Tuberculosis – metabolism and respiration in the absence of

growth-- prepared by Shenghua Liang

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

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

Tuberculosis

Tuberculosis

• 1st infection - pulmonary macrophages• 2nd infection - lymph nodes, kidneys, brain,

and even bone.• Granuloma

– T/B cell, macrophages– Necrosis/cell death– Bacteria go dormant

• Tissue destruction• Caseation, scars…

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.

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

Animal models - primates

• The most suitable, but expensive

• Infection by bronchial instillation

• Granuloma, with caseation

• Probably similar immune response

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

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

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

The signal for persistence

• Nitric oxide inhibit mycobacterial growth• In mice, DosR, the dormancy regulon regulatory, is up-re

gulated under microaerobic condition• Nitric oxide and oxygen deprivation have similar poisonin

g 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.

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

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.

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.

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 (NO3

-) reductase

• Nitrate reductase might simply be required for restoring redox balance during growth on fatty acids.

Does M. tuberculosis ferment?

• Fermentation – the energy-yielding anaerobic metabolic breakdown of a nutrient molecule without net oxidation; yields lactate, acetic acid, ethanol, etc.