Amino acid sensing is an intracellular function that supports nutrient homeostasis, largely through controlled release of amino acids from lysosomal pools. The intracellular pathogen Leishmania resides and proliferates within human macrophage phagolysosomes. Here we describe a new pathway in Leishmania that specifically senses the extracellular levels of arginine, an amino acid that is essential for the parasite. During infection, the macrophage arginine pool is depleted due to its use to produce metabolites (NO and polyamines) that constitute part of the host defense response and its suppression, respectively. We found that parasites respond to this shortage of arginine by up-regulating expression and activity of the Leishmania arginine transporter (LdAAP3), as well as several other transporters. Our analysis indicates the parasite monitors arginine levels in the environment rather than the intracellular pools. Phosphoproteomics and genetic analysis indicates that the arginine-deprivation response is mediated through a mitogen-activated protein kinase-2-dependent signaling cascade.

Arginine homeostasis in lysosomes is critical for the growth and metabolism of mammalian cells. Phagolysosomes of macrophages are the niche where the parasitic protozoan Leishmania resides and causes human leishmaniasis. During infection, parasites encounter arginine deprivation, which is monitored by a sensor on the parasite cell surface. The sensor promptly activates a mitogen-activated protein kinase 2 (MAPK2)-mediated arginine deprivation response (ADR) pathway, resulting in upregulating the abundance and activity of the Leishmania arginine transporter (AAP3). Significantly, the ADR is also activated during macrophage infection, implying that arginine levels within the host phagolysosome are limiting for growth. We hypothesize that ADR-mediated upregulation of AAP3 activity is necessary to withstand arginine starvation, suggesting that the ADR is essential for parasite intracellular development. CRISPR/Cas9-mediated disruption of the AAP3 locus yielded mutants that retain a basal level of arginine transport but lack the ability to respond to arginine starvation. While these mutants grow normally in culture, they were impaired in their ability to develop inside THP-1 macrophages and were ∼70 to 80% less infective in BALB/c mice. Hence, inside the host macrophage, Leishmania must overcome the arginine “hunger games” by upregulating the transport of arginine via the ADR. We show that the ability to monitor and respond to changes in host metabolite levels is essential for pathogenesis. IMPORTANCE In this study, we report that the ability of the human pathogen Leishmania to sense and monitor the lack of arginine in the phagolysosome of the host macrophage is essential for disease development. Phagolysosomes of macrophages are the niche where Leishmania resides and causes human leishmaniasis. During infection, the arginine concentration in the phagolysosome decreases as part of the host innate immune response. An arginine sensor on the Leishmania cell surface activates an arginine deprivation response pathway that upregulates the expression of a parasite arginine transporter (AAP3). Here, we use CRISPR/Cas9-mediated disruption of the AAP3 locus to show that this response enables Leishmania parasites to successfully compete with the host macrophage in the “hunger games” for arginine.

The aim of the present study was to investigate the feasibility of targeting Leishmania transporters via appropriately designed chemical probes. Leishmania donovani, the parasite that causes visceral leishmaniasis, is auxotrophic for arginine and lysine and has specific transporters (LdAAP3 and LdAAP7) to import these nutrients. Probes 1–15 were originated by conjugating cytotoxic quinone fragments (II and III) with amino acids (i.e. arginine and lysine) by means of an amide linkage. The toxicity of the synthesized conjugates against Leishmania extracellular (promastigotes) and intracellular (amastigotes) forms was investigated, as well their inhibition of the relevant amino acid transporters. We observed that some conjugates indeed displayed toxicity against the parasites; in particular, 7 was identified as the most potent derivative (at concentrations of 1 µg/mL and 2.5 µg/mL residual cell viability was reduced to 15% and 48% in promastigotes and amastigotes, respectively). Notably, 6, while retaining the cytotoxic activity of quinone II, displayed no toxicity against mammalian THP1 cells. Transport assays indicated that the novel conjugates inhibited transport activity of lysine, arginine and proline transporters. Furthermore, our analyses suggested that the toxic conjugates might be translocated by the transporters into the cells. The non-toxic probes that inhibited transport competed with the natural substrates for binding to the transporters without being translocated. Thus, it is likely that 6, by exploiting amino acid transporters, can selectively deliver its toxic effects to Leishmania cells. This work provides the first evidence that amino acid transporters of the human pathogen Leishmania might be modulated by small molecules, and warrants their further investigation from drug discovery and chemical biology perspectives.

Arginine homeostasis in lysosomes is critical for growth and metabolism of mammalian cells. They employ a specific sensor (SLC38A9) that monitors intra-lysosome arginine sufficiency and subsequently up-regulates cellular mTORC1 activity. Lysosomes of macrophages (phagolysosomes) are the niche where the parasitic protozoan Leishmania resides and causes important human disease. Several years ago, we discovered that upon arginine starvation, cultured Leishmania parasites promptly activate a MAPK2-mediated Arginine Deprivation Response (ADR) pathway, resulting in up-regulation of the Leishmania arginine transporter (AAP3), as well as a small group of other transporters. Significantly, ADR is also activated during macrophage infection, implying that the intracellular parasite actively depletes arginine within the host phagolysosome, likely to prevent mTORC1 activation and enhance intracellular development. We hypothesize that ADR-mediated upregulation of AAP3 activity is necessary to withstand the resultant arginine starvation. Both copies of the AAP3 genes are located (in tandem) on a tetrasomic chromosome (chr31), but only one (AAP3.2) is responsive to arginine deprivation. CRISPR/Cas9-mediated disruption of the AAP3 locus yielded mutants that retain a basal level of arginine transport (mediated by AAP3.1), but lack a functional copy of AAP3.2 and are therefore not responsive to arginine starvation. While these mutants grow normally in culture as promastigotes, they were impaired in their ability to develop inside THP1 macrophages grown under physiological concentrations of arginine (0.1 mM). However, flooding the macrophage growth medium with arginine (1.5 mM) restored parasite infectivity and intracellular growth to that of wild type. The results indicate that inside the host macrophage, Leishmania must overcome the arginine "Hunger Games" by up-regulating transport of arginine via the ADR. Furthermore, the AAP3.2 mutants were ~70-80% less virulent in Balb/C mice, showing, for the first time,

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