pneumo
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2
Q

What is the fundamental problem with forward and reverse genetic approaches?

A

Genetic redundancy.

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3
Q

Why is genetic redundancy a problem when studying essential cellular processes?

A

Because essential processes require the actions of many essential proteins in a pathway, and if any part of the pathway is blocked, the essential process is stopped, leading to cell death.

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4
Q

When does genetic redundancy occur?

A

> In real life, genetic pathways don’t just contain one linear pathway, there are many parallel pathways leading to the same outcome

> So genetic redundancy occurs when there are multiple different parallel genetic pathways leading to the same outcome, so when knocking out one gene will stop one pathway but the outcome will still occur due to the other pathways.

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5
Q

What occurs when there are multiple different parallel genetic pathways leading to the same outcome?

A

Genetic redundancy, where knocking out one gene stops one pathway, but the outcome still occurs due to the other pathways.

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6
Q

How can pathways which contain genetic redundancy be studied by conventional genetics and what is this called ?

A

By collapsing the redundancy, making other pathways redundant so there is just one pathway left, making it essential. This allows forward and reverse genetics to be used. This is synthetic lethality

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7
Q

What is synthetic lethality?

A

The combination of genetic mutations that are lethal to cells, where the relationship between genetic loci on different pathways interact, causing cell death when both mutations are present simultaneously.

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8
Q

What is the mechanism of synthetic lethality?

A

It represents a relationship where the genetic locus on one pathway and the genetic locus on another complementary pathway interact. The combination of mutations in these pathways disrupts essential cellular functions, leading to lethality.

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9
Q

Why are synthetic lethal pairs significant?

A

Identifying synthetic lethal pairs indicates that the two pathways involved converge on a critical biological outcome, useful in understanding cellular processes and developing targeted therapies.

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10
Q

What is an example of the outcome when two parallel pathways leading to an essential cellular process are both mutated?

A

Cell death when both mutations are present simultaneously.

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11
Q

How common and useful are synthetic lethal combinations?

A

These genetic interactions are rare but highly significant. Identifying synthetic lethal pairs is powerful because it indicates that the two pathways involved converge on a critical biological outcome. This can be particularly useful in understanding cellular processes and developing targeted therapies.

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12
Q

What is a great example of using synthetic lethal genetics?

A

The discovery of PBP regulatory factors in E.coli (gram negative)

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13
Q

What is the purpose of targeting PBP regulatory factors in bacteria?

A

To kill bacteria.

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14
Q

Which PBPs in E. coli are synthetically lethal to each other?

A

PBP1A (in elongation complex) and PBP1B (in division complex).

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15
Q

What happens if either PBP1A or PBP1B is non-functional?

A

The enzymes themselves and their regulators for the other enzymes become essential for growth and survival.

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16
Q

What biochemical activities do both Class A enzymes like PBP1A and PBP1B contain?

A
  1. Transglycosylase (TG) domain - polymerizes lipid into glycan strands.
  2. Transpeptidase (TP) domain - cross-links glycan strands together.
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17
Q

What is the role of Class A PBPs in synthesizing peptidoglycan?

A

They contain both biochemical activities required to synthesize peptidoglycan from the lipid II precursor.

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18
Q

What happens in E. coli if PBP1B is knocked out?

A

PBP1A becomes essential for the cell to grow and divide.

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19
Q

What does it mean if PBP1A has any activators or regulators required for its function in the context of synthetic lethality?

A

These activators or regulators would also become essential.

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20
Q

What happens when PBP1B is removed from E. coli cells?

A

It collapses the genetic redundancy and allows for a forward genetic approach on the PBP1A pathway, including its regulators.

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21
Q

How is synthetic lethality discovered?

A

Through synthetic lethal screens using unstable plasmids paired with transposon mutagenesis.

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22
Q

Describe the first step in a synthetic lethal screen.

A

Use an E. coli strain that lacks the LacZYA genes (ΔlacZYA strain), which are responsible for blue/white selection.

