Leishmania amazonensis Arginase Compartmentalization in the Glycosome Is Important for Parasite Infectivity

Abstract In Leishmania, de novo polyamine synthesis is initiated by the cleavage of L-arginine to urea and L-ornithine by the action of arginase (ARG, E.C. 3.5.3.1). Previous studies in L. major and L. mexicana showed that ARG is essential for in vitro growth in the absence of polyamines and needed for full infectivity in animal infections. The ARG protein is normally found within the parasite glycosome, and here we examined whether this localization is required for survival and infectivity. First, the localization of L. amazonensis ARG in the glycosome was confirmed in both the promastigote and amastigote stages. As in other species, arg 2 L. amazonensis required putrescine for growth and presented an attenuated infectivity. Restoration of a wild type ARG to the arg 2 mutant restored ARG expression, growth and infectivity. In contrast, restoration of a cytosoltargeted ARG lacking the glycosomal SKL targeting sequence (argDSKL) restored growth but failed to restore infectivity. Further study showed that the ARGDSKL protein was found in the cytosol as expected, but at very low levels. Our results indicate that the proper compartmentalization of L. amazonensis arginase in the glycosome is important for enzyme activity and optimal infectivity. Our conjecture is that parasite arginase participates in a complex equilibrium that defines the fate of L-arginine and that its proper subcellular location may be essential for this physiological orchestration

Leishmania amazonensis Arginase Compartmentalization in the Glycosome Is Important for Parasite Infectivity

In Leishmania, de novo polyamine synthesis is initiated by the cleavage of L-arginine to urea and L-ornithine by the action of arginase (ARG, E.C. 3.5.3.1). Previous studies in L. major and L. mexicana showed that ARG is essential for in vitro growth in the absence of polyamines and needed for full infectivity in animal infections. The ARG protein is normally found within the parasite glycosome, and here we examined whether this localization is required for survival and infectivity. First, the localization of L. amazonensis ARG in the glycosome was confirmed in both the promastigote and amastigote stages. As in other species, arg− L. amazonensis required putrescine for growth and presented an attenuated infectivity. Restoration of a wild type ARG to the arg− mutant restored ARG expression, growth and infectivity. In contrast, restoration of a cytosol-targeted ARG lacking the glycosomal SKL targeting sequence (argΔSKL) restored growth but failed to restore infectivity. Further study showed that the ARGΔSKL protein was found in the cytosol as expected, but at very low levels. Our results indicate that the proper compartmentalization of L. amazonensis arginase in the glycosome is important for enzyme activity and optimal infectivity. Our conjecture is that parasite arginase participates in a complex equilibrium that defines the fate of L-arginine and that its proper subcellular location may be essential for this physiological orchestration

Responsible AI governance: A response to UN interim report on governing AI for humanity

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International genome-wide meta-analysis identifies new primary biliary cirrhosis risk loci and targetable pathogenic pathways.

Primary biliary cirrhosis (PBC) is a classical autoimmune liver disease for which effective immunomodulatory therapy is lacking. Here we perform meta-analyses of discovery data sets from genome-wide association studies of European subjects (n=2,764 cases and 10,475 controls) followed by validation genotyping in an independent cohort (n=3,716 cases and 4,261 controls). We discover and validate six previously unknown risk loci for PBC (Pcombined<5 × 10(-8)) and used pathway analysis to identify JAK-STAT/IL12/IL27 signalling and cytokine-cytokine pathways, for which relevant therapies exist

ARG cytosolic localization in <i>arg</i>⁻/+<i>arg</i>ΔSKL add-back opposed to ARG glycosomal compartmentalization in WT and <i>arg</i>⁻/+<i>ARG</i> add-back.

<p>Cytological preparations probed for ARG immunolocalization via electron microscopy of <i>L. amazonensis</i> wild type (WT), <i>ARG</i> knockout (<i>arg</i>⁻), and the add-backs <i>arg</i>⁻/+<i>ARG</i> and <i>arg</i>⁻/+<i>arg</i>ΔSKL promastigotes. Images are representative of at least 8 different sections. Black arrows indicate membrane-surrounded organelles that present the same microscopic pattern as glycosomes. Parasite nuclei (N). Scale bar: 250 nm.</p

<i>L. amazonensis</i> ARG protein level and enzymatic activity are impaired by arginase mislocation.

<p>(A) <i>ARG</i> mRNA copies were determined by real time PCR and normalized by <i>GAPDH</i> mRNA copies for <i>L. amazonensis</i> wild type (WT), <i>ARG</i> knockout (<i>arg</i>⁻), and the add-backs <i>arg</i>⁻/+<i>ARG</i> and <i>arg</i>⁻/+<i>arg</i>ΔSKL strains. The obtained values are the means (+/− SD) of at least 2 independent experiments each performed in duplicate. The values for <i>arg</i>⁻/+<i>ARG</i> and <i>arg</i>⁻/+<i>arg</i>ΔSKL are significantly different from those for <i>arg</i>⁻ and WT (p<0.0015*, t test). (B) ARG enzymatic activity from protein extracts of the same strains was determined and is expressed as nmol/min/mg. The obtained values are the means (+/− SD) of 3 independent experiments each performed in triplicate. The values for <i>arg</i>⁻/+<i>ARG</i> are significantly different from those for <i>arg</i>⁻ and <i>arg</i>⁻/+<i>arg</i>ΔSKL (p<0.0001**, t test). (C) ARG and Orc1 (loading control) levels were determined by western blotting of total cell lysates from the same strains before and after treatment with MG-132 50 µM for 24 hours.</p

ARG absence or improper sub-cellular location impairs <i>L. amazonensis in vitro</i> infectivity.

<p>Peritoneal macrophages infected with <i>L. amazonensis</i> wild type (WT, white bars), <i>ARG</i> knockout (<i>arg</i>⁻, black bars) and the add-backs <i>arg</i>⁻/+<i>ARG</i> (grey bars) and <i>arg</i>⁻/+<i>arg</i>ΔSKL (dashed bars) mutants for 48 and 72 hours. (A) Means (+/− SD) of the rate of macrophage infection from two experiments. (B) Means (+/− SD) of amastigotes per infected macrophage from two experiments. At least 150 macrophages were analyzed for each experiment. ^# indicates values with no significant difference (One-way ANOVA). * p<0.005 and ** p<0.05 (t test).</p

L-arginine cellular concentration is increased in <i>arg</i>⁻ and <i>arg</i>⁻/+<i>arg</i>ΔSKL compared with WT and <i>arg</i>⁻/+<i>ARG</i>.

<p>L-arginine cellular concentration in <i>L. amazonensis</i> wild type (WT), <i>ARG</i> knockout (<i>arg</i>⁻) and the add-backs <i>arg</i>⁻/+<i>ARG</i> and <i>arg</i>⁻/+<i>arg</i>ΔSKL, determined by HPLC analyses of cellular extracts from 10⁷ parasites. Values are the means (+/−SD) of a representative experiment performed in triplicate.</p>*<p>indicates values significantly different compared with WT (p<0.05, t test).</p>§ and #<p>indicates values with no significant difference compared with WT and <i>arg</i>⁻, respectively.</p