1. Transcriptional profiling and mRNA stability – don’t shoot the messenger David R. Sherman Seattle Biomedical Research Institute Grand Challenge of Latent TB Mtg. Cape Town February 25, 2012

2. Landscape of TB latency CONFIDENTIAL 4.4 billion PPD(-) 1.8 billion PPD(+) 16 million active infections 1.5 million deaths

3. Landscape of TB latency CONFIDENTIAL 4.4 billion PPD(-) 1.8 billion PPD(+) 16 million active infections 1.5 million deaths Calcified lesions Few viable bacteria Caseous lesions Low numbers of bacteria Evolving lesions Bacterial replication Latency to Disease Active disease

4. Landscape of TB latency CONFIDENTIAL 4.4 billion PPD(-) 1.8 billion PPD(+) 16 million active infections 1.5 million deaths Calcified lesions Few viable bacteria Caseous lesions Low numbers of bacteria Evolving lesions Bacterial replication Latency to Disease Active disease GC-11

5. Using gene expression to probe latent TB Concept – TB gene expression in vivo will reveal the conditions that it experiences. - Physiology - Drug targets Infected tissue Gene regulatory network

6. Using gene expression to probe latent TB Infected tissue Gene regulatory network Assumes TF binding = Tx initiation = mRNA abundance

7. mRNA abundance balance Shalem et al. 2008 Transcription Rate Degradation Rate

8. Talk outline Characterize MTB mRNA decay – Global mRNA half life (T 1/2 ) – Most stable/labile messages – Factors affecting stability mRNA decay in stress response – Mild cold shock – Hypoxia Summary

9. Measuring mRNA degradation Rifampicin Log Phase 01060 RNA Cy dye label Microarray 2030515 Custom array design: 100,000 TB oligos 30,000 control oligos

10. T0 T20 T5T10 T15 mRNA decay by microarray

11. Individual decay curve Rifampicin (rif)

12. Individual decay curve Rifampicin (rif) T 1/2

13. Data filtered for reproducibility and R 2 Inclusion criteria: – T 1/2 with R 2 >0.7 – Starting [RNA] > 4x background – Valid measures at > half replicates 2139 genes met criteria

14. TB mRNA is very stable Average MTB T 1/2 = 9.5 minutes

15. TB mRNA is very stable

16. Functional Category Mean half-life (minutes) information pathways8.7 * virulence, detoxification, and adaptation9.3 lipid metabolism9.4 cell wall and cell processes9.4 metabolism and respiration9.5 hypothetical protein9.6 Regulatory proteins9.6 insertion seqs and phages10.4 ** PE/PPE11.1 ** *=significantly lower **=significantly higher mRNA T 1/2 by functional category

17. Functional subcategory Specified Gene % Whole Genome % P Value Functional Enrichment for the genes with the shortest 1/2 life Posttranslational modification, protein turnover, chaperones12.82.30 Translation, ribosomal structure and biogenesis21.43.00 Energy production and conversion14.25.20.002 Intracellular trafficking, secretion, and vesicular transport2.80.30.025 Functional Enrichment for the genes with the longest 1/2 life Replication, recombination and repair12.84.80.004 Amino acid transport and metabolism11.44.50.01 PE/PPE2.80.20.014 Energy production and conversion11.45.20.019

18. Physical characteristics and mRNA stability

19. mRNA abundance and stability R 2 = 0.8

20. mRNA abundance and stability Inherent to the mRNA? Inherent to abundance? Test: DosR regulon: ~48 genes induced by hypoxia, etc. Place dosR under tet control. Induce regulon in log phase

21. Induced transcripts degrade faster >2000 transcripts did not change stability

22. Modified mRNA decay in response to stress conditions Are specific transcripts (de)stabilized? Does the global mRNA ½ life change? Is mRNA decay regulated to change transcript abundance? Temperature Hypoxia

23. mRNA degradation at 20C After 5 hrs: Only 55 genes decayed 2x or more. Degradation not measurable. T 1/2 very sensitive to temp. T = 5 hrs

24. mRNA stability in hypoxia Rifampicin Log Phase 0 10 60 20 30 Hypoxia 120+ RNA Cy dye label Microarray

25. mRNA stability in altered O 2

26. MTB mRNA decay characterization Reliable mRNA half lives for >2000 genes. Average half life ~9.5 minutes. Half life inversely correlated with transcript concentration. Transcripts stabilized by cold, hypooxia.

27. Questions to explore Why is TB mRNA very stable? – RNases or mRNA secondary structure? Hypoxia and low temp: Transcription decreased to balance decay? Mechanism? How to repress genes? What are the consequences? – Systems modeling

28. Thank you! Kyle Minch Tige Rustad Debbie Whitley Bill Brabant Jessica Winkler Paul G Allen Family Foundation Bill and Melinda Gates Foundation NIAID contract # HHSN272200800059C

29. Rifampicin is stopping transcription No rif induced genes Lux assay to look for induction after rif treatment – Hypoxia sensitive promoter driving lux Total degradation by array