The Role of the Novel Exopolyphosphatase MT0516 in Mycobacterium tuberculosis Drug Tolerance and Persistence

Inorganic polyphosphate (poly P) has been postulated to play a regulatory role in the transition to bacterial persistence. In bacteria, poly P balance in the cell is maintained by the hydrolysis activity of the exopolyphosphatase PPX. However, the Mycobacterium tuberculosis PPX has not been characterized previously. Here we show that recombinant MT0516 hydrolyzes poly P, and an MT0516-deficient M. tuberculosis mutant exhibits elevated intracellular levels of poly P and increased expression of the genes mprB, sigE, and rel relative to the isogenic wild-type strain, indicating poly P-mediated signaling. Deficiency of MT0516 resulted in decelerated growth during logarithmic-phase in axenic cultures, and tolerance to the cell wall-active drug isoniazid. The MT0516-deficient mutant showed a significant survival defect in activated human macrophages and reduced persistence in the lungs of guinea pigs. We conclude that exopolyphosphatase is required for long-term survival of M. tuberculosis in necrotic lung lesions

Constitutive smooth muscle tumour necrosis factor regulates microvascular myogenic responsiveness and systemic blood pressure

Tumour necrosis factor (TNF) is a ubiquitously expressed cytokine with functions beyond the immune system. In several diseases, the induction of TNF expression in resistance artery smooth muscle cells enhances microvascular myogenic vasoconstriction and perturbs blood flow. This pathological role prompted our hypothesis that constitutively expressed TNF regulates myogenic signalling and systemic haemodynamics under non-pathological settings. Here we show that acutely deleting the TNF gene in smooth muscle cells or pharmacologically scavenging TNF with etanercept (ETN) reduces blood pressure and resistance artery myogenic responsiveness; the latter effect is conserved across five species, including humans. Changes in transmural pressure are transduced into intracellular signals by membrane-bound TNF (mTNF) that connect to a canonical myogenic signalling pathway. Our data positions mTNF ‘reverse signalling' as an integral element of a microvascular mechanosensor; pathologic or therapeutic perturbations of TNF signalling, therefore, necessarily affect microvascular tone and systemic haemodynamics

Inorganic Polyphosphate and Energy Metabolism in Mammalian Cells*

Inorganic polyphosphate (poly P) is a polymer made from as few as 10 to several hundred phosphate molecules linked by phosphoanhydride bonds similar to ATP. Poly P is ubiquitous in all mammalian organisms, where it plays multiple physiological roles. The metabolism of poly P in mammalian organisms is not well understood. We have examined the mechanism of poly P production and the role of this polymer in cell energy metabolism. Poly P levels in mitochondria and intact cells were estimated using a fluorescent molecular probe, 4′,6-diamidino-2-phenylindole. Poly P levels were dependent on the metabolic state of the mitochondria. Poly P levels were increased by substrates of respiration and in turn reduced by mitochondrial inhibitor (rotenone) or an uncoupler (carbonyl cyanide p-trifluoromethoxyphenylhydrazone). Oligomycin, an inhibitor of mitochondrial ATP-synthase, blocked the production of poly P. Enzymatic depletion of poly P from cells significantly altered the rate of ATP metabolism. We propose the existence of a feedback mechanism where poly P production and cell energy metabolism regulate each other

CFTR Therapeutics Normalize Cerebral Perfusion Deficits in Mouse Models of Heart Failure and Subarachnoid Hemorrhage

Heart failure (HF) and subarachnoid hemorrhage (SAH) chronically reduce cerebral perfusion, which negatively affects clinical outcome. This work demonstrates a strong relationship between cerebral artery cystic fibrosis transmembrane conductance regulator (CFTR) expression and altered cerebrovascular reactivity in HF and SAH. In HF and SAH, CFTR corrector compounds (C18 or lumacaftor) normalize pathological alterations in cerebral artery CFTR expression, vascular reactivity, and cerebral perfusion, without affecting systemic hemodynamic parameters. This normalization correlates with reduced neuronal injury. Therefore, CFTR therapeutics have emerged as valuable clinical tools to manage cerebrovascular dysfunction, impaired cerebral perfusion, and neuronal injury