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23
Q

Describe the whole “4 step” process of the old synthetic lethal screen in E.coli for discovering LpoA/B

A
  1. Strain that lacks LacZYA gene (ΔlacZYA strain)
    >This LacZ gene is necessary for turning cells blue in media.
  2. Put LacZYA gene onto unstable plasmid
    >Also delete one synthetic lethal gene in the strain, e.g. PBP1b and the functional gene for this this on unstable plasmid too, so PBP1b can act as a selectable marker (meaning the unstable plasmid contains the functioning PBP1b gene)
    >So this strain contains a functional PBP1A gene which is now essential as the PBP1b gene has been removed and is just on the plasmid.
  3. Carry out Transposon mutagenesis on this ΔlacZYA strain
    >Giving multiple different transponents each of which with a random transposon insertion into the genome.
  4. Then plate the cells
  5. The genetic relationship
    >If the transposon has jumped into a random gene nothing to do with the synthetic lethal phenotype one of two outcomes occur:
    i. No selection maintained for
    plasmid, lost from population
    completely (fully white)
    ii. Or sectored colony (some maintain
    plasmid by chance so remain blue,
    while outer colonies lose it and
    turn white).
    >If the transposon jumps into a gene that is synthetically lethal, if this strain loses the unstable plasmid, the cell will die as needs a functioning PBP1b enzyme to survive. As this strain cannot lose this plasmid:
    i. The colony will be small, as if by
    chance one of the daughter cells
    loses this plasmid they die; so
    progeny is constantly being lost
    ii. Also colonies appear as solid blue
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24
Q

Explain the genetic relationship in a synthetic lethal screen involving PBP1A and PBP1B.

A

PBP1A and PBP1B form a synthetic lethal pair. If one gene is knocked out, the other becomes essential for survival, allowing the identification of essential genes and their regulators.

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25
Q

What is the LacZYA gene and what happens when it is knocked out

A

> LacZYA gene: This is a name given to a set of genes involved in the metabolism of lactose in bacteria. Specifically, LacZ encodes β-galactosidase, which is responsible for breaking down lactose into glucose and galactose. ΔlacZYA strain: The “Δ” symbol denotes a deletion.

> Therefore, “ΔlacZYA strain” refers to a bacterial strain that has had the LacZYA genes deleted or knocked out. This deletion prevents the bacteria from turning blue in the presence of X-gal, as they lack the necessary LacZ enzyme to cleave X-gal and produce the blue color.

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26
Q

What happens when the ΔlacZYA strain with the unstable plasmid is grown in media containing ampicillin and X-gal?

A

The cells form blue colonies because the plasmid is maintained due to the selection pressure from ampicillin.

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27
Q

What is observed when ampicillin is removed from the media in a synthetic lethal screen?

A

Without ampicillin, the selection pressure is removed, causing the plasmid to be lost over time, leading to a sectored phenotype: blue at the center (plasmid maintained) and white at the edges (plasmid lost).

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28
Q

What is the role of the LacZYA gene in a synthetic lethal screen?

A

The LacZYA gene allows for blue/white selection in the presence of X-gal. The ΔlacZYA strain, which lacks this gene, is used to monitor plasmid maintenance in the screen.

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29
Q

What does a blue colony indicate in a synthetic lethal screen?

A

A blue colony indicates that the cell must maintain the unstable plasmid to survive, showing that a gene involved in synthetic lethality with PBP1B (LpoA, as it is a regulator of PBP1A) has been disrupted by the transposon.

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30
Q

What does it mean if a colony is small and solid blue in a synthetic lethal screen?

A

It means the cell cannot lose the plasmid because it contains the only functional copy of PBP1B. This means the tranpsoson hit the LpoA gene (regulator of PBP1A) making the PBP1B on the plasmid essential for survival. The colony appears small because cells that lose the plasmid die, leading to reduced colony growth.

