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

Katrina J. Binger,Berin A. Boughton,Eleanor C. Saunders,Sean O’Callaghan,Malcolm J. McConville,Joachim Kloehn,Barbara Burleigh

doi:10.1128/mbio.00129-21

Abstract

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.

Highlights

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
We have used a universal stable isotope labeling approach coupled with ultra high-resolution imaging mass spectrometry (IMS) to detect metabolically distinct subpopulations of Leishmania mexicana protists in acutely infected animal tissue and define the physiological state of parasite populations that persist after drug treatment
We have coupled in vivo 2H2O stable isotope labeling with imaging mass spectrometry (IMS) to measure the temporal-spatial dynamics of parasite and host lipid dynamics in Leishmania granulomas. 2H2O rapidly equilibrates across all tissues, with concomitant incorporation of deuterium into a wide range of primary and secondary metabolites, as well as major cellular macromolecules [31, 42]

Summary

Introduction

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. Quiescent parasites 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. Recent studies have shown that both M1 and M2 polarized macrophages can coexist in the same tissues, even in the presence of a strong TH1 response due to local production of IL-4/IL-13 by granuloma eosinophils [21] Other factors, such as gradients of diffusible NO [25, 26], hypoxia [27], or salt [28] and nutrient levels, can modulate host cell polarization, the extent to which variability in host cell activation leads to heterogeneity in parasite growth and metabolism in vivo remains poorly defined

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Conclusion