Clinical Disease through to Scientific Understanding

Question 1

What are the 3 ways of handling data relating to an unusual microbial related phenotype/genotype?

Answer 1

Clinical/Epidemiological observation
Population genetics
Phenotypic study

Question 2

What are the 4 reasons we study unusual microbial phenotype/genotype?

Answer 2

Unusual phenotype teaches us something we do not yet know about the pathogen
Bacteria of the same species may not be the same genetically and/or phenotypically
The more we understand how something causes disease, the more able we are to tackle it
Important to public health, drug research etc.

Question 3

What are the 2 initial unusual microbial clinical observations?

Answer 3

Unusual disease progression or presentation
Outbreak situation

Question 4

What are the 2 initial unusual microbial diagnostic observations?

Answer 4

Bacterial colonies look different
Mixed phenotype

Question 5

What are the 2 initial unusual microbial epidemiological observations?

Answer 5

Increase in disease in a specific region or during a specific time
Increase in a certain or unusual bacterial genotype

Question 6

What are the 4 things we must consider when working back from initial clinical/diagnostic/epidemiological observation?

Answer 6

Phenotype
Protein and its translation
Protein function
Gene and its transcription

Question 7

In clinical phenotype from (3 - p.2), neutrophils are not migrating towards site of infection. Why may this be? (hint - IL-8)
What bacteria was causing infection?

Answer 7

Bacteria may be actively preventing neutrophil migration
IL-8 is a key factor for attracting neutrophils - Bacteria may be targeting this

Streptococcus pyogenes (Strep A)

Question 8

Explain concept of ELISA assay in context of IL-8
Look at (3 - p.2) and explain what ELISA graph shows

Answer 8

You have antibodies specific to what you want to measure the amount of (e.g. IL-8)
- You have a capture antibody that binds IL-8 to coat the bottom of the plate
- You introduce secondary antibody that binds IL-8 (sometimes a 3rd antibody too); Either one has a label that reacts with substrate to produce change in colour or fluorescence etc.

Graph shows that overall, isolates from the throat incubated with IL-8 return more IL-8 than those from the blood
- Isolates from the blood are doing something with the IL-8

Question 9

What does western blot of H292 and IL-8 show? (look at (3 - p.3))
What is used as control and why?

Answer 9

Smaller band in samples containing both H292 and IL-8 compared to those with just IL-8 which have a larger band
- In the presence of H292, there is less IL-8 present; H292 releases something to cleave IL-8 – Bands are not disappearing, but rather become smaller

H292 and no IL-8 samples is used as control; See if H292 just secretes something at 6 kDa

Question 10

Where does H292 cleave IL-8?
Why does this affect western blot results?
How did we discover this?

Answer 10

C-terminus α-helix is cleaved off

Antibody for western blot binds this helix; Explains why band is thinner

Reverse-phase high-performance liquid chromatography (HLPC) and mass spectrometry analysis

Question 11

What is the role of IL-8?
Activated neutrophils shed CD62L; CD62L levels positively correlate with mean fluorescent intensity (MFI) - Subtract MFI from unstimulated neutrophils = ΔMFI
What does decrease in ΔMFI represent?
Look at graph on (3 - p.4) and explain it

Answer 11

Activates neutrophils

Decrease in ΔMFI represents neutrophil activation

Incubation with IL-8 activates neutrophils
Incubation with bacterial supernatant does not activate neutrophils
Neutrophils with IL-8 and bacterial supernatant does not activate neutrophils
- Whatever is in the bacterial supernatant prevents activation

Question 12

What do we know about whatever is cleaving IL-8 (hint - supernatant and exponential)
Look at graph on (3 - p.4-5)
What is this ‘thing’ likely to be?

Answer 12

It’s present in the culture supernatant
Produced by bacteria during exponential growth

Likely to be an enzyme

Question 13

How can we determine what type of enzyme is cleaving IL-8?
Based on graph in (3 - p.5), what type of enzyme is it?
How can we determine which gene encodes this enzyme?; Which gene is it?

Answer 13

Use inhibitors of different types of enzymes and see with which one(s), IL-8 concentration is at the same level as without supernatant

Inhibited by Pefabloc, which is a serine protease inhibitor; Enzyme is a serine protease

Fractionate supernatant by size and test IL-8 degrading activity
Must be 100-150 kDa therefore must be SpyCEP

Question 14

How did we confirm SpyCEP was likely to be the one cleaving IL-8? (hint - degraders vs non-degraders)

Answer 14

Used western blot to detect SpyCEP in culture supernatant of degraders and non-degraders
SpyCEP was only present in isolates that degraded, and absent in non-degraders of IL-8

Question 15

How could we prove SpyCEP is responsible for the degrading phenotype?

Answer 15

Knockout SpyCEP gene, and test to see if IL-8 degrading ability is lost; Then reintroduce SpyCEP gene through complementation to see if degrading function is restored

Several other ways to prove it

Question 16

Look at A and B in figure on (3 - p.6); What do they show?
Why do we grow both control vector and pcepA in antibiotics?

Answer 16

By putting SpyCEP gene into SpyCEP mutant (doesn’t have gene) and also into another species (doesn’t have gene), IL-8 degradation ability is present and it looks like the WT

These plasmids will contain an antibiotic resistance gene so it is essential for the bacteria to keep these to survive

Question 17

Look at C in figure on (3 - p.6); Why is there less neutrophil migration in pcepA than WT?

Answer 17

Plasmid replicates more than chromosome, so you get more copies of SpyCEP so more expression

Question 18

How can we see what could be the underlying cause behind the Degraders vs Non-Degraders phenotype?
What did we see (look at graph in (3 - p.7))
What did we compare expression of cepA of against and what did we see?

Answer 18

See if they express the gene differently via qPCR

Non-degraders have much lower expression of SpyCEP compared to degraders

Another virulence factor (hasA) to see if they share regulators
Their expression is positively correlated so they are controlled by the same thing

Question 19

hasA is controlled by CovR/S system; What is it, where is it and how does it work?

Answer 19

2 component regulator

CovS sits in membrane, CovR in cytoplasm

CovS senses something and phosphorylates CovR
CovR then dimerises and binds promoter of gene and affects its transcription; Regulates expression

Question 20

How do we know that CovR/S is a negative regulator of SpyCEP?
What 2 things about degraders can we now conclude?

Answer 20

Took a strain we knew didn’t produce SpyCEP (can’t detect SpyCEP activity or protein) and deleted CovR/S
Deletion causes presence of SpyCEP and IL-8 degrading activity
- WT has no SpyCEP expression or activity

CovR/S downregulates expression of SpyCEP

Only degraders express and produce the SpyCEP gene

Question 21

By sequencing CovR/S genes in degraders and non-degraders, what did we find?
What does this suggest happens during infection?

Answer 21

Degraders had mutated CovS and non-degraders didn’t

Suggests mutations in this gene can arise during infection resulting in strains with high expression of virulence factors like SpyCEP

Question 22

What does phylogenetic tree show?

Answer 22

Genetic relationship between isolates

Question 23

How do we identify phylogenetic differences between isolates? (hint - WGS)
How do we draw genetic relationships on a phylogenetic tree?

Answer 23

WGS isolates to get alignment of isolates with reference genome
Look for variations/SNPs

Lineages break off at nodes and genetic distance (i.e. number of SNPs) is represented by horizontal lines and their length

Question 24

Differences between rooted and unrooted tree?

Answer 24

In a rooted tree, vertical branches are not related to genetic distance (can be as big as you want)
In an unrooted tree, length of all branches is important

Question 25

What does a phylogenetic tree give you an idea of? (hint - strains)

Answer 25

How strains are related BUT this must be explored further
How bacteria cause infection and factors that are important – Vaccine or therapy targets

Question 26

Phylogenetic tree will give you an idea of how strains are related but this must be explored further; What 4 things must be considered?

Answer 26

• Differences in expression of factors
• Differences in whether they even carry the genes
• Differences in ability to cause disease
• Differences that have occurred during growth/passage; Artificially introducing changes when isolating from patient and treating them badly when passaging them between plates

Question 27

How can epidemiology be used to observe initial observation?

Answer 27

Gives you an overall picture of the population
Monitor changes in disease
- Increases or decreases in frequency
Important because we may have to intervene if there’s an increase

Question 28

How can disease case graphs be useful? (hint - 2 ways)
How can disease variant graphs be useful?

Answer 28

Helpful for seeing if vaccines are effective; See if number of cases drops after vaccine introduction
- If we start to see higher cases than normal, we can then begin to think about preparations in hospitals and other areas

Number of cases data can be combined with genomics to see the number of cases across different variants; Helpful to see if vaccines are no longer effective against new strains
- If you start to see increases in disease you could relate it back to potential emergence of new variant

Question 29

What genotype do we use to classify Strep A into groups?
If total number of invasive GAS disease greatly correlates and essentially mirrors the number of invasive GAS disease caused by emm3, what does this suggest?

Answer 29

Sequence of emm gene

Suggests emm3 encodes in a large portion of invasive GAS diseases

Question 30

We would want to do whole genome sequencing on isolates around upsurge in iGAS to determine changes in the population
We should sequence isolates from the same genotype; Why?
We should sequence isolates from both outside and inside upsurge period; Why?

Answer 30

Limited changes within a population of the same genotype, so if we see an increase in infections caused by a genotype, it suggest something has happened within that genotype to make it more successful – Looking to sequence emm3 in different isolates

See what has changed between these isolates

Question 31

What information should we include on a phylogenetic tree about this infection?

Answer 31

Type of infection caused by the isolate
Region of the country the patient/isolate was from
Year/month the isolate was taken; Compare with upsurge

Question 32

A lot of the isolates that were taken from patients during the period of upsurge in disease were associated with Lineage C
How was this lineage different? (hint - virulence factors)

Answer 32

Gained 2 new virulence factors:
Superantigen – speC
DNase – spd1

But lost:
Superantigen – ssa

Question 33

What is a super antigen?
What is a DNase
- How can these be measured?
Look at graph in (4 - p.6) about DNA and superantigen; What does it show?

Answer 33

Has the ability to activate T cells non-specifically; Massive T-cell activation can cause scarlet fever and toxic shock syndrome
- Incubate it with T cells and they proliferate; Measure ∆Cpm to see how many T cells have proliferated due to super antigen

Degrades DNA
- Measure how much DNA is degraded

Question 34

What can we do to compare virulence factors (superantigens) between lineage C and others? (2 ways)

Answer 34

Compare the 2 superantigens directly in an assay; Express them recombinantly

• Western blot to compare protein expression levels of the 2 superantigens; Isolates are different lineages so different factors need considering
• Quantitative real time PCR to compare gene expression of the 2 superantigens

Question 35

What did we see after WGS of emm89 isolates from upsurge?

Answer 35

Emergence of new genetically divergent clade within emm89

Question 36

What did the clustering of the 229 SNPs in the new clade indicate?
How many regions of recombination?

Answer 36

Normally get random SNPs occurring and distributed in the genome; In this case we saw regions with lots of SNPs together – Unusual for this to happen in a short period of time

Indicates the SNP cluster originates from an external source, like another bacteria with same genes and different sequence, and has recombined with the host bacteria chromosome

6 regions of recombination

Question 37

What has changed in the isolates of the new emm89 clade? (hint - 2 regions of recombination)

Answer 37

Loss of the genes required to make the hyaluronic acid capsule

Change in a region surrounding 2 toxins; NADase and Streptolysin

Question 38

emm89 isolates have greater growth in blood and greater adherence than non-clade emm89; Why is this?

Answer 38

Greater growth due to not having capsule; Strange
Greater adherence as without capsule they are more likely to adhere; emm89 variation shows capsule production level correlates with adherence

Question 39

What does NADase do?
What does NADase assay involve?
- Why is it not very accurate?
Look at the graph in (4); What does it show?

Answer 39

Cleaves NAD+

Add in something to make it glow in the dark with UV – Then just scale it relative to eachother e.g. high activity is 5 and low activity is 2
- Measuring by eye

Question 40

What does Streptolysin O do?
What does SLO assay involve?
- Why is it more reliable than NADase assay?
Look at the graph in (4); What does it show?

Answer 40

Lyses red blood cells

incubating culture supernatant with red blood cells and measuring the optical density; As the red blood cells lyse the OD will increase
- This assay is more complex than NADase assay so this may explain why there’s more variation; Actually measuring a value

Question 41

Do extra practice on (4)