Metabolomic Profile of BALB/c Macrophages Infected with Leishmania amazonensis: Deciphering L-Arginine Metabolism

Abstract

Background: Leishmaniases are neglected tropical diseases that are caused by Leishmania, being endemic worldwide. L-arginine is an essential amino acid that is required for polyamines production on mammal cells. During Leishmania infection of macrophages, L-arginine is used by host and parasite arginase to produce polyamines, leading to parasite survival; or, by nitric oxide synthase 2 to produce nitric oxide leading to parasite killing. Here, we determined the metabolomic profile of BALB/c macrophages that were infected with L. amazonensis wild type or with L. amazonensis arginase knockout, correlating the regulation of L-arginine metabolism from both host and parasite. Methods: The metabolites of infected macrophages were analyzed by capillary electrophoresis coupled with mass spectrometry (CE-MS). The metabolic fingerprints analysis provided the dual profile from the host and parasite. Results: We observed increased levels of proline, glutamic acid, glutamine, L-arginine, ornithine, and putrescine in infected-L. amazonensis wild type macrophages, which indicated that this infection induces the polyamine production. Despite this, we observed reduced levels of ornithine, proline, and trypanothione in infected-L. amazonensis arginase knockout macrophages, indicating that this infection reduces the polyamine production. Conclusions: The metabolome fingerprint indicated that Leishmania infection alters the L-arginine/polyamines/trypanothione metabolism inside the host cell and the parasite arginase impacts on L-arginine metabolism and polyamine production, defining the infection fate.Int. J. Mol. Sci. 2019, 20, 6248 2 of 20 forms found in the proboscis of the phlebotomine sand fly host, to amastigote forms in the interior of macrophage phagolysosomes in the mammalian host [3]. Leishmania infection results in the regulation of pathways that are involved in the inflammatory response and leishmanicidal mechanisms, such as nitric oxide (NO) production via nitric oxide synthase 2 (NOS2), which is induced by Th1-cytokines [4][5][6][7][8][9]. However, Leishmania can subvert these mechanisms, which impacts on Th1/Th2 cytokine balance and induces macrophage arginase 1 (ARG1) activity to produce polyamines, putrescine, spermidine, and spermine, leading to Leishmania survival [6,[10][11][12].L-arginine is an essential amino acid and a precursor in the synthesis of proteins, urea, ornithine, citrulline, NO, creatinine, agmatine, glutamate, proline, putrescine, spermidine, and spermine, supporting the proline, glutamate, and polyamine metabolism at the whole organism level or cellular level in mammals. Its availability and metabolization can modulate inflammation and immune response regulation during infections and also recover the physiological steady-state [13,14].In mammalian cells, the endogenous synthesis of L-arginine from proline or glutamate via ornithine is not sufficient for supplying several pathways that need this amino acid as a precursor. L-arginine uptakes occur via cationic amino acid transporters (CAT1, CAT2A, ...

Dual transcriptome analysis reveals differential gene expression modulation influenced by Leishmania arginase and host genetic background

“…The upregulation of these amino acid transporters () corroborates previous metabolomic profiling data showing increased levels of arginine and citrulline in BALB/c_ La -arg - compared to BALB/c_ La -WT [57].…”

“…In general, amino acid starvation in mammalian cells leads to increased CAT1 activity and arginine uptake [55, 56]. In addition, the absence of parasite arginase activity leads to reduced levels of ornithine and increased levels of l -arginine [57, 58]. The similarity between CAT1 and amino acid permease 3 (AAP3), an exclusively Leishmania amino acid transporter [2, 59–62], indicates that both may respond similarly to amino acid starvation [59].…”

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

“…In addition, changes in the host plasma metabolite abundance, including pyruvate, taurine and N -acetylglutamine, can serve as an indicator of response to CL treatment [ 19 ]. Several studies previously investigated Leishmania and host metabolism during in vitro macrophage infection [ 20 , 21 , 22 , 23 ], or in amastigotes purified from mouse granulomatous lesions [ 24 ], but there is still a lack of knowledge of host metabolic responses during in vivo infection.…”

Differential expression of polyamine biosynthetic pathways in skin lesions and in plasma reveals distinct profiles in diffuse cutaneous leishmaniasis

“…has been described to benefit from host-derived polyamines and several studies have indicated that genetic or pharmacological suppression of parasite enzymes involved in polyamine pathways results in impairment of parasite growth and establishment of host cell infection 22,27,28,29 . More recently, it has been shown that during infection, L. amazonensis is able to alter the host metabolism, inducing polyamine production 30 . Although in our analyses we could not find a specific AAP3 gene, due to the lack of genome annotation, we retrieved the functional annotation in other Leishmania putative genes that are involved A hierarchical clustering analysis (Ward's method) was employed to illustrate the overall expression profile of Leishmania genes in DCL and LCL lesion and its correlation with parasite loads (normalized leishmania transcripts as described in "Methods" section).…”