1. Inhibition of respiration by nitric oxide induces a Mycobacterium tuberculosis dormancy program Voskuil, M.I., Schappinger, D., Visconti, K.C., Harrell, M.I., Dolganov, G.M., Sherman, D.R., and Schoolnik, G.K. (2003). J. Exp. Med. 198(5), 705- 713. doi:10.1084/jem.20030205. Journal Club Presentation Isabel Gonzaga BIOL 398: Bioinformatics Laboratory November 12, 2014

2. Outline Tuberculosis latency period is crucial for disease control Nitric Oxide may be an immune factor crucial for dormancy Low concentrations of NO signal induction Dormancy regulon determined by NO, dormancy and hypoxia response NO binding promotes the signal transduction program

3. Tuberculosis infection has three developmental stages TB is a pulmonary infection caused by Mycobacterium tuberculosis 3 stage pathogenic sequence Inhalation of infection aerosol Latency: Cell-mediated immunity in granulamatous lesions Unimpeded bacterial replication (onset of disease) 1/3 of the world is latently infected and the most aggressive TB cases exist in latent form Factors improving latency need to be investigated

4. O 2 depletion improves M. tuberculosis latent period Gradual O 2 depletion leads to nonreplicating, persistant state; leads to structural, metabolic and chromosomal changes to the bacteria Reduced O 2 tensions lead to resistance to antimicrobials Introduction of O 2 allows for easy conversion to an active form of the bactria

5. Nitric Oxide (NO) is an immune factor in certain concentrations High doses of NO is toxic for bacteria NO inhibits aerobic respiration in mitochondria and bacteria Important signaling agent for eukaryotes Present study: investigates role of NO in inducing latent period program Hypothesis: NO controls M. tuberculosis growth by inhibiting aerobic respiration

6. NO induces gene expression for 48 genes A DETA/NO generated NO and rapidly induced 48 genes B Response not desensitized to subsequent doses NO dissipation returned induction to basal levels C qRT-PCR measured induction magnitude of five sentinel NO induced genes mRNA levels up to 140x increase

7. Dormancy regulon determined by coinduction by NO, low O 2 and adaptation to an in vitro dormant state

9. Dormancy regulon increases overall M. tuberculosis fitness in vitro

14. Cyanide blocks expression of dormany regulon genes by NO and low O 2 Heme binds to NO and O 2 ; compeitivie inhibitor Cyanide: heme-protein inhibitor Found to block dormancy regulon gene expression without affecting overall transcription levels Indicates that a heme-containing protein is likely to be a component of the NO/low O 2 signal transductio system CN - +HYP HYP CN - +NO CN - NO

15. O 2 inhibits NO mediated regulon induction

16. Cytochrome Oxidase proposed for the NO/Low O 2 CcO is shown to be reversibly inhibited by low concentrations of NO This proposal must be supported by further functional studies comparing purified wild type and CcO mutant Decreasing respiration initiates transcriptional response, and the pathogen is transformed to stabilize the virus. This lets the pathogen endure longer latency periods NO thus serves as an environmental signal for activation of the bacteria by the immune system

17. Low NO concentrations induce 48 gene regulon using the DosR regulator. This inhibits aerobic respiration and slows replication Regulon is used to increase fitness in latency Predicted roles of genes within the dormancy regulon are supported by previous research of the physiological properties in the dormant state See: Crowe et al (1992), Yuan et al. (1996), Garbe et al. (1995) and Narberhaus (2002) Literature has yet to prove in vivo functioning's of M. tuberculosis in humans

18. Citations Voskuil, M.I., Schappinger, D., Visconti, K.C., Harrell, M.I., Dolganov, G.M., Sherman, D.R., and Schoolnik, G.K. (2003). Inhibition of respiration by nitric oxide induces a Mycobacterium tuberculosis dormancy program. J. Exp. Med. 198(5), 705-713. doi:10.1084/jem.20030205.

19. Acknowledgements Loyola Marymount University Kam Dahlquist, Ph. D TA: Stephen Louie