1. Tuberculosis – metabolism and respiration in the absence of growth -- prepared by Shenghua Liang

2. Table of contents Introduction Animal models of latency In vitro models of latency and persistence The signal for persistence Redox balance during beta-oxidation Does M. tuberculosis ferment? The role of F420 in persistence Conclusions

3. Tuberculosis Caused by aerobic bacteria mycobacterium tuberculosis Top infectious killing diseases. Each year, –HIV/AIDS 3 million –Tuberculosis kills 2 million –Malaria kills 1 million Widely spreaded world-wide –1/3 carriers, among which 10% dev. disease No effective vaccine

4. Tuberculosis

5. 1 st infection - pulmonary macrophages 2 nd infection - lymph nodes, kidneys, brain, and even bone. Granuloma –T/B cell, macrophages –Necrosis/cell death –Bacteria go dormant Tissue destruction Caseation, scars…

9. Animal models - murine Low cost, genetically well-studied, extensive literature on mouse immu., and availability of reagents. Similar immune control including T-helper 1 response Similar granuloma formation, but not progress to caseation and liquefaction. Becomes chronic. Main immune containment depends on nitric oxide and other reactive nitrogen intermediates.

10. Animal models – guinea pig and rabbit Very similar disease progression –Granuloma and caseation Rabbit –Liquefaction, and cavity formation Guinea pig –Before immune onset, bacteria kills –BCG vaccine helps

11. Animal models - primates The most suitable, but expensive Infection by bronchial instillation Granuloma, with caseation Probably similar immune response

12. In vitro models of latency and persistence Upon oxygen depletion, M. tuberculosis becomes dormant in two steps NRP-1 – non-replicating persistence stage 1, oxygen lower than 1% –Cell division stops NRP-2 – non-replicating persistence stage 2, oxygen lower than 0.06% –Shutdown of metabolism

13. In vitro models of latency and persistence Up-regulates bd-type menaquinol oxidase, which has higher oxygen affinity. NADH dehydrogenase –Type I, proton pumping, down-regulated –Type II, non-proton pumping, up-regulated ATP synthase units are down-regulated ? An energized membrane is maintained –Survive without external terminal electron acceptors

14. In vitro models of latency and persistence Certain nutrients, but not all, are limited in intraphagosomal environment Ribonucleotide reductase is upregulated Triacylglycerol synthases are upregulated Isocitrate lyase and glycine dehydrogenase are upregulated Stringent response and polyphosphate metabolism might be crucial for the adaptation

15. The signal for persistence Nitric oxide inhibit mycobacterial growth In mice, DosR, the dormancy regulon regulatory, is up- regulated under microaerobic condition Nitric oxide and oxygen deprivation have similar poisoning effect on cytochrome DosR is required for dormancy regulon activation and is essential for anaerobic survival of M. bovis and M. tuberculosis in vitro. dosR mutant is not attenuated for growth and survival in mouse tissues. – chronic murine granulomas are not anoxic. dosR is required for virulence in guinea pigs. – oxygen is limited in the caseous lesions in this animal model.

17. The signal for persistence Acellular caseous material that characterize some human lesions is produced due to reduced survival of cells in the increasingly anaerobic interior of such granulomas or due to immune-mediated tissue destruction is unknown. The availability of nutrients might be limited for M. tuberculosis that are located in hypoxic granuloma. Carbon might be obtained from intracellular triglyceride stores, or from lipids in the surrounding host tissues. Stringent response, regulated by RelA, might have a role during the onset of dormancy. –Produce ppGpp, which in turn affects ~60 genes

18. The signal for persistence In mice, the primary trigger for chronic TB is nitric oxide; and in human, anaerobiasis might be the primary trigger. The metabolic state that is induced by nitric oxide might have important differences from that induced by hypoxic conditions.

19. The role of beta-oxidation and gluconeogenesis Carbon utilization by M. tuberculosis during infection depend on the activation state of macrophages. Activated macrosome is glucose-deficient but replete in fatty acids. During macrosomal survival, enzymes involved in beta-oxidation, the glyoxylate shunt and gluconeogensis are induced.

20. Redox balance during beta- oxidation Beta-oxidation – the process by which fats are broken into Acetyl-CoA. Beta-oxidation is limited by the availability of terminal electron acceptors. In resting and activated macrophages, genes in alternative electron-transport pathways are up-regulated. –Fumarate reductase –Non-proton pumping type II NADH dehydrogenase –Nitrate (NO 3 - ) reductase Nitrate reductase might simply be required for restoring redox balance during growth on fatty acids.

22. Does M. tuberculosis ferment? Fermentation – the energy-yielding anaerobic metabolic breakdown of a nutrient molecule without net oxidation; yields lactate, acetic acid, ethanol, etc.