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31
Q

What was the outcome of screening over 30,000 colonies in the synthetic lethal screen for PBP regulatory factors?

A

Only two synthetically lethal hits were identified, both of which were transposons that jumped into the lpoA gene.

32
Q

Explain why a colony might shrink in a synthetic lethal screen.

A

The plasmid has an unstable origin of replication, leading to its loss over time. Cells that lose the plasmid also lose the essential PBP1B gene, resulting in cell death and smaller colony size.

33
Q

How does a synthetic lethal screen help in identifying PBP regulatory factors?

A

By using the unstable plasmid system, researchers can identify genes that are essential when the plasmid is lost, highlighting the interactions and dependencies between genes like PBP1A and PBP1B.

34
Q

What must be done after identifying synthetic lethal ‘hits’ to confirm the genetic relationship?

A

Follow up and verify the hits by stripping out all other machinery to check if the identified genes are synthetically lethal for each other, using an inducible promoter (IPTG).

35
Q

How can the synthetic lethality of PBP1B and LpoA be tested?

A

Knock out both genes, reintroduce one using an inducible promoter (IPTG), and observe if the strain grows in the presence of IPTG and doesn’t in its absence.

36
Q

Where are LpoA and LpoB found in E. coli?

A

Both LpoA and LpoB are found in the outer membrane.

37
Q

What is the role of LpoA in E. coli (extra reading)?

A

LpoA is synthetically lethal with PBP1B and interacts with the N terminus of PBP1A, stimulating its enzymatic activity, particularly activating the transpeptidase (TP) activity.

38
Q

What is the role of LpoB in E. coli (extra reading)?

A

LpoB is synthetically lethal with PBP1A and interacts with the UvrB domain 2 of PBP1B. It plays a role in the length distribution of glycan chains, suggesting involvement in initiating and terminating glycan synthesis.

39
Q

Describe the new model of peptidoglycan synthesis after the discovery of LpoA and LpoB

A

If a hole appears in peptidoglycan, due to random chance or growth, these proteins can fall through the peptidoglycan and stimulate PBP1A and PBP1B enzymes which make peptidoglycan on their own (de novo) and they fill in the gap and therefore prevent their own activation. (model: everywhere there is a hole these enzymes (LpoA/B) can reach through, if they can reach through the enzymes are activated and fill in the hole).

40
Q

What was the traditional belief about the control of PBP enzymes, and how has it changed?

A

Traditionally, it was believed that these enzymes were controlled from the cytoplasmic side. However, it is now understood that outer membrane proteins like LpoA and LpoB regulate them from the outer side.

41
Q

Why are outer membrane proteins like LpoA and LpoB considered good drug targets?

A

They don’t have to cross barriers to reach the target, making them more accessible and potentially effective drug targets.

42
Q

Are Class A PBP complexes in E. coli independent of the Cell Elongation and Cell Division Machinery?

A

Yes, to an extent, they can synthesize peptidoglycan alone due to regulation by outer membrane proteins like LpoA and LpoB, independent of the coordination with MreB and FtsZ.

43
Q

Why is the model for how class A PBP enzymes are regulated in gram-positive bacteria different from gram-negative bacteria?

A

Gram-positive bacteria like S. pneumoniae lack an outer membrane, so the regulatory mechanisms must differ from those in gram-negative bacteria like E. coli.

44
Q

What synthetic lethal pair was identified in S. pneumoniae, and what does it imply?

A

PBP1A and PBP2A form a synthetic lethal pair, implying parallel genetic pathways leading to the same essential process (the same as in E.coli).

45
Q

How has next-generation sequencing impacted forward genetic approaches and what is one issue with it?

A

> Next-generation sequencing has revolutionized forward genetic approaches by allowing the detection of the absence of cell signatures and the identification of essential genes without the need for cells to survive.

> Need to have a lot of confidence in the sequencing technology.

46
Q

What tools are now unnecessary for synthetic lethal screens due to next-generation sequencing?

