An arginine deprivation response pathway is induced in Leishmania during macrophage invasion

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

Potential members of the ADR pathway.

<p>Potential members of the ADR pathway.</p

Time course of Arginine Deprivation Response (ADR).

<p>A) Time-course of LdAAP3 protein abundance increase as function of arginine deprivation time in wild type (left four lanes, lower bands) and <i>Δldaap24</i> (right four lanes, lower bands) <i>L</i>. <i>donovani</i> promastigotes. Mid-log phase parasites were suspended at a density of 0.5–1×10⁷ cells/ml in medium 199 without arginine and further incubated at 26°C. Aliquots were collected at 0, 10, 30 and 60 minutes, cellular proteins separated on 9% SDS-PAGE, transferred to nitrocellulose paper and subsequently probed with anti LdAAP3 antiserum. B) Rate of arginine pool depletion following arginine deprivation of <i>L</i>. <i>donovani</i> promastigotes wild type and <i>Δldaap24</i>. Aliquots of wild type (●) and <i>Δldaap24</i> (■) collected in (A) were subjected to amino acid analyses as described in Materials and Methods. Values are mean ± S.D. (n = 3). Inset is a table that indicates intracellular arginine concentration values at zero and 60 minutes. C) Northern analysis of <i>LdAAP3</i> mRNA abundance change during first 30 minutes of arginine deprivation. As a probe we used LdAAP3.2 (LinJ.31.0910). D) Arginine (0.45 mM) added to promastigotes after 2 hours of arginine deprivation induced rapid degradation of LdAAP3. Degradation was inhibited by the addition of 1 mM MG132 (Last two right lanes).</p

Arginine deprivation-induced protein phosphorylation in <i>L</i>. <i>donovani</i> promastigotes.

<p>Arginine deprivation-induced protein phosphorylation in <i>L</i>. <i>donovani</i> promastigotes.</p

LdAAP3 expression increases during macrophage infection.

<p>LdAAP3.2 mRNA (LinJ.31.0910) abundance in axenic <i>L</i>. <i>donovani</i> promastigotes and amastigotes, before and after 2 hours arginine deprivation (indicated as "axenic culture") and THP1-derived amastigotes (indicated as "intracellular amastigotes"). Total RNA was extracted from axenic parasites and <i>L</i>. <i>donovani</i> infected-THP1 macrophages 48 hours after infection. THP1 macrophages were infected with late-log phase <i>L</i>. <i>donovani</i> promastigotes at a ratio of 10:1 parasites per macrophage. 48 hours after infection macrophages (30% infection, a mean of 3 parasites per macrophage) were collected and total RNA was extracted as described in Materials and Methods. <i>LdAAP3</i>.<i>2</i> was used as probe. Band abundance relative to mRNA of non-deprived promastigotes was calculated using TINA algorithm and are illustrated in the graphs below the gel.</p

LdAAP3 localizes to both flagella surface and glycosome membranes.

<p>A) Indirect immunofluorescence of LdAAP3. Axenic <i>L</i>. <i>donovani</i> promastigotes were stained using anti LdAAP3 IgG (red) and the DNA stain DAPI (blue). The latter stained the nucleus and kinetoplast. The two immunofluorescence images were merged. Immunofluorescence analysis was performed using the inverted cell observer Zeiss Axiovert 2. B) Subcellular distribution of LdAAP3. Axenic <i>L</i>. <i>donovani</i> promastigotes before and after 4 hours of arginine deprivation were extracted, non-soluble membranes were separated and subsequently subjected to sucrose gradient (20–60%) as described in Materials and Methods. Aliquots of plasma and glycosome membrane fractions before and after arginine deprivation were subjected to western blot analysis. Anti proline/alanine transporter (LdAAP24) and PEX14 were used as plasma and glycosome membrane markers, respectively. LdAAP3 molecular mass is 46 kDa. Note that length of exposure to peroxidase chemiluminescense was calibrated to starved cells. Bands in non-starved cells were visible after longer exposure (not shown).</p