Sensing Host Arginine Is Essential for Leishmania Parasites’ Intracellular Development

Abstract

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.

Evidence that a naturally occurring single nucleotide polymorphism in the RagC gene of Leishmania donovani contributes to reduced virulence

“…The CASTOR1/2 protein complex and solute carrier family38 member 9 (SLC38A9) (a lysosomal amino acid transporter) were identified as intracellular arginine sensors [ 14 , 18 , 27 ].Our bioinformatic analysis has shown that Leishmania has the homologs for SLC38A9 and components for the GATOR2 complex, normally downstream of CASTOR1/2 but we did not identify any proteins with significant CASTOR homology in Leishmania . It has been recently reported that in order to survive arginine deprivation in macrophages, Leishmania up regulates expression of its arginine transporter (AAP3) through a MAPK2-mediated arginine deprivation response (ADR) pathway [ 28 ]. As the identity of the arginine sensor that triggers the ADR pathway is unknown, it will be interesting to investigate whether the ADR pathway shares the same potential arginine sensors (SLC38A9 and a possibly unidentified protein upstream of GATOR2) with mTORC1 signaling pathway and whether the ADR pathway is downstream of or parallel to the mTORC1 pathway.…”

Metabolite Biomarkers of Leishmania Antimony Resistance

“…This profile has been also detected in L. infantum promastigotes [ 37 ], while an arginine downregulation has been associated with sensitive parasites growing under antimony pressure [ 36 ]. It has been suggested that Leishmania parasites can sense arginine availability regulating the expression of Leishmania arginine transporter (AAP3) under stress conditions [ 67 ]. Interestingly, there is evidence that the AAP3 transporter is upregulated at the mRNA level in Sb III -resistant parasites derived from clinical isolates [ 68 ], which could explain the tendency toward a higher arginine intracellular concentration in Sb III -resistant phenotypes.…”

Leishmania parasite arginine deprivation response pathway influences the host macrophage lysosomal arginine sensing machinery

“…Leishmania -adapted CRISPR/Cas9 system (24) to expedite disruption of the AAP3 genes, was used to create ADR mutants as described by Goldman-Pinkovich et al (23). These knockouts were obtained from the Prof. Zilberstein group (Technion-Israel Institute of Technology, Israel).…”

“…We have previously reported that the Leishmania donovani arginine sensor responds to arginine deprivation by activating a MAPK2-mediated signalling pathway, known as the arginine deprivation response (ADR), resulting in the up-regulation of expression and activity of its amino acid transporter 3 ( Ld AAP3) (21). Significantly, ADR is also activated during macrophage infection (21, 22, 23), indicating a role for intra-phagolysosomal arginine sensing in intracellular (amastigote) parasite survival and pathogenesis. In the present study, we have investigated the involvement of the host arginine sensing pathway by which L. donovani can gain access to the macrophage L-arginine pool.…”