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

Extensive interaction between the host and pathogen metabolic networks decidedly shapes the outcome of infection. Infection with Leishmania donovani, an intracellular protozoan parasite, leads to a competition for arginine between the host and the parasite. L. donovani transports arginine via a high-affinity transporter LdAAP3, encoded by the two genes LdAAP3.1 and LdAAP3.2. Earlier reports show that upon arginine starvation, cultured Leishmania parasites promptly activate an Arginine Deprivation Response (ADR) pathway, resulting in the stoichiometric up-regulation of LdAAP3.2 mRNA, protein and activity. Lysosomes, on the other hand, are known to employ a specific sensor and an arginine-activated amino acid transporter, solute carrier family 38 member 9 (SLC38A9) that monitors intra-lysosome arginine sufficiency and subsequently up-regulates cellular mTORkinase activity. The present study investigates the interaction between Leishmania and macrophage-lysosome arginine sensing machinery. We show that infection with L. donovani activates SLC38A9 arginine sensing in the human monocyte like-macrophage cell line (THP-1) when grown under physiological concentrations of arginine (0.1 mM). However, supplementing the macrophage growth medium with excess arginine (1.5 mM) followed by infection led to the down-regulation of SLC38A9. Similarly, THP-1 cells infected with LdAAP3.2 null mutants grown in 0.1 mM arginine resulted in reduced expression of SLC38A9 and mTOR. These results indicate that inside the host macrophage, Leishmania overcome low arginine levels by up-regulating the transport of arginine via LdAAP3 and SLC38A9 signalling. Furthermore, while LdAAP3.2 null mutants were impaired in their ability to develop inside THP-1 macrophages, their infectivity and intracellular growth were restored in SLC38A9 silenced macrophages. This study provides the first identification of regulatory role of SLC38A9 in the expression and role of LdAAP3.Author SummaryLeishmania donovani, the causative agent of kala-azar, exhibits a digenetic life cycle. Following infection of the mammalian host, promastigotes differentiate into intracellular amastigotes within the phagolysosome of macrophages. Arginine is a central point of competition between the host and the pathogen. L. donovani senses lack of arginine in the surrounding micro-environment and activates a unique ADR pathway, thus upregulating the expression of the arginine transporter (LdAAP3). The arginine-activated amino acid transporter SLC38A9 localizes to the lysosome surface of mammalian cells and acts as a sensor that transmits information about arginine levels in the lysosome lumen to the mechanistic target of rapamycin (mTOR) kinase. In the present study, we identified the functional interaction of host SLC38A9 and parasite LdAAP3 in macrophages infected with L. donovani. We report that host SLC38A9 upregulation is critical for enhancing and maintaining high LdAAP3 levels in intracellular L. donovani. Our results decode crucial information regarding the molecular mechanism involved in the arginine sensing response in L. donovani-infected host cells. These findings increase our understanding of the interaction of signalling intermediates during Leishmania infection which may lead to the discovery of novel therapeutic interventions.

Dual-lipid metabolomics revealed the communication of MTB or MB with Bovine alveolar macrophages in lipid metabolisms

M. tuberculosis(MTB) and M. bovis(MB) of the Mycobacterium tuberculosis complex (MTBC) are the causative agents of the notorious infectious disease tuberculosis(TB) in a range of mammals, including cattle and human. The lipid composition of MTB/MB performed imperative function as invading host macrophage. In the present study, a dual-lipid metabolomics were used to elucidate the differences in lipid composition of MTB and MB and the different responses in lipid metabolisms of bovine alveolar macrophage challenged by MTB/MB. The lipid metabolomics of MTB and MB indicated that there were significant differences in lipid composition of both bacteria that the level of various lipids belonged to Glycerophospholipids, Sterol Lipids, Fatty Acyls and Polyketides exhibited differences between MTB and MB. Meanwhile, both MTB and MB with different lipid composition could invoked different responses in lipid metabolisms of the host macrophage. MTB infection mainly induced the increase in content of Polyketides and Glycerophospholipids in macrophages, whereas MB infection induced the level of Glycerophospholipids and Sterol Lipids of macrophages. Furthermore, we identified TAG 13:0-18:5-18:5 of MTB and PC(16:1(9E)/0:0), PI(20:2(11Z,14Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), 4,6-Decadiyn-1-ol isovalerate and LacCer(d18:1/24:1(15Z)) of MB caused the different variations in lipid metabolisms of macrophage following MTB/MB attacks, respectively. Finally, we proposed MTB and MB with different lipid compositions could successfully colonize in macrophage by different mechanisms that MTB could promote the formation of foam cells of macrophage for its colonization and development, while MB mainly through suppressing the macrophage autophagy to escape the immune responses of host.

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.