A

The complex 4-step process involving unstable plasmids and extensive genetic manipulation is no longer necessary; only transposon mutagenesis is required (CRISPR), which can be done in almost all bacteria

47
Q

How does next-generation sequencing aid in synthetic lethal screens?

A

It captures millions of independent transposon insertion events in one dataset, allowing mapping to the genome and identification of essential genes by observing gaps where lethal insertions occurred.

(In this case, if having a known synthetic lethal combination, a cell lacking PBP1A due to knockout strain and PBP2A due to transposon insertion, this cell will die.
So if we took a massive populations of transposon insertions and then mapped them to the genome, so we have many insertion sites, we can look at PBP2A and see a gap as the transposon insertion died and therefore not available to be sequenced).

48
Q

What is In-seq a different name for?

A

TraDIS

49
Q

What are the steps to identify genes synthetically lethal with PBP1A using next-generation sequencing (TraDIS)?

A

Build large transposon libraries with a WT control and a PBP1A strain, grow them, perform next-gen sequencing, and map the insertions to the genome to identify synthetic lethal genes.

50
Q

What did the synthetic lethal screen between WT and Δpbp1A (PBP1A knockout) strain reveal?

A

It confirmed PBP2A as conditionally essential by showing fewer insertions in the Δpbp1A strain than in the WT, indicating cell death when PBP2A is non-functional without PBP1A through less transposon insertions into PBP2A gene as the cells die.

51
Q

What does it mean if a bacteria cannot tolerate transposon insertion even in the WT?

A

It is an essential gene, as transposon insertion (disrupting function of the gene) kills the cell even when all other genes are functioning (if it was a synthetic lethal gene/ conditionally essential, it would survive in WT as has the other pathway for survival but die when that other pathway is knocked out)

52
Q

What new regulators of class A PBP enzymes were identified in S. pneumoniae using TraDIS sequencing of synthetic lethal pathways?

A

CozE and MacP were identified as new regulators.

53
Q

How does MacP differ from Lpo proteins in its regulatory mechanism? (extra reading)

A

MacP is oriented to associate with PBP2a via its TM domain or N-terminal cytoplasmic tail and is regulated by phosphorylation via the kinase StkP, unlike the outer membrane proteins of Lpo which bind to C-terminus and is not regulated by phosphorylation.

54
Q

Describe what MacP activity in S. pneumo is dependent on (extra reading)

A

MacP activity is dependent on phosphorylation, so is subject to kinase regulation. The serine/threonine kinase StkP phosphorylates MacP at residue T32. This phosphorylation event allows MacP to activate the penicillin-binding protein PBP2a, which is involved in bacterial cell wall synthesis. Furthermore, this phosphorylation by StkP is influenced by external signals that are detected by StkP’s PASTA domains, integrating environmental cues into cellular responses.

55
Q

What role does CozE play in cell wall synthesis by PBP1a?

A

CozE coordinates cell wall synthesis, preventing inappropriate cell wall activity that can be lethal.

56
Q

What happens to CozE’s essentiality in a Δpbp1a strain?

A

CozE becomes non-essential in the absence of PBP1a, indicating its role in regulating PBP1A and preventing its toxic activity.

57
Q

What occurs when both CozE and PBP1A are knocked out, and PBP1A is reintroduced?

A

The reintroduction of PBP1A leads to cell swelling and lysis due to inappropriate peptidoglycan synthesis, highlighting potential drug targets for overstimulating PBP1A by reducing CozE regulation of the enzyme.

58
Q

Why might targeting CozE for therapeutic purposes be beneficial?

A

Removing CozE’s regulation on PBP1A can trigger PBP1A’s toxic activity, leading to the death of pneumococcal cells.

59
Q

What is MacP’s role in S. pneumoniae?

A

MacP was discovered as a regulator of Class A PBPs (like CozE) and specifically regulates PBP2A, functioning similarly to LpoA and B in gram-negative bacteria.

60
Q

What are the screening results for MacP in a synthetic lethal screen with Δpbp1A?

A

WT strains show MacP transposon hits, indicating cells can survive without MacP when PBP1A is present. In strains lacking PBP1A, there are no insertions in MacP, demonstrating it is a synthetic lethal pair with PBP1A, as cells die without MacP when PBP1A is absent. (expected results, unlike CozE which had extra regulatory function for PBP1A).

61
Q

Why must synthetic lethal ‘hits’ be followed up and verified?

A

To confirm the genetic relationship, it’s essential to verify that the pair is conventionally synthetic lethal. This requires ensuring cells die when either PBP1A or MacP is turned off, confirming their conditional essentiality.

62
Q

What happens when MacP is removed from S. pneumoniae cells?

A

Cells wither and die as the cell wall isn’t made every generation, leading to cell lysis over time.

63
Q

Should MacP be targeted for therapeutic gain in S. pneumoniae?

A

No, triggering the removal of MacP for therapeutic gain is not effective as it doesn’t kill fast enough, giving bacteria too many opportunities to mutate and escape the phenotype.

64
Q

How are CozE and MacP similar to LpoA and LpoB in E. coli?

A

CozE and MacP in S. pneumoniae have similar functions to LpoA and LpoB in E. coli, regulating Class A PBP enzymes and maintaining peptidoglycan homeostasis.

65
Q

What does the synthetic lethal screen reveal about MacP and PBP1A interaction?

A

The screen shows that MacP is conditionally essential when PBP1A is absent, indicating a synthetic lethal relationship crucial for cell survival.

66
Q

How is synthetic lethality confirmed in genetic relationship studies?

A

By turning off one gene (PBP1A) and observing if the cells die when the other gene (MacP) is switched off, verifying the synthetic lethal relationship.

67
Q

What is the effect of MacP removal on cell wall synthesis in S. pneumoniae?

A

Removing MacP disrupts proper cell wall synthesis, causing cells to wither and die due to lack of cell wall production, eventually leading to cell lysis.

68
Q

What is the importance of verifying synthetic lethal hits?

A

Verifying synthetic lethal hits ensures accurate identification of genetic relationships, confirming that certain gene pairs are essential for survival when one is inactive.

69
Q

What is my overview point for the use of bacterial genetics?

A

Bacterial genetics is a potent tool to understand fundamental biology and drive identification of new points of weakness in bacterial metabolism towards therapeutic gain

70
Q

What is RodA and why is it under investigation? (extra reading)

A

RodA is a member of the SEDS (shape, elongation, division, and sporulation) protein family (found in Bacillus and S. pneumo) and is under investigation because it has enzymatic activity as a transglycosylase. It is targeted by compound 654/A.

71
Q

How is RodA studied in the context of bacterial cell processes? (extra reading)

A

RodA is studied using reverse genetics, which helps identify its role and function in bacterial cells, particularly in cell wall elongation.

72
Q

What complex is RodA integral to in bacterial cells? (extra reading)

A

RodA is integral to the Rod complex, which governs cell wall elongation.

73
Q

What role does RodA play within the Rod complex? (extra reading)

A

RodA drives the dynamic movements within the Rod complex due to its role as the principal peptidoglycan glycosyltransferase (PGT).

74
Q

How does RodA interact with Class B PBPs? (extra reading)

A

RodA complements Class B PBPs, helping to ensure proper cell wall synthesis and maintenance.

75
Q

What does the investigation of RodA involve? (extra reading)

A

The investigation of RodA involves targeting it with compound 654/A, studying its transglycosylase activity, and using reverse genetics to understand its role in the SEDS protein family and its involvement in cell wall elongation.

76
Q

Why is synthetic lethal screening essential now?

A

To find new stuff about bacteria is through synthetic lethality, as all the easy stuff has bene found, strip away of one layer of genetic redundancy to reveal new genetic relationships underneath (synthetic lethality)

Bacterial Pathogenicity (11 decks)

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