Strep. pneumoniae

Question 1

What are the characteristics of strep. pneumo?

Answer 1

Pathogen: Streptococcus pneumoniae
Classification: Gram-positive bacteria
Disease: Pneumonia, meningitis, sepsis, otitis media
Symptoms: Shaking chill, fever, cough Discomfort, heavy breathing Symptoms can be very subtle! Onset of severe illness is abrupt.
Virulence: Causes at least 1.2 million deaths worldwide every year. Mainly in patients >65 or <5 years or immunocompromised. Underreported figure.
Reservoir/Transmission: ‘Direct contact’ with respiratory secretions containing the organism through coughing and sneezing etc.
Treatment/Prevention strategies: Antibiotics such as penicillin or related compounds. Vaccination against capsule types.
Major virulence factors: Polysaccharide capsule, pneumolysin and many others.

Question 2

What are the niches for Strep. pneumo?

Answer 2

Nasopharynx = Mixture of 60% squamous (‘flat’) epithelial cells and 40% ciliated columnar cells. Lymphocytes buried in the submucosa along with seromucous glands that produce mucus. Mucus - ≈(complex mixture of glycoproteins) Glycoproteins on the surface of epithelial cells.
Brain = Causes meningitis
Blood = Causes sepsis
Lungs = Causes pneumonia
Middle ear= Causes otitis media

Question 3

What are the primary treatments for Strep. pneumo?

Answer 3

Penicillin-related drugs such as ampicillin, cephalosporins and other beta-lactams.

Question 4

What challenges do cell walls face?

Answer 4

Building the cell wall from the inside out, maintaining its shape without compromising its integrity all while completing it at the maximum possible rate.

Question 5

What is the role of transglycosylase in peptidoglycan biosynthesis?

Answer 5

Polymerises glycan strands

Question 6

What is the role of transpeptidases in peptidoglycan biosynthetic pathway?

Answer 6

Cross-linked the peptidoglycan.

Question 7

What is penicillin?

Answer 7

Penicillin is a chemical mimic of the D-ala, D-ala leaving group and blocks transpeptidation through irreversible competitive inhibition. This inhibits the use of transpeptidases.

Question 8

What did penicillin-binding proteins do?

Answer 8

Allowed enzymes which build/remodel the cell to be identified biochemically as they are all covalently modified through penicillin binding.

Question 9

What did Van der Poll and Opal, 2009 say?

Answer 9

“Versatility of the genome of pneumococci and the bacteria’s polygenic virulence capabilities show that a multifaceted approach with many vaccine antigens, antibiotic combinations, and immunoadjuvant therapies will be needed to control this microbe.”

Question 10

What dedicated machinery is present for each growth mode?

Answer 10

MreB a protein holding elongation complex in place and FtsZ coordinates the division complex in E.coli. In S.pneumoniae there is no MreB but there is an FtsZ.

Question 11

What is forward genetics?

Answer 11

Mutagenesis -> Phenotype -> Genotype. Random mutagenesis can come from chemical agents, UV light, transposons or CRISPR.

Question 12

What is the problem with studying growth processes in bacteria?

Answer 12

The fundamental problem with studying essential growth processes in bacteria is that cells must survive to be available for study. Conditional phenotypes are needed to study essential processes. Permissive conditions mean cells live, and Non-permissive conditions mean cells die. Can be done by adding Mg2+ to the media, changing the temp or +/- antibiotics. Forward genetics was used in FtsZ discovery.

Question 13

Who used forward genetics in Strep. pneumo?

Answer 13

In the 1970/80s Jo Lukenhaus used a forward genetic approach to search for genes involved in cell division (E.coli). Filament Temperature Sensitive “FTS.”
Dr Lukenhaus knew he had strains containing mutations in essential division processes, but he did not know which genes were responsible for the phenotype. The ‘fts’ genes were identified using phage complementation

Question 14

How did an ordered assembly model of cell division emerge?

Answer 14

Using a combination of GFP-tagged ‘Fts proteins’ and the ‘fts’ temperature sensitive strains an ordered-assembly model of cell division emerged:
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 15

What has FtsZ done as a therpeutic target?

Answer 15

Targeting FtsZ has led to the development of many anti-microbial compounds. Issue is FtsZ is a tubulin homologue and found in eukaryotic cells. Needed a compound that bound FtsZ but not tubulin. Became more profitable to make it toxic and target tubulin, marketing it as cancer chemotherapy. FstZ inhibitors can work by enhancing penicillin activity.

Question 16

What is reverse genetics?

Answer 16

Genotype -> Phenotype <-> Mutagenesis. This is more targeted site-directed mutagenesis through PCR modification of specific codons. The best example is in the characterisation of MreB.

Question 17

What is Mre?

Answer 17

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. Mre = Murein (Cell wall) formation Gene cluster E - mreA had been re-classified leaving: mreB, mreC and mreD. All essential genes in B. subtilis.

Question 18

What did tagged proteins show?

Answer 18

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

Question 19

What do current models of MreB show?

Answer 19

Current models suggest, MreB patches move to sites of ‘high’ cell curvature, promoting cell wall synthesis at these sites to drive shape homeostasis. Given its essential function, MreB was pursued as a drug target.

Question 20

Why is it hard to make drugs for MreB?

Answer 20

MreB is an actin homologue so need to inhibit MreB and not actin.

Question 21

Along with forward and reverse genetics, what’s another analysis?

Answer 21

Another approach use is to use phylogenetic analysis. The components highlighted by this approach seem to target cell wall turnover enzymes.

Question 22

What did Typas et al, 2011 say?

Answer 22

Growth of the sacculus is sensitive to mechanical force and nutritional status, and describe the roles of peptidoglycan hydrolases in generating cell shape and of D-amino acids in sacculus remodelling

Question 23

What is genetic redundancy?

Answer 23

The fundamental problem with forward / reverse genetic approaches is genetic redundancy. Genetic redundancy occurs when a function of the cell is carried out by two (or more) parallel pathways. Synthetic lethality is the combination of genetic mutations which are lethal to cells -> rare interactions but very powerful.

Question 24

What is a good example of using synthetic lethal genetics?

Answer 24

The discovery of PBP regulatory factors such as Class A PBP regulators in E.coil. In E.coli PBP1A (elongation) and PBP1B (division) form a synthetic lethal pair. Class A PBPs contain both biochemical activities (transglycosylase + transpeptidase) required to synthesis peptidoglycan from the lipid II precursor.

Question 25

What does removing PBP1B from E.coli do?

Answer 25

Collapses the Genetic Redundancy and allows for a Forward Genetic Approach. Removing PBP1B makes PBP1A and its regulators/activators conditionally essential.

Question 26

What is synthetic lethal screen?

Answer 26

One approach to identifying synthetic lethal pairs in E. coli used unstable plasmids paired with transposon mutagenesis. PBP1A is conditionally essential.

Question 27

What are the four parts of conditional essentiality?

Answer 27

1. ɅLacZYA strain
2. Unstable plasmid (lacZYA+)
3. Transposon mutagenesis.
4. The synthetic lethality

Question 28

What is the model of conditional essentiality?

Answer 28

ɅLacZYA strain allows for blue-white selection. These genes are taken and put into an unstable plasmid. Removing ampicillin removes the selection for the plasmid and so eventually the plasmid gets lost – can also give sectored colonies. Transposons are short pieces of DNA that jump into DNA at random – causing gene inactivation by physical disruption.
In the experiment, lacZYA and deleted PBP1B is added to the plasmid meaning PBP1B can be selected for in the plasmid. The cells are then plated. If transposon jumps into a gene not synthetically lethal, the plate is white. If the gene is synthetic lethal, the plasmid is conditionally essential and so shows a blue colony. LpoA interacts with PBP1A whilst LpoB interacts with PBP1B. As they are outer membrane proteins, they can become drug targets.

Question 29

Why can’t Strep. pneumo follow the same conditional essentiality model as in E.coli?

Answer 29

Gram-positive do not have an outer membrane

Question 30

What has changed forward genetics for the better?

Answer 30

The use of “next-generation” sequencing has completely changed forward genetic approaches and allowed hits to be found in S.pneumoniae for PBP activators. Genetic sequencers can be Tn-seq, Hits, TraDIS or In-seq etc.
To get around the fundamental problems of requiring cell survival and synthetic lethal screens requiring a sophisticated set of genetic tools, we can now detect the “absence” of cell signatures and only require transposon mutagenesis.
Next-gen sequencing allows us to capture (in one dataset) millions of independent transposon insertion events.

Question 31

What can be targeted for therapeutic gain?

Answer 31

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

Question 32

What mediates cell wall synthesis?

Answer 32

cozE = Coordinator Of Zonal Elongation. CozE coordinates cell wall synthesis by PBP1a. Lack of CosE causes delocalised cell wall synthesis and morphology changes including lysis.
MacP = Membrane-Associated Cofactor of PBP2a. CozE and MacP regulate Class A PBP enzymes in pneumo. Cells wither and die if PBP2A activity is reduced. Can’t target MacP for therapeutic gain as it doesn’t kill fast enough and so bacteria could acquire a mutation to escape this phenotype.

Question 33

What did Paradis-Bleau et al, 2010 say?

Answer 33

Because they are the targets of penicillin and related antibiotics, the structure and biochemical functions of the PBPs have been extensively studied