Dysregulation of Glycerophosphocholines in the Cutaneous Lesion Caused by Leishmania major in Experimental Murine Models

Abstract

Cutaneous leishmaniasis (CL) is the most common disease form caused by a Leishmania parasite infection and considered a neglected tropical disease (NTD), affecting 700,000 to 1.2 million new cases per year in the world. Leishmania major is one of several different species of the Leishmania genus that can cause CL. Current CL treatments are limited by adverse effects and rising resistance. Studying disease metabolism at the site of infection can provide knowledge of new targets for host-targeted drug development. In this study, tissue samples were collected from mice infected in the ear or footpad with L. major and analyzed by untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS). Significant differences in overall metabolite profiles were noted in the ear at the site of the lesion. Interestingly, lesion-adjacent, macroscopically healthy sites also showed alterations in specific metabolites, including selected glycerophosphocholines (PCs). Host-derived PCs in the lower m/z range (m/z 200-799) showed an increase with infection in the ear at the lesion site, while those in the higher m/z range (m/z 800-899) were decreased with infection at the lesion site. Overall, our results expanded our understanding of the mechanisms of CL pathogenesis through host metabolism and may lead to new curative measures against infection with Leishmania.

Keywords: Leishmania major; cutaneous leishmaniasis; glycerophosphocholines; host metabolism; neglected tropical diseases; untargeted metabolomics.

Figure 1

Effect of in vivo L. major infection on host metabolite profile. (A) Sites of infection and sample collection. Lesion at the center of the infected ear is circled in blue. (B) PCoA analysis of aqueous extraction from infected and uninfected ear samples, showing overall differences in metabolite profiles between sampling sites: PERMANOVA p = 0.004, R² = 0.288. (C) PCoA analysis of organic extraction from infected and uninfected ears, showing differences in global metabolite profiles between sampling sites: PERMANOVA p = 0.003, R² = 0.248. (D) Representative metabolite decreased by infection at the site of the lesion: glutamine (Wilcoxon rank-sum test comparing infected ear center vs infected ear edge p = 0.008). (E) Representative metabolite increased only at infection-adjacent sites: PC(O-34:1), Wilcoxon rank-sum test comparing infected ear center vs infected ear edge p = 0.008. Non-overlapping boxplot notches indicate significantly different medians between groups.

Figure 2

Top 15 infection-modulated metabolites as identified by random forest. (A) Footpad aqueous extraction. (B) Footpad organic extraction. (C) Ear aqueous extraction. (D) Ear organic extraction.

Figure 3

PC subnetworks. PC family metabolites in the footpad organic extraction (A), footpad aqueous extraction (B), ear organic extraction (C) and ear aqueous extraction (D) molecular networks. Relative metabolite abundance in the presence of infection is shown in red, and absence of infection is blue.

Figure 4

(A–F) PCs in the m/z range 200–299, 400–499, 500–599, 600–699, 700–799 and 800–899, respectively, change with infection and sampling position in the ear. (G) Total PC levels were increased at the site of infection in the ear. Non-overlapping boxplot notches indicate significantly different medians between groups.