Development of the WT, Cas9 and BTN1 KO strains (labeled KO) in sand flies.

<p>A, Intensity of infection was assayed on days 2–3 and 7–8 p.i. and defined as weak (less than 100 promastigotes), moderate (100–1,000 promastigotes), or heavy (over 1,000 promastigotes), depending on the number of parasites per gut. Data are summarized from five independent biological replicates, numbers above each bar indicate the total number of dissected females. B, Quantitative PCR analysis of the <i>L</i>. <i>mexicana</i> load in the insect gut 7–8 days p.i. Boxplots are from five independent biological replicates and show 1st quartile, median, 3rd quartile, and 1.5× interquartile range values. C, Localization of parasites in sand fly gut 7–8 days p.i. (SV, stomodeal valve; TH,ABM, both thoracic and abdominal midgut; ABM, abdominal midgut). Numbers above each bar indicate the number of dissected females. D, Morphological analysis of <i>Leishmania mexicana</i> cells from thoracic midgut and stomodeal valve of infected sand fly females 7–8 days p.i. (LN, long nectomonade; SN, short nectomonade; ME, metacyclic promastigote).</p

Ablation of <i>LmxM</i>.<i>22</i>.<i>0010</i> by conventional approach.

<p>A, Schematic representation of the WT and recombined alleles after replacement with Sat-, Hyg-, Neo-, and Ble-resistant genes. Annealing positions of the probes and expected fragment sizes are shown. B, Southern blot analysis of the <i>Bst</i>E II digested <i>L</i>. <i>mexicana</i> genomic DNA of the WT and BTN1 ablated strains with Sat, Hyg, Neo, Ble, 5' UTR, and <i>LmxM</i>.<i>22</i>.<i>0010</i> ORF probes.</p

Ablation of <i>LmxM</i>.<i>22</i>.<i>0010</i> by CRISPR-Cas9.

<p>A, Schematic representation of the WT and recombined alleles after replacement with Puro resistant gene. Annealing positions of the probes and expected fragment sizes are shown. B, Southern blot analysis of the <i>Bst</i>E II digested <i>L</i>. <i>mexicana</i> genomic DNA of the WT, Cas9, and BTN1 ablated strains (labeled KO) with <i>LmxM</i>.<i>22</i>.<i>0010</i> 5' UTR, <i>LmxM</i>.<i>22</i>.<i>0010</i> 3' UTR and Puro probes.</p

Experimental infection of <i>Lutzomyia longipalpis</i> with WT and ALD1 KO <i>Leishmania mexicana</i>.

<p>A, Intensity of infection was assayed on days 1–2 and 7 p.i. and defined as light (less than 100 promastigotes, white bar), moderate (100–1,000 promastigotes, grey bar), or heavy (over 1,000 promastigotes, black bar) depending on the number of parasites per gut. Numbers above each bar indicate the number of dissected females. B, Localization of <i>L</i>. <i>mexicana</i> in the insect gut 7 d.p.i. ABM, abdominal midgut; TH, thoracic midgut, SV, stomodeal valve. Numbers above each bar indicate the number of dissected females. C, Quantitative PCR analysis of the <i>L</i>. <i>mexicana</i> load in the insect gut 7 d.p.i. Boxplots in C are from three independent biological replicates and show 1st quartile, median, 3rd quartile, and 1.5 x interquartile range values. T-test statistical value is symbolized by asterisks: **** (≤ 0.0001).</p

A putative ATP/GTP binding protein affects <i>Leishmania mexicana</i> growth in insect vectors and vertebrate hosts

