The S. pneumoniae pathogen and the bacterial cell wall

Question 1

Classification

Answer 1

Gram positive, rugby balled shape (essential for its growth as its virulence factors are in the middle)

Question 2

Diseases it causes

Answer 2

Pneumonia, Meningitis, Sepsis, Otitis media

Question 3

Symptoms

Answer 3

Pneumonia only: Shaking chill, fever, cough, discomfort, heavy breathing.

Symptoms can be very very subtle onset of severe illness is abrupt

Question 4

Virulence

Answer 4

Causes at least 1.2 million deaths worldwide every year. Mainly in patients >65, <5 years olds or immunocompromised but it’s an unreported figure

Question 5

Major virulence factors

Answer 5

Polysaccharide capsule
Pneuomolysin
+many others

Question 6

Reservoir/ Transmission

Answer 6

“Direct contact with respiratory secretions containing the organism

Coughing/ sneezing onto surfaces

Question 7

How many deaths does it cause in children under 5

Answer 7

causes 16% of all deaths of children under 5 years old

The most common cause of infection - related deaths in the UK and the USA “old mans friend”

Question 8

Where does Pneumo grow and what does it eat

Answer 8

Grows within the Nasopharynx (at the back of nose/ top of throat)

At the site, there’s a mixture of squamus epithelial cells and ciliated columnar cells which generate and move mucous. There’s a set of seromucous glands making mucus which is important because it tells us what pneumo eats (carbon and nitrogen sources)

It can eat the mucous directly as its being produced or it trims off the sugars that are on the membranes on the cells within the nasopharynx.

This explains why pneumos genome is full of sugar metabolite enymes and sugar transporters

Question 9

How does S. pneumoniae cause invasive diseases

Answer 9

Brain meningitis: there’s a hole in the bottom of the skull/ no hard cavity so nothing to stop bacteria getting through. Lymphocytes patrol the nasopharynx, they phagocytose the pneumo and deposit pneumo deeper into the tissue

Middle ear infections: If there’s a big change in pressure, you get an opening to the outside. The tubes open into the grove and pneumo can get access

Blood/ sepsis: nasopharynx is covered in blood vessels so you get direct access to the blood there. In sepsis, you get localed pneumonia infection because the lungs are full of vasulature

Question 10

Why do pneumo become invasive?

Answer 10

Resevoir: 10% adults and up to 60% of infants are colonised at one time. Carriage only lasts weeks or months

S. pneumoniae does not have a stable niche

They move aggressively between humans in order to stay alive because humans have adapted good immune systems

Pneumo falls between a parasite and a commensal

Question 11

When would pneumo become invasive

Answer 11

If the person is coinfected with another bacteria or if the immune system is in a suppressed state e.g. immunocompromised:

• viral infections (cold/ flu), HIV patients, age (old/ young)

Question 12

S. pneumoniae - vaccination

Answer 12

The S. pneumoniae vaccine primes the immune
response to clear carriage, removing the risk of
invasive disease

Block Colonisation = Prevent Invasive Infection

Vaccine target= capsule. 92 capsule types

Question 13

2006 vaccine

Answer 13

A vaccine was employed that covered 7 of the serotypes (7 valent- 4, 6B, 9V, 14, 18C, 19F and 23F)

It was effective for a short period of time until it displaced carriage of those particular serotypes and lowered invasive diseases of those particular serotypes

Vaccine reduced invasive disease in targeted strains, but left the niche ‘open’ for other strains. It took 2-3 years for invasive disease to return to pre-vaccine levels and started a runaway chain reaction

Question 14

2010 vaccine

Answer 14

13-valent, added the different serotypes that had been brought in (PCV7 + 1, 3, 5, 6A, 7F and 19A)

Same thing happened again

Question 15

2014 vaccine

Answer 15

23 valent (PCV13 + 2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20, 22F, 33F) - 6a

Every time we try and displace new serotypes, the amount of immunogenicity in patients after you’ve given them this vaccine decreases

Question 16

S. pneumoniae - treatment

Answer 16

The primary treatment approaches for S. pneumoniae are penicillin related drugs such as ampicillin, cephalosporin(s) and other β-lactams- cell wall targeting antibiotics

Question 17

What makes the cell wall such a good antibiotic target?

