Analysis of the Physiological and Metabolic State of Leishmania Using Heavy Water Labeling

Kloehn, Joachim; McConville, Malcolm J.

doi:10.1007/978-1-0716-0294-2_35

Cited by 3 publications

(4 citation statements)

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“…Mice were metabolically labeled with 2 H 2 O during the acute phase of infection when rump lesions were ;5 mm in diameter. Labeling was initiated by intraperitoneal injection with 99.9% 2 H 2 O made up to 0.9% NaCl (35 ml of 2 H 2 O/g of body weight), and then mice were provided with 9% 2 H 2 O in their drinking water for the duration of the experiment (33). This regime results in a stable enrichment of the body water at 5% 2 H 2 O (31,33,55).…”

Section: Methodsmentioning

confidence: 99%

“…Labeling was initiated by intraperitoneal injection with 99.9% 2 H 2 O made up to 0.9% NaCl (35 ml of 2 H 2 O/g of body weight), and then mice were provided with 9% 2 H 2 O in their drinking water for the duration of the experiment (33). This regime results in a stable enrichment of the body water at 5% 2 H 2 O (31,33,55). Acutely infected mice (5-mm lesions) were treated with miltefosine, which was administered orally by gavage (40 mg/kg in 200 ml of H 2 O) for short treatments (single dose or 6 days of treatment) or given in the drinking water for longer treatments (40 mg/kg/day, assuming an average water intake of 5 ml per day per mouse [56]).…”

Section: Methodsmentioning

confidence: 99%

“…2 H 2 O-PBS (final concentration, 5% [vol/vol]) was added 1 h after initiation of drug treatment, and the cells were incubated for 30 h. Cells were quenched and lysed, and metabolites were extracted using a chloroformmethanol-water extraction, as described previously (59). Fatty acids and DNA derived deoxyribose were prepared and analyzed by gas chromatography-mass spectrometry (GC-MS), and 2 H labeling was quantified as the excess molar enrichment of the M1 mass isotopologue over the parental M0 ion for each fatty acid, as described previously (31,33).…”

Section: Methodsmentioning

confidence: 99%

“…In the presence of low concentration of 2 H 2 O, deuterium ( 2 H) is incorporated into a wide range of metabolites, including nucleotides, amino acids, sugars, and fatty acids, that, in turn, are precursors for membrane lipids and macromolecules. 2 H 2 O rapidly equilibrates across tissues and membranes and can be used to measure rates of DNA, RNA, and protein turnover, as well as key metabolic pathways (carbohydrate and lipid synthesis) in intracellular parasite stages in acute-phase granulomas (31)(32)(33). Using this approach, we have shown that the bulk population of L. mexicana amastigotes in granulomatous tissues have a very low growth rate (half-life [t 1/2 ], ;12 days) and switch to a metabolically quiescent state, characterized by low rates of RNA, protein, and lipid synthesis (31).…”

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confidence: 99%

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Identification of Metabolically Quiescent Leishmania mexicana Parasites in Peripheral and Cured Dermal Granulomas Using Stable Isotope Tracing Imaging Mass Spectrometry

Kloehn

Boughton

Saunders

et al. 2021

mBio

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Leishmania are sandfly-transmitted protists that induce granulomatous lesions in their mammalian host. Although infected host cells in these tissues can exist in different activation states, the extent to which intracellular parasites stages also exist in different growth or physiological states remains poorly defined. Here, we have mapped the spatial distribution of metabolically quiescent and active subpopulations of Leishmania mexicana in dermal granulomas in susceptible BALB/c mice, using in vivo heavy water labeling and ultra high-resolution imaging mass spectrometry. Quantitation of the rate of turnover of parasite and host-specific lipids at high spatial resolution, suggested that the granuloma core comprised mixed populations of metabolically active and quiescent parasites. Unexpectedly, a significant population of metabolically quiescent parasites was also identified in the surrounding collagen-rich, dermal mesothelium. Mesothelium-like tissues harboring quiescent parasites progressively replaced macrophage-rich granuloma tissues following treatment with the first-line drug, miltefosine. In contrast to the granulomatous tissue, neither the mesothelium nor newly deposited tissue sequestered miltefosine. These studies suggest that the presence of quiescent parasites in acute granulomatous tissues, together with the lack of miltefosine accumulation in cured lesion tissue, may contribute to drug failure and nonsterile cure. IMPORTANCE Many microbial pathogens switch between different growth and physiological states in vivo in order to adapt to local nutrient levels and host microbicidal responses. Heterogeneity in microbial growth and metabolism may also contribute to nongenetic mechanisms of drug resistance and drug failure. In this study, we have developed a new approach for measuring spatial heterogeneity in microbial metabolism in vivo using a combination of heavy water (2H2O) labeling and imaging mass spectrometry. Using this approach, we show that lesions contain a patchwork of metabolically distinct parasite populations, while the underlying dermal tissues contain a large population of metabolically quiescent parasites. Quiescent parasites also dominate drug-depleted tissues in healed animals, providing an explanation for failure of some first line drugs to completely eradicate parasites. This approach is broadly applicable to study the metabolic and growth dynamics in other host-pathogen interactions.

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