1. Streptococcus pneumoniae pathogenesis
Sam King
CMP and BCMM meeting

2. Projects
Does pneumococcal genomic variation contribute to development of different disease states
Structure function analysis of pneumococcal transporters

3. Pneumococcal diversity
Pneumococcal bacteremia Hemolytic uremic syndrome (HUS) Is there something different about these isolates? Can we use that knowledge to develop tests to identify patients at risk or therapeutics?

4. To Identify pneumococcal sequences that contribute to HUS

5. Aims
Identify pneumococcal loci that potentially correlate with development of HUS
Identify pneumococcal HUS enriched sequences
Determine the biological contribution of these sequences to pathogenesis

6. Identify pneumococcal loci that potentially correlate with development of HUS
Sequence six pneumococcal HUS isolates
Two methodologies
Next generation (Biomedical Genomics Core)
30 million reads for each strain
Short (~100bp), high accuracy
Has to be aligned to a sequenced genome
Third generation sequencing (EA sequencing)
Longer reads (~3kb)
Accuracy low
Will allow generation of de novo sequence

7. Analysis of sequence data
Biomedical Genomics Core (Post doctoral scientist)
Will generate accurate de novo sequence using info from both techniques
Compare sequence with 16 sequenced genomes
King lab
Prioritize sequence variants for analysis
Conserved in the majority of HUS isolates
Absent in the majority of non-HUS isolate
Introduce or remove open reading frames
Change promoter or coding sequence of predicted extracellular proteins

8. Identify pneumococcal HUS enriched sequences
Screen presence of up to 50 variants
In up to:
50 HUS isolates
50 non-HUS blood isolates
Determine if there is a significant correlation with HUS

9. Identify pneumococcal HUS enriched sequences
Will 50 strains be enough?
If pneumococcal HUS isolates are more closely related genetically how will that affect our data? Can we take account of that?

10. Biological consequences of these HUS enriched sequences
Correlation but not causation
Contribution to pathogenesis
Bioinformatics
Biochemical assays
Genetic approaches

11. Structure function analysis of pneumococcal transporters
ATP binding cassette transporters
Conserved protein family
Five components
Substrate binding protein
Permease proteins
Interested in understanding the interaction of the atpase with these different components.
Structures of ATPases and complete transporters are known in other organisms so we can model based on this.
Do the differences in the amino acid sequences change the strengths of interaction can they help us understand this protein family
ATPase (nuclear binding domains)
ATP
ADP + Pi
ADP + Pi
ATP