Answer 17

Bacterial cells are preprimed to catastrophically explode when under high internal osmotic pressure

If the tensile forces (which resist outwards forces) are compromised, water rushes into the bacterial cell and pushes the membrane outwards. The cell starts to balloon which kills the bacteria and cures the patients

Question 18

The bacterial cell wall faces the following challenges:

Answer 18

to build their cell wall from the inside out, maintaining its shape without compromising its integrity while all at the maximum possible rate so they’re not outcompeted by other bacteria

Question 19

What is peptidoglycan composed of

Answer 19

Repeating disaccharide units cross linked together by short peptides

This material is resisting the internal osmotic pressure. It consists of long glycan strands which are cross linked together

Question 20

The peptidoglycan biosynthetic pathway

Answer 20

Peptidoglycan is made from a lipid linked precursor - lipid 2

Lipid 2 has two important features: a pentapeptide stem and a terminal d-alanine (only two things that are really important)

The precursor is turned into peptidoglycan via two ways:

Using enzymatic activity known as transglycosylase activity which polymerises it into glycan strands. This needs a second precursor known as transpeptidase which crosslinks those strands together in order to make new peptidoglycan

Terminal D-alanine is important because that is used as a leaving group for this reaction. You get the transfer of one amide bond from one peptide to another forming the cross link

Question 21

Penicillin

Answer 21

Antibiotics can be used to inhibit or kill other microbes growing in patients

Sir Howard Florey, Dr Ernst, Boris chain are responsible for discovery and application. It saved millions of lives

Question 22

How does penicillin work on the peptidoglycan biosynthetic pathway

Answer 22

1. Penicillin works by specifically inactivating enzymatic activity of the bacterial cell (transpeptidase activity)
2. It chemically mimics the leaving group, penicillin binds to all its enzymes
3. Transpeptidase activity recognises it as substrate and will try to do transpeptidation but will get stuck half way through
4. You end with penicillin covalently linked to the transpeptidases and the enzyme is completely useless
5. Peptidoglycan synthesis still continues and it will incorporate broken peptidoglycan into the structure

Question 23

What are the Penicillin Binding Proteins (PBPs)

Answer 23

The enzymes which build/ remodel the cell wall were identified biochemically as they are all covalently modified through penicillin binding

Question 25

How many binding proteins

Answer 25

6 and we know them from clinical experiments as they are good drug targets

Question 26

How do bacterial cells divide

Answer 26

Bacterial cells grow and divide through repeating cycles of cell elongation and division

They elongate untul they become twice their cell volume then switch into division mode and divide in the centre, creating two daughter cells

How much freedom you give your elongation complex is what dictates the size and shape of your cell

Question 27

Whats the elongation and division complex normally and whats missing in pneumo

Answer 27

Elongation: mreB

ftsZ: cell division complex

Pneumo is missing mreB in this complex and it operates on a different mechanism but mostly have remained the same

Missing factors: EzrA, SepF, PcsB, StkP, PhpP

Question 28

Whats forward genetics

Answer 28

Trying to find a gene thats responsible for a phenotype before you know what gene it is

1. Start with mutagenesis, force this in a lab by adding a chemical agent, UV light etc
2. this will generate a particular phenotype then you identify what genes are responsible for that phenotype

Problem: cells must survive to be available to study and the cells usually die

Question 29

Discovery of ftsZ

Answer 29

Uses conditional phenotypes (needed to study essential processes)
Cells were grown at low temperatures, adding magnesium to the medium can reduce defects because magensium stabilises the cell membranes

Question 30

Forward genetic approach for ftsZ

Answer 30

Lukenhaus used forward genetic appraoch to search for genes involved in cell division - did this in E.coli adding mutagenic agent called nitrosoguanidine

He screened those random mutants for phenotypes: he looked for cells which grew and divided well at 30 degrees (permissive conditions) but when the temperature shifted to 42 degrees, they lost the ability to divide so would continue to elongate until they became so large, would rupture

At the time these were called filament sensitive mutants or fts sensitive mutants

Question 31

How were the fts genes identifed

Answer 31

The ‘fts’ genes were identified using phage complementation

Take random sections of the E. coli chromosome, package that into a phage. If the phage contains the genes which are defective in your strain, then adding the phage in will cure it of that defect and it will cure that temperature sensitive phenotype and strain will be able to grow and divide

Through this approach an essential set of genes were identified

Question 32

Using a combination of GFP-tagged ‘Fts proteins’ and the ‘fts’ temperature sensitive strains an ordered-assembly model of cell
division emerged.

What was this?

Answer 32

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

Question 33

Forward Genetic Approach – FtsZ as a drug target?

Answer 33

2011/2012 - Divinocell tried to generate small molecules that could specifically inhibit ftsZ

ftsZ which is found in tubulin - responsible for moving around chromatids in eukaryotic cell division - generate a compound accidentally reacts with ftsZ which isn’t this magic bullet- that’s okay- more successful in generating drugs that could kill you but could also be used to treat cancer

Question 34

Reverse Genetic Approach

Answer 34

Reverse is the reverse of forward

You start with the genotype and try to identify what’s going on with the phenotype, and then you try and identify what’s going on with the phenotype

Usually you do some sort of mutagensis to try and probe the function- unclear what the relationship exactly is.

