An Arginine Deprivation Response Pathway Is Induced in Leishmania during Macrophage Invasion

Goldman-Pinkovich, Adele; Balno, Caitlin; Strasser, Richard; Michal, Zeituni-Molad; Keren, Bendelak; Rentsch, Doris; Ephros, Moshe; Wiese, Martin; Jardim, Armando; Myler, Peter J.; Zilberstein, Dan

doi:10.1371/journal.ppat.1005494

Cited by 76 publications

(105 citation statements)

References 55 publications

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“…3B, modules 20 and 126; Data Set S1, NP versus PP). aATP11 and AAP3 were previously associated with a response to purine and amino acid starvations, respectively (36–38), reinforcing the suggestion that the NP stage is associated with one or a number of stress conditions.…”

Section: Resultsmentioning

confidence: 58%

The Transcriptome ofLeishmania majorDevelopmental Stages in Their Natural Sand Fly Vector

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Hughitt

Dillon

et al. 2017

mBio

144

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The life cycle of the Leishmania parasite in the sand fly vector involves differentiation into several distinctive forms, each thought to represent an adaptation to specific microenvironments in the midgut of the fly. Based on transcriptome sequencing (RNA-Seq) results, we describe the first high-resolution analysis of the transcriptome dynamics of four distinct stages of Leishmania major as they develop in a natural vector, Phlebotomus duboscqi. The early transformation from tissue amastigotes to procyclic promastigotes in the blood-fed midgut was accompanied by the greatest number of differentially expressed genes, including the downregulation of amastins, and upregulation of multiple cell surface proteins, sugar and amino acid transporters, and genes related to glucose metabolism and cell cycle progression. The global changes accompanying post-blood meal differentiation of procyclic promastigotes to the nectomonad and metacyclic stages were less extensive, though each displayed a unique signature. The transcriptome of nectomonads, which has not been studied previously, revealed changes consistent with cell cycle arrest and the upregulation of genes associated with starvation and stress, including autophagic pathways of protein recycling. Maturation to the infective, metacyclic stage was accompanied by changes suggesting preadaptation to the intracellular environment of the mammalian host, demonstrated by the amastigote-like profiles of surface proteins and metabolism-related genes. Finally, a direct comparison between sand fly-derived and culture-derived metacyclics revealed a reassuring similarity between the two forms, with the in vivo forms distinguished mainly by a stronger upregulation of transcripts associated with nutrient stress.

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Section: Resultsmentioning

confidence: 58%

The Transcriptome ofLeishmania majorDevelopmental Stages in Their Natural Sand Fly Vector

Inbar

Hughitt

Dillon

et al. 2017

mBio

144

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“…Western blot analysis was done as described previously (Darlyuk et al, 2009) using a 1:1000 dilution of rabbit anti AAP3 N-terminus antisera (Goldman-Pinkovich et al, 2016).…”

Section: Western Blot Analysesmentioning

confidence: 99%

“…Uptake of 25 μM L-[ 3 H] arginine (600 mCi/mmol) into L. donovani promastigotes was determined using the rapid filtration technique described previously (Goldman-Pinkovich et al, 2016). Briefly, transport reaction mixture contained 1×10 8 cells/ml suspended in Earle's balanced salt solution at pH 7 supplemented with 5 mM glucose and 1% dialyzed heatinactivated fetal calf.…”

Section: Transport Assaysmentioning

confidence: 99%

“…Recently, our laboratory discovered that an MAPK2-mediated Arginine Deprivation Response (ADR) pathway is triggered immediately upon arginine starvation of the cultured extracellular insect-form (promastigote) Leishmania, resulting in up-regulation of arginine transporter (AAP3) mRNA and protein levels, with a concomitant increase in arginine transport efficiency (Goldman-Pinkovich et al, 2016). Significantly, the ADR is also activated during Leishmania infection of macrophages (Goldman-Pinkovich et al, 2016) , (Pawar et al, 2019), suggesting a role for intra-lysosomal arginine sensing in intracellular (amastigote) parasite survival and pathogenesis. In this study, we sought to…”

Section: Introductionmentioning

confidence: 99%

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The hunger games: sensing host arginine is essential for Leishmania parasite virulence

Goldman-Pinkovich

Kannan

Nitzan-Koren

et al. 2019

Preprint

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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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“…It has been suggested that LdAAP3 contributes to parasite virulence by increased arginine uptake [6]. The arginine-deprivation response resulting in LdAAP3 up-regulation was recently shown to be mediated through a MAPK2-dependent signaling pathway [7]. While arginine is important for polyamine biosynthesis in Leishmania , the absence of a functional arginase homolog and the essentiality of ornithine decarboxylase (ODC) in T .…”

Section: Introductionmentioning

confidence: 99%

Arginine and Lysine Transporters Are Essential for Trypanosoma brucei

et al. 2017

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For Trypanosoma brucei arginine and lysine are essential amino acids and therefore have to be imported from the host. Heterologous expression in Saccharomyces cerevisiae mutants identified cationic amino acid transporters among members of the T. brucei AAAP (amino acid/auxin permease) family. TbAAT5-3 showed high affinity arginine uptake (Km 3.6 ± 0.4 μM) and high selectivity for L-arginine. L-arginine transport was reduced by a 10-times excess of L-arginine, homo-arginine, canavanine or arginine-β-naphthylamide, while lysine was inhibitory only at 100-times excess, and histidine or ornithine did not reduce arginine uptake rates significantly. TbAAT16-1 is a high affinity (Km 4.3 ± 0.5 μM) and highly selective L-lysine transporter and of the compounds tested, only L-lysine and thialysine were competing for L-lysine uptake. TbAAT5-3 and TbAAT16-1 are expressed in both procyclic and bloodstream form T. brucei and cMyc-tagged proteins indicate localization at the plasma membrane. RNAi-mediated down-regulation of TbAAT5 and TbAAT16 in bloodstream form trypanosomes resulted in growth arrest, demonstrating that TbAAT5-mediated arginine and TbAAT16-mediated lysine transport are essential for T. brucei. Growth of induced RNAi lines could partially be rescued by supplementing a surplus of arginine or lysine, respectively, while addition of both amino acids was less efficient. Single and double RNAi lines indicate that additional low affinity uptake systems for arginine and lysine are present in T. brucei.

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An Arginine Deprivation Response Pathway Is Induced in Leishmania during Macrophage Invasion

Cited by 76 publications

References 55 publications

The Transcriptome ofLeishmania majorDevelopmental Stages in Their Natural Sand Fly Vector

The Transcriptome ofLeishmania majorDevelopmental Stages in Their Natural Sand Fly Vector

The hunger games: sensing host arginine is essential for Leishmania parasite virulence

Arginine and Lysine Transporters Are Essential for Trypanosoma brucei

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