The S. pneumoniae pathogen and the bacterial cell wall Flashcards
Classification
Gram positive, rugby balled shape (essential for its growth as its virulence factors are in the middle)
Diseases it causes
Pneumonia, Meningitis, Sepsis, Otitis media
Symptoms
Pneumonia only: Shaking chill, fever, cough, discomfort, heavy breathing.
Symptoms can be very very subtle onset of severe illness is abrupt
Virulence
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
Major virulence factors
Polysaccharide capsule
Pneuomolysin
+many others
Reservoir/ Transmission
“Direct contact with respiratory secretions containing the organism
Coughing/ sneezing onto surfaces
How many deaths does it cause in children under 5
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”
Where does Pneumo grow and what does it eat
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
How does S. pneumoniae cause invasive diseases
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
Why do pneumo become invasive?
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
When would pneumo become invasive
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)
S. pneumoniae - vaccination
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
2006 vaccine
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
2010 vaccine
13-valent, added the different serotypes that had been brought in (PCV7 + 1, 3, 5, 6A, 7F and 19A)
Same thing happened again
2014 vaccine
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
S. pneumoniae - treatment
The primary treatment approaches for S. pneumoniae are penicillin related drugs such as ampicillin, cephalosporin(s) and other β-lactams- cell wall targeting antibiotics
What makes the cell wall such a good antibiotic target?
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
The bacterial cell wall faces the following challenges:
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
What is peptidoglycan composed of
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
The peptidoglycan biosynthetic pathway
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
Penicillin
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
How does penicillin work on the peptidoglycan biosynthetic pathway
- Penicillin works by specifically inactivating enzymatic activity of the bacterial cell (transpeptidase activity)
- It chemically mimics the leaving group, penicillin binds to all its enzymes
- Transpeptidase activity recognises it as substrate and will try to do transpeptidation but will get stuck half way through
- You end with penicillin covalently linked to the transpeptidases and the enzyme is completely useless
- Peptidoglycan synthesis still continues and it will incorporate broken peptidoglycan into the structure
What are the Penicillin Binding Proteins (PBPs)
The enzymes which build/ remodel the cell wall were identified biochemically as they are all covalently modified through penicillin binding
How many binding proteins
6 and we know them from clinical experiments as they are good drug targets
How do bacterial cells divide
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
Whats the elongation and division complex normally and whats missing in pneumo
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
Whats forward genetics
Trying to find a gene thats responsible for a phenotype before you know what gene it is
- Start with mutagenesis, force this in a lab by adding a chemical agent, UV light etc
- 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
Discovery of ftsZ
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
Forward genetic approach for ftsZ
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
How were the fts genes identifed
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
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?
- FtsZ (tubulin homologue) polymerises into a dynamic structure at the new division site. It does this in combination with other protein factors. (Z-ring)
- This structure recruits all other cell division proteins to the division site to drive cell division/PG synthesis.
Forward Genetic Approach – FtsZ as a drug target?
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
Reverse Genetic Approach
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
Reverse Genetic Approach – MreB function
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
What was the reverse genetics approach for working out mreB function
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.
- the cells stop dividing properly and become spherical- short term
- 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
Reverse Genetic Approach – Drug Target?
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
Reverse Genetic Approach – MreB as a Drug Target?
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
Whats the fundamental problem with forward/ reverse genetics
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
What happens if you block one pathway and what is it known as
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
What is a good example of using synthetic lethal genetics
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
What forms a synthetic lethal pair in peptidoglycan
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.
What happens when you remove one of the synthetic lethal pair
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
Unstable plasmids paired with transposon mutagenesis to identify synthetic lethal pairs in E. coli- what does it entail:
There are 4 moving parts :
- LacZYA phenotypes
- Unstable plasmids (give sectored colonies)
- Transposon mutagenesis
- The synthetic lethality
Synthetic lethal hits must be followed up and verified to confirm the genetic relationship
Synthetic lethal screen- the strain background
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
Synthetic lethal screens- screening for PBP regulatory factors
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
How are synthetic lethal hits followed up
We’ve identified a synthetic lethal combination, PBP1B and LpoA
- Strip out all the rest of that machinery and check these two genes are synthetically lethal with eachother
- 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
What are LpoA and LpoB
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
How are LpoA and LpoB controlled
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
What factors regulate Class A PBP enzymes in s. pneumoniae
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)
Synthetic lethal screen in S. pneumoniae
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
Building transposon libraries
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
Synthetic lethal hit with PBP1A and PBP2A
- 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
What genes regulate Class A PBP enzymes in s. pneumoniae
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
What does CozE do
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
What does MacP do in S. pneumoniae
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
Synthetic lethal hit with Δpbp1A – macP
There are no insertions in this gene at all
Synthetic lethal hits must be followed up and verified to confirm the genetic relationship
What happens if there’s no CozE or MacP
No macP - the cells shrink until they can’t anymore and burst - doesn’t kill the cells fast enough to be used for therapeutics
Final summary
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
Forward Genetic Approach – Cell division genes: summary
Cell division proteins identified using Forward-Genetic approaches
Most of the components of the division complex were identified relatively early (1980s- 1990s)
Reverse Genetic Approach – Cell Elongation : summary
Cell elongation proteins characterised using Reverse Genetic approaches
Class A PBP regulators in E. coli: Summary
Class A PBP regulators were identified in first E. coli using a sophisticated synthetic lethal screening approach
Class A PBPs in S. pneumoniae: summary
Class A PBP regulators were identified in S. pneumoniae using next-generation sequencing approach (Tn-Seq)
S. pneumoniae treatment: summary
We know that in Pneumo, penicillin and vancomycin are useful antimicrobial compounds because they target these processes