Vast majority of genetics that takes place in the lab is reverse genetics - mutagenesis is much more targeted

Mutagenesis is usually done through PCR or you can do specific modifications of certain codons- changing the surface of that protein is going to have a certain effect on that phenotype

Question 35

Reverse Genetic Approach – MreB function

Answer 35

Previous Forward Genetic studies had identified a ‘mre’
cluster of genes, which were linked to cell growth,
however no follow up studies had been carried out. So while it was understood that mreB had some role in cell wall biosynthesis it wasn’t exactly sure what

Mre = Murein(Cell wall) formation Gene cluster E (bad name)
mreA had been re-classified leaving: mreB, mreC and mreD

Used powerful conditional expression system in B. subtilis - had a strain that lacked mreB but was placed back under the control of the xylose inducible promoter- this in the expression of xylose mimics wildtype

Question 36

What was the reverse genetics approach for working out mreB function

Answer 36

In the permissive condition when you have xylose, the mreB is on and therefore your cells grow and divide fine but in the non permissive conditions when you lack the xylose that the mreB is slowly being removed from these cells certain things happen.

1. the cells stop dividing properly and become spherical- short term
2. Long term, there’s so much damage to the peptidoglycan, the collapse cells and lyse

Using tagged-proteins to study MreB localisation, a model of multiple ‘small’ protein-complexes driving diffuse cell wall synthesis/ elongation emerged

MreB monomers- actin like protein can polymerise like actin can

Current models suggest, MreB patches move to sites of ‘high’ cell curvature, promoting cell wall synthesis at these sites to drive shape homeostasis over time

Question 37

Reverse Genetic Approach – Drug Target?

Answer 37

Given its essential function, MreB was pursued as a drug target- connected to the pbp enzymes which are good drug targets

A22 - used to treat e.coli cells

If you treat that cell with A22 a couple of things happen- patches completely disapear, the cells lose the ability to elongate and they start to round up

Eventually the cells are on a one way ticket to cell lysis

Question 38

Reverse Genetic Approach – MreB as a Drug Target?

Answer 38

MreB is an actin like homolog and as a result, you want to create a compound that’s capable of inhibiting mreB but not of actin.

Actin inhibitors kill humans - can’t be used for chemotherapies- higher bar to get over than it was with the ftsZ

Question 39

Whats the fundamental problem with forward/ reverse genetics

Answer 39

Genetic redundancy which occurs when a function of the cell is carried out by two or more parallel pathways

There has to be a way of collapsing that redundancy so that other genetic pathways become conditionally essential and therefore carry out forward and reverse genetics in that context

Question 40

What happens if you block one pathway and what is it known as

Answer 40

Everything will become essential, known as synthetic lethality (the combination of genetic mutations which are lethal to cells)

Synthetic lethality: the relationship between one genetic locus on one pathway and the other genetic locus on the other complementary pathway

Question 41

What is a good example of using synthetic lethal genetics

Answer 41

The discovery of PBP regulatory factors (want to study these regulatory factors because we target these in order to kill bacteria and generate new therapies

Question 42

What forms a synthetic lethal pair in peptidoglycan

Answer 42

PBP1A and PBP1B

They’re both class A PBP enzymes, containing both biochemical activities required to make the cell wall from a lipid 2 linked precursor molecule

They contain the transglycosylase domain to polymerise the lipids into glycan strands

This cross links them together.

Question 43

What happens when you remove one of the synthetic lethal pair

Answer 43

The other becomes conditionally essential and the cell would absolutely need the activity of this enzyme in order to grow and divide

If we remove PBP1B from E. coli cells, collapses the genetic redundancy and allows for a forward genetic approach, therefore making PBP1A conditionally essential. If PBP1A has any activators or regulators that are required for its function, these would also now be conditionally essential

Question 44

Unstable plasmids paired with transposon mutagenesis to identify synthetic lethal pairs in E. coli- what does it entail:

Answer 44

There are 4 moving parts :

1. LacZYA phenotypes
2. Unstable plasmids (give sectored colonies)
3. Transposon mutagenesis
4. The synthetic lethality

Synthetic lethal hits must be followed up and verified to confirm the genetic relationship

Question 45

Synthetic lethal screen- the strain background

Answer 45

One approach to identifying synthetic lethal pairs in E. coli used unstable plasmids paired with transposon mutagenesis

You need an E. coli strain that lacks lacZYA

Take those genes and put them onto an unstable plasmid, if you don’t select for them (remove ampicillin), they will gradually be lost so over time, the plasmid will be lost

If you give this strain a plasmid and grow it with ampicllin and X-gal, you’ll get a blue colony

Question 46

Synthetic lethal screens- screening for PBP regulatory factors

Answer 46

One approach to identifying PBP regulatory factors is to use a synthetic lethal screen

If the transposont has jumped into a gene that is synthetically lethal and loses its plasmid, it will die because it needs functional PBP1A and PBP1B enzymes in order to grow

