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 PresentationIsabel Gonzaga

BIOL 398: Bioinformatics LaboratoryNovember 12, 2014

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

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

O2 depletion improves M. tuberculosis latent period

• Gradual O2 depletion leads to nonreplicating, persistant state; leads to structural, metabolic and chromosomal changes to the bacteria

• Reduced O2 tensions lead to resistance to antimicrobials

• Introduction of O2 allows for easy conversion to an active form of the bactria

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

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

Dormancy regulon determined by coinduction by NO, low O2 and adaptation to an in vitro dormant state

Dormancy regulon determined by coinduction by NO, low O2 and adaptation to an in vitro dormant state

Dormancy regulon increases overall M. tuberculosis fitness in vitro

Cyanide blocks expression of dormany regulon genes by NO and low O2

• Heme binds to NO and O2; 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 O2 signal transductio system

CN-+HYPHYP

CN-+NO

CN-

NO

O2 inhibits NO mediated regulon induction

Cytochrome Oxidase proposed for the NO/Low O2

• 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

• 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

CitationsVoskuil, 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.

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