Clinical Disease through to Scientific Understanding Flashcards
What are the 3 ways of handling data relating to an unusual microbial related phenotype/genotype?
Clinical/Epidemiological observation
Population genetics
Phenotypic study
What are the 4 reasons we study unusual microbial phenotype/genotype?
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.
What are the 2 initial unusual microbial clinical observations?
Unusual disease progression or presentation
Outbreak situation
What are the 2 initial unusual microbial diagnostic observations?
Bacterial colonies look different
Mixed phenotype
What are the 2 initial unusual microbial epidemiological observations?
Increase in disease in a specific region or during a specific time
Increase in a certain or unusual bacterial genotype
What are the 4 things we must consider when working back from initial clinical/diagnostic/epidemiological observation?
Phenotype
Protein and its translation
Protein function
Gene and its transcription
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?
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)
Explain concept of ELISA assay in context of IL-8
Look at (3 - p.2) and explain what ELISA graph shows
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
What does western blot of H292 and IL-8 show? (look at (3 - p.3))
What is used as control and why?
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
Where does H292 cleave IL-8?
Why does this affect western blot results?
How did we discover this?
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
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
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
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?
It’s present in the culture supernatant
Produced by bacteria during exponential growth
Likely to be an enzyme
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?
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
How did we confirm SpyCEP was likely to be the one cleaving IL-8? (hint - degraders vs non-degraders)
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
How could we prove SpyCEP is responsible for the degrading phenotype?
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
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?
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
Look at C in figure on (3 - p.6); Why is there less neutrophil migration in pcepA than WT?
Plasmid replicates more than chromosome, so you get more copies of SpyCEP so more expression
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?
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
hasA is controlled by CovR/S system; What is it, where is it and how does it work?
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
How do we know that CovR/S is a negative regulator of SpyCEP?
What 2 things about degraders can we now conclude?
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
By sequencing CovR/S genes in degraders and non-degraders, what did we find?
What does this suggest happens during infection?
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
What does phylogenetic tree show?
Genetic relationship between isolates
How do we identify phylogenetic differences between isolates? (hint - WGS)
How do we draw genetic relationships on a phylogenetic tree?
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
Differences between rooted and unrooted tree?
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
What does a phylogenetic tree give you an idea of? (hint - strains)
How strains are related BUT this must be explored further
How bacteria cause infection and factors that are important – Vaccine or therapy targets
Phylogenetic tree will give you an idea of how strains are related but this must be explored further; What 4 things must be considered?
- 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
How can epidemiology be used to observe initial observation?
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
How can disease case graphs be useful? (hint - 2 ways)
How can disease variant graphs be useful?
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
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?
Sequence of emm gene
Suggests emm3 encodes in a large portion of invasive GAS diseases
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?
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
What information should we include on a phylogenetic tree about this infection?
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
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)
Gained 2 new virulence factors:
Superantigen – speC
DNase – spd1
But lost:
Superantigen – ssa
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?
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
What can we do to compare virulence factors (superantigens) between lineage C and others? (2 ways)
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
What did we see after WGS of emm89 isolates from upsurge?
Emergence of new genetically divergent clade within emm89
What did the clustering of the 229 SNPs in the new clade indicate?
How many regions of recombination?
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
What has changed in the isolates of the new emm89 clade? (hint - 2 regions of recombination)
Loss of the genes required to make the hyaluronic acid capsule
Change in a region surrounding 2 toxins; NADase and Streptolysin
emm89 isolates have greater growth in blood and greater adherence than non-clade emm89; Why is this?
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
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?
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
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?
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
Do extra practice on (4)