<div><p>Background</p><p><i>Leishmania</i> virulence factors responsible for the complicated epidemiology of the various leishmaniases remain mainly unidentified. This study is a characterization of a gene previously identified as upregulated in two of three overlapping datasets containing putative factors important for <i>Leishmania</i>’s ability to establish mammalian intracellular infection and to colonize the gut of an insect vector.</p><p>Methodology/Principal findings</p><p>The investigated gene encodes <u><b>A</b></u>TP/GTP binding motif-containing protein related to <u><b><i>L</i></b></u><i>eishmania</i> <u><b>d</b></u>evelopment <u><b>1</b></u> (ALD1), a cytosolic protein that contains a cryptic ATP/GTP binding P-loop. We compared differentiation, growth rates, and infective abilities of wild-type and ALD1 null mutant cell lines of <i>L</i>. <i>mexicana</i>. Loss of ALD1 results in retarded growth kinetics but not defects in differentiation in axenic culture. Similarly, when mice and the sand fly vector were infected with the ALD1 null mutant, the primary difference in infection and colonization phenotype relative to wild type was an inability to achieve maximal host pathogenicity. While ability of the ALD1 null mutant cells to infect macrophages <i>in vitro</i> was not affected, replication within macrophages was clearly curtailed.</p><p>Conclusions/Significance</p><p><i>L</i>. <i>mexicana</i> ALD1, encoding a protein with no assigned functional domains or motifs, was identified utilizing multiple comparative analyses with the related and often experimentally overlooked monoxenous flagellates. We found that it plays a role in <i>Leishmania</i> infection and colonization <i>in vitro</i> and <i>in vivo</i>. Results suggest that ALD1 functions in <i>L</i>. <i>mexicana</i>’s general metabolic network, rather than function in specific aspect of virulence as anticipated from the compared datasets. This result validates our comparative genomics approach for finding relevant factors, yet highlights the importance of quality laboratory-based analysis of genes tagged by these methods.</p></div

Ablation of the <i>LmxM</i>.<i>30</i>.<i>2090</i> in <i>L</i>. <i>mexicana</i>.

<p>A, Schematic representation of the WT and recombined alleles. Annealing positions of the probes and expected fragment sizes are shown. B, Southern blot analysis of the <i>Nco</i> I digested <i>L</i>. <i>mexicana</i> genomic DNA of the WT and ALD1 KO strains with Sat, Hyg, 5' UTR, 3' UTR, and <i>LmxM</i>.<i>30</i>.<i>2090</i> ORF probes.</p

ALD1 primary structure and localization.

<p>A, Sequence alignment of the <i>L</i>. <i>mexicana</i> ALD1 protein and its orthologs found in the subfamily Leishmaniinae. All the columns containing insertions relative to the <i>L</i>. <i>mexicana</i> protein sequence are hidden and the sites of such insertions are shown with black vertical lines. The Walker motifs A (aa 113–118) and B (aa 250–260) are highlighted in grey; crucial residue in the P-loop represented by K117 of the motif A is shown in dark grey. The numbers on top correspond to the <i>L</i>. <i>mexicana</i> protein. <i>Lmex</i> = <i>Leishmania mexicana</i>; <i>Ldon</i> = <i>Leishmania donovani</i>; <i>Linf</i> = <i>Leishmania infantum</i>; <i>Lmaj</i> = <i>Leishmania major</i>; <i>Lpyr</i> = <i>Leptomonas pyrrhocoris</i>; <i>Lsey</i> = <i>Leptomonas seymouri</i>; <i>Cfas</i> = <i>Crithidia fasciculata</i>. B, Cytoplasmic localization of HA-tagged ALD1 analyzed by confocal immunofluorescence microscopy with anti-HA antibodies and Alexa Fluor 488—conjugated secondary antibodies (green). The nuclear and kinetoplast DNAs were stained with DAPI (blue), and mitochondria were stained with MitoTracker Red CMXRos (red). Scale bar is 5 μm.</p

Growth kinetics and differentiation of <i>Leishmania</i> strains <i>in vitro</i>.

<p>A, Growth curves of WT and ALD1 KO <i>L</i>. <i>mexicana</i>. B, C, D, quantification by RT-qPCR of <i>Pfr1D</i>, <i>Sherp</i>, and