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

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.

Mass spectrometry imaging identifies altered hepatic lipid signatures during experimental Leishmania donovani infection

“…A recent study by Kloehn et al. ( 41 ) showed unique lipids distributed across the lesion induced by L. mexicana and surrounding host tissue. However, to our knowledge, there is no evidence that highlights lipid compositional changes between inflammatory microenvironments at different time points of infection.…”

“…Earlier research demonstrated the ability of MSI to visualize candidate peptides derived from visceralising Leishmania in mice (55) and to relate peptides to cutaneous Leishmania infection in mice (56). A recent study by Kloehn et al (41) showed unique lipids distributed across the lesion induced by L. mexicana and surrounding host tissue. However, to our knowledge, there is no evidence that highlights lipid compositional changes between inflammatory microenvironments at different time points of infection.…”

“…Subsequently, we structurally identified lipids using tandem MS identifying specific lipid classes at each time point studied. We here focused on ionizing lipids in negative mode, as this allows detection of lipid classes that are highly related to more generalizable host-pathogen inflammatory interactions ( 14 , 41 , 57 ).…”

“…Several reviews highlight how leishmaniasis affects and causes aberrant lipid compositions ( 10 , 11 , 54 ). In addition, the role of lipids in these cascades are prominent as phospholipids (PLs), glycolipids (GLs) and cholesterol were shown to influence host-pathogen interactions following infection with L. donovani ( 41 , 58 ). Interestingly, while most lipids exhibited either an increased or decreased intensity across infected compared to naïve liver, we also identified lipids that were mainly expressed in d14 and d20 parasitized livers ( viz.…”

“…A recent study applied MSI to evaluate lipid metabolism in the cutaneous lesion caused by L. mexicana infection in susceptible BALB/c mice, describing altered lipid profiles in quiescent and replicative amastigotes in a spatially defined way ( 41 ). In contrast to the macroscopic skin lesion caused by L. mexicana , where multiple granulomas may coalesce with associated necrosis and other cellular infiltrates, the hepatic granulomatous response to L. donovani infection in both resistant and susceptible strains of mice provides for a more discrete identification of individual granulomas, each formed around an initiating infected Kupffer cell and embedded within the hepatic parenchyma ( 42 , 43 ).…”