Because it can’t lose this plasmid, it’ll be a small colony because if by chance one of the daughters lose this plasmid, it dies due to the synthetical lethality - meaning the progeny are constantly being lost from the population

Question 47

How are synthetic lethal hits followed up

Answer 47

We’ve identified a synthetic lethal combination, PBP1B and LpoA

1. Strip out all the rest of that machinery and check these two genes are synthetically lethal with eachother
2. Knock out these genes, put one of them under an inducible promoter, this strain in the presence of IPTG grows and in the absence it doesn’t if the synthetic lethal relationship is true

Question 48

What are LpoA and LpoB

Answer 48

They’re found in the outer membrane that interacts with a domain on the back of PBP1A and causes it to stimulate its enzyme activity and therefore its function

When the cell is growing, and a hole appears in the peptidoglycan by either random chance or just growth, these proteins can fall through the peptidoglycan, stimulate these enzymes which can then make peptidoglycan de novo and fill in the gap

Question 49

How are LpoA and LpoB controlled

Answer 49

They were thought to be controlled from the cytoplasmic side of the membrane up (part of multi domain complexes, all controlled from MIB up)

These are actually controlled from the top down. Outer membrane proteins are much more accessible to drugs, they don’t have to pass through all of these different layers of the bacterial cell

Question 50

What factors regulate Class A PBP enzymes in s. pneumoniae

Answer 50

They looked for factors that looked for enzymes that regulated PBP enzymes in pneumo knowing they couldn’t be the same as in E. coli

They knew that PBP1A and PBP2A formed a synthetic lethal pair (parallel genetic pathways pointing to the same genetic process)

Question 51

Synthetic lethal screen in S. pneumoniae

Answer 51

The use of next generation sequencing has completely changed forward genetic approaches

Synthetic lethal screens require a sophisticated set of genetic tools (only require transposon mutagenesis)

Have a known synthetic lethal combination - plotted them all onto the same genome. Then took a massive population of transposon insertions and mapped them all to genes so had lots of different insertion sites

Question 52

Building transposon libraries

Answer 52

Built a transposon library in a wild type as a control and a PBP1A strain as a test, grew them out and did NGS to find all the locations of transposon insertions

Question 53

Synthetic lethal hit with PBP1A and PBP2A

Answer 53

• Want to have a look at the known synthetic lethal hit (PBP1A)
• Lots of insertions in one condition and no insertions in another, telling us this gene is conditionally essential

Question 54

What genes regulate Class A PBP enzymes in s. pneumoniae

Answer 54

CozE and MacP

• Identified two new regulators of Class A PBP enzymes called CozE and MacP
• CozE teaches us that inappropriate cell wall activity can be lethal in cells

Question 55

What does CozE do

Answer 55

CozE coordinates cell wall synthesis by PBP1A

If you express coze and PBP1A over time, the cells start to swell because there’s inappropriate peptidoglycan being generated and eventually the cells burst

This opens up a new way of PBP peptidoglycan inhibition

If your cells lack CozE, the PBP1A enzyme is not localised, making the cell wall over the place and this delocalises cell wall synthesis

Inappropriate cell wall synthetic activity can be lethal to the bacterial cell

Question 56

What does MacP do in S. pneumoniae

Answer 56

It regulates class a PBP enzymes in S. pneumoniae

MacP is a regulator of PBP2A- this functions similarly to LpoA and LpoB. insertions in it in the wildtype

Question 57

Synthetic lethal hit with Δpbp1A – macP

Answer 57

There are no insertions in this gene at all

Synthetic lethal hits must be followed up and verified to confirm the genetic relationship

Question 58

What happens if there’s no CozE or MacP

Answer 58

No macP - the cells shrink until they can’t anymore and burst - doesn’t kill the cells fast enough to be used for therapeutics

Question 59

Final summary

Answer 59

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

How bacterial cells grow and divide: Most bacteria have two broad growth modes: elongation and division

PBPs were discovered before molecular genetics through the covalent modification by penicillin

Question 60

Forward Genetic Approach – Cell division genes: summary

Answer 60

Cell division proteins identified using Forward-Genetic approaches

Most of the components of the division complex were identified relatively early (1980s- 1990s)

Question 61

Reverse Genetic Approach – Cell Elongation : summary

Answer 61

Cell elongation proteins characterised using Reverse Genetic approaches

Question 62

Class A PBP regulators in E. coli: Summary

Answer 62

Class A PBP regulators were identified in first E. coli using a sophisticated synthetic lethal screening approach

Question 63

Class A PBPs in S. pneumoniae: summary

Answer 63

Class A PBP regulators were identified in S. pneumoniae using next-generation sequencing approach (Tn-Seq)

Question 64

S. pneumoniae treatment: summary

Answer 64

We know that in Pneumo, penicillin and vancomycin are useful antimicrobial compounds because they target these processes