S. pneumoniae and the Cell Wall Flashcards

Question

How does penicillin kill bacteria? Covalent bonds? Transglycosylase? (hint - lysis)

Answer 1

Inhibits transpeptidase activity, as it is a chemical mimic of D-Ala - D-Ala and blocks transpeptidation through irreversible competitive inhibition PBP forms a covalent bond with penicillin, locking it in inactive state; New protein is required Transglycosylation is still occurring, generating further glycan chains Generates weakness in the cell wall and the cell eventually lyses

Answer 2

Biochemically as they are all covalently modified through penicillin-binding - Radioactive or fluorescent penicillin used for gel electrophoresis Gels improved and some bands split in 2, identifying further proteins 6 PBPs in S. pneumoniae These are all targeted by drugs to treat infections

Answer 3

Grow and divide through repeating cycles of elongation and division Bacterial cells have dedicated protein machinery for each growth mode

Answer 4

Both are multimeric and contain some PBP enzymes Elongation - Anchored by an actin like protein called MreB Division - Slightly more complicated complex and anchored by FtsZ

Answer 5

Lost MreB coordination factor in elongation complex Slight changes in the certain membrane proteins involved in each complex; Missing certain factors If we can find ways to deregulate either one of the complexes, we might find a way of perturbing the function and incorporate damage into the cell wall

Answer 6

FtsZ and its role in cell division machinery MreB and its role in cell elongation machinery LpoA/LpoB and their role in cell wall regulation

Answer 7

Forward genetics to identify FtsZ Reverse genetics to identify MreB and further understand cell elongation Next gen sequencing and synthetic lethality to identify LpoA/LpoB

Answer 8

Mutagenesis --> Phenotype --> Genotype - Perform some form of random mutagenesis; Chemical agent, UV light, transposons, CRISPR - That mutagenesis reveals a phenotype; Either selecting for that phenotype or identifying subjects with that phenotype - Then identify genotype responsible for that phenotype

Answer 9

Cells must survive to be available for study Mutagenesis could cause phenotype of cell death; Nothing to then study Conditional phenotypes are needed to study essential processes - Permissive – Cells live - Non-Permissive – Cells die - Can be achieved in multiple ways; Changing media composition (e.g. add salt, Mg2+, temperature, +/- antibiotics etc.) e.g. FtsZ

Answer 10

Mutated E. coli using nitrosoguanadine and selected cells displaying phenotype of not dividing, and only elongating in non-permissive (higher temperature) conditions Phage complementation

Answer 11

Use λ phage which contains random chunks of wild type E. coli genome and treat mutant cells, then grow them at 42°C Anything that survived would have been complemented with the wild type gene, allowing it to survive Many phages all at once; Don’t know how much genome has been packaged into your λ phage so you often get a readout of not just one gene, but many - Need to compare multiple different complimenting phage and find the gene they all have in common, as this is the one responsible for the phenotype

Answer 12

Use of phage complementation on different fts mutant strains - Multiple temperature sensitive E. coli - Multiple different complementary phage ZapAB was missed as they're so small; Not likely to get mutation

Answer 13

Combination of GFP-tagged fts proteins and the fts temperature sensitive strains

Answer 14

1. FtsZ (tubulin homologue) polymerises into a dynamic structure (Z-ring) at the new division site (middle of cell) - It does this in combination with other protein factors 2. This structure recruits all other cell division proteins to the division site to drive cell division/PG synthesis

Answer 15

Helps division site selection Provides ‘protein platform’ for further assembly FtsQLB comes in, then FtsK, then FTSW and PBP enzymes; Now getting PG synthesis Finally, FtsEX and FTSN join; Now getting hydrolysis to ensure PG is cut to insert newly synthesised material

Answer 16

Mostly conserved - Most components have the same names - Order of components is also very similar Any bacteria has an FtsZ ring on which all of the cell division proteins are recruited; FtsZ could be a good antibiotic target

Answer 17

FtsZ is a tubulin homologue; Tubulins are important for the function of eukaryotic cells and cell division - Hard to stop a compound from hitting tubulin when targeting FtsZ - Targeting FtsZ/tubulin leads to cell death, meaning an inhibitor would be an effective cancer chemotherapy drug

Answer 18

Momentary inhibition, but cell count then recovers and continues to increase Combined with Cefdinir (penicillin like) to increase potency of both and produce more cell lysis

Answer 19

Mutagenesis <----> Phenotype <-- Genotype Start with a genotype (gene of interest) May or may not know the phenotype Carry out mutagenesis to understand the phenotype in more detail - Mutagenesis is more targeted (not random); Site directed mutagenesis e.g. PCR modification of specific codons

Answer 20

Use of inducible promoters - In presence of inducer, inducible promoter is expressed and cell lives - In its absence, cell dies

Answer 21

MreB MreC MreD Needed for cell growth

Answer 22

Deleted mreB and put it back on an inducible promoter, induced by xylose (xylose gives WT) By removing xylose, you can see what happens to phenotype without mreB Cell morphology changed and they lyse; Osmotic pressure

Answer 23

Used tagged-MreB to study MreB localisation Model of multiple ‘small’ protein-complexes driving diffuse cell wall synthesis - Monomers formed 'patches' of MreB that navigate around circumference of bacteria - Move around in a helical structure in a circular pattern

Answer 24

Sites of 'high' cell curvature Promote cell wall synthesis at these sites to drive shape homeostasis and 'smooth out' the cell wall

Answer 25

A22 Causes a loss of mreB localisation as it causes it to become diffuse - Causes cell swelling and osmotic lysis; Cell wall damage

Answer 26

mreB is an actin like protein Potential drug would need to inhibit mreB but not actin Actin is even more important than tubulin; Harder to develop a drug targeting mreB selectively

Answer 27

Phylogenetic analysis Actinomycetes family of bacteria (known to make many antimicrobial compounds) ≈ Reverse genetics

Answer 28

They ignored any actinomycete genome which already created vancomycin; These already have an effective antimicrobial compound Other organisms that don’t produce vancomycin must be producing other compounds; Clearly still competitive as they still exist in nature Following this assumption, they discovered 2 completely novel antibiotics - Both seem to target cell wall turnover enzymes (specifically hydrolases; Produces characteristic phenotype of twists)

Answer 29

Genetic redundancy

Answer 30

No redundancy - Every gene in the chain is essential; Can knock out gene to see that it is essential Genetic redundancy occurs when a function of the cell is carried out by 2 (or more) parallel pathways; Especially important processes Deliberately block one pathway (e.g. with deletion) Now restored having 1 pathway with essential genes

Answer 31

Combination of genetic mutations which are lethal to cells Discovery of PBP regulatory factors

Answer 32

Contain both biochemical activities required to synthesise PG from the lipid II precursor; Transpeptidase (TP) and transglycosylase (TG)

Answer 33

Knock out PBP1B to collapse genetic redundancy and make PBP1A essential - Any upstream or downstream processes/genes required for this enzyme (PBP1A) to function are now also essential

Answer 34

Used unstable plasmids paired with transposon mutagenesis Need a ∆lacZYA E. coli strain; LacZYA is responsible for blue-white screening - LacZ+ cells grown in presence of X-gal will turn blue - In absence they will be white Need unstable lacZYA+ plasmid with defective origin of replication, as well as ampicillin resistance cassette for selection; As cells grow and divide, likely the daughter cells will not have this plasmid

Answer 35

White colony Blue colonies Sectored colony (gradient from blue to white) - Population initially has plasmid but as they divide they lose the plasmid and turn white This process is paired with transposon mutagenesis to disrupt genes at random

Answer 36

∆pbp1b ∆lacZYA E. coli deletion strain Unstable lacZYA+ pbp1b+ plasmid; Possible for pbp1b to be lost over time as cells lose the plasmid If we carry out transposon mutagenesis on this strain, we will get disruption of random genes in the genome; Whole population of these bacteria, each with a random gene deleted

Answer 37

No selection for plasmid as transposon has jumped into into gene that is unrelated - White colonies Transposon inserts into a gene that is synthetically lethal with pbp1b; Plasmid is now essential as the cell needs pbp1b - Blue colonies Take solid blue colonies and work out where the transposon inserted - 2 colonies were selected; Both had transposon insertions in a gene called LpoA

Answer 38

Inducible promoters Knockout both LpoA and PBP1B, put LpoA back in on an inducible promoter - Expression with IPTG - Death without IPTG Spot dilution plates of mutant strains with both permissive and non-permissive conditions verify this synthetically lethality with PBP1B

Answer 39

Outer membrane Activate either PBP1a or PBP1B by reaching through a potential hole in the PG layer - PBP1A/B then fills in the hole to achieve homeostasis This is like a PG damage repair system

Answer 40

Regulate PBP1A/B enzymes Much easier to access than MreB and FtsZ; Only have to pass through 1 membrane rather than 2

Answer 41

E. coli is gram-negative; Outer membrane S. pneumoniae is gram-positive; No outer membrane for activators

Answer 42

Cells must survive to be available to study Synthetic lethal screen require a sophisticated set of genetic tools Can now detect ‘absence’ of cell signatures; Cells don’t need to survive anymore - Sequence a sample so much we can be reasonably sure that if we don’t detect something, it isn’t there Now only require transposon mutagenesis tools, rather than sophisticated sets of tools; Can make transposon mutagenesis work in any bacterial species

Answer 43

Insertions which are synthetically lethal - Insertions in pbp2a of a ∆pbp1a S. pneumoniae are non-permissive and the cell dies Observe a gap in the synthetically lethal gene (proves it if we're sure of methods)

Answer 44

Compare mapped library of insertions between WT and ∆pbp1a Looking for genes that can take insertions in the WT (but not ∆pbp1a) but die when there is an absence of pbp1a

Answer 45

CozE – Regulates PBP1A MacP – Regulates PBP2A

Answer 46

CozE has the opposite genetic relationship than expected - Essential in the WT - Essentiality disappears if you knockout pbp1a; You can get insertions in CozE PBP1A must be toxic - Toxicity only released in WT, but can’t be released in mutant strain as it doesn’t have that enzyme

Answer 47

In permissive conditions, cells swell and produce light; Characteristic of cell wall damage and lysis

Answer 48

Keeps PBP1A in a regulated state at cell division site Without CozE, these enzymes can escape - They then continually make cell wall being produced all over the cell, which is lethal Works as drug in lab - However, combined with penicillin, these 2 would cancel each other out Need to prove CozE drug inhibitors kill just as effectively as penicillin which is a high bar to meet

Answer 49

PBP1A Needed for PBP2A activation

Answer 50

Knockout either one, and put back the other with an inducible promoter (zinc) Both should have same pattern of cell death or survival between permissive and non-permissive conditions

Answer 51

Cell wither, shrink and lyse Easier to drug, but harder to use as its not deadly or effective enough when compared with deregulating CozE Slower than deregulating cozE; Need a drug that kills bacteria very quickly, otherwise they can respond rapidly and effectively

S. pneumoniae and the Cell Wall Flashcards

(75 cards)