WGS of Pathogens

Question 1

What is most abundant protein on Strep A surface?
Conservation?

Answer 1

M protein

Highly conserved

Question 2

What region of M protein is hypervariable
What are the 2 ways of classifying the M protein and thus strep A?

Answer 2

N-terminal

Serotype using antibodies
Sequence region of emm gene that encodes M protein for genotype

Question 3

Most emm-types are single lineage. What does this mean within and across emm-types?

Answer 3

Isolates within lineages are closely related
Each emm-type lineage is quite different to a neighbouring emm-type

Question 4

Difference in emm-type between LICs and HICs?

Answer 4

Higher diversity of isolates in LICs

HICs isolates are very similar to eachother

No clustering of HIC isolates with LIC isolates; Very different

Question 5

What has genome sequencing of emm-types taught us? (hint - HICs vs LICs)
What must we do?

Answer 5

emm-type gives you an idea of what the genetic content is
HICs and LICs isolates are very different so same vaccines may not work for both

Need to find other conserved factors

Question 6

What did WGS of emm89 show us?
What did this imply?

Answer 6

Emergent lineage separated by 229 clustered SNPs

Suggests horizontal gene transfer event as SNPS should be randomly distributed

Question 7

What were 2 of the 6 key regions of recombination?

Answer 7

Loss of hyaluronic capsule which was though to be essential for infection

Changes around the genes encoding NADase and Slo; Caused high expression of these factors

Question 8

What were the 2 key traits emergent, higher activity genotypes showed?

Answer 8

Acapsular and high toxin variant

Question 9

How has convergent evolution been seen in different emm genotypes?

Answer 9

Different emm genotypes showed varying length and regions of recombination, but all achieved same acapsular and high toxin activity through different mechanisms

Question 10

How is C. difficile opportunistic?

Answer 10

Only causes disease when gut microbiome is disrupted and it colonise, typically due to broad spectrum antibiotics
Otherwise it doesn’t cause disease

Question 11

What does natural microbiome restoration do to C. difficile?

Answer 11

Drive its decline

Question 12

Although most people have self-resolving infections of C. difficile, what are treatments for those who have it more severe?

Answer 12

Vancomycin is a common antibiotic with no known resistance

Faecal transplant to restore natural healthy microbiome

Question 13

How does vancomycin work? (hint - D-Ala)
How is resistance emerging?

Answer 13

Vancomycin binds to the terminal D-Ala-D-Ala on peptidoglycan precursors and sterically inhibits transpeptidation

Bacteria acquiring genes through horizontal gene transfer to replace last D-Ala with another amino acid

Question 14

What are 2 ways helping to diagnose C. difficile infection?

Answer 14

ELISA assays that detect the toxin in faecal sample

qPCR assays that amplify C. difficile genes and directly detect presence of organism

Question 15

How do we assay for potential vancomycin resistance pathways? (hint - gradient broth evolution)

Answer 15

Use hyper mutating C. difficile and grow them in a gradient broth evolution with varying levels of MIC
Take well with highest antibiotic conc. and bacterial growth and repeat with same vancomycin gradient

Causes vancomycin tolerance to increase from each passage to the next

Question 16

What was observed in hypermutating C. difficile with high vancomycin resistance? (3 things)

Answer 16

Cant grow as fast
Produces fewer spores so cant transmit as well
Produce odd morphologies

Question 17

How are pipelines used to analyse C. difficile samples?

Answer 17

Software used to identify SNPs and insertions/deletions

Question 18

There is lots of parallel evolution between different hypermutating populations. How do we identify which mutations are causing certain traits such as vancomycin resistance? (2 ways)

Answer 18

Recapitulate mutations in isolation and combinations; Introduce amino acid change and see if it changes resistance

Design assays based on predicted function (BLAST or structural homology), assay protein activities (WT and variants) in vitro etc.

Question 19

What can we do instead of looking at combinations of mutations that co-occur in a particular cell?
What do we then adjust?

Answer 19

Look at and sequence all of the mutations present in an evolving population
Do deep sequencing to see low frequency alleles

Adjust pipeline to look at allele frequency rather than if a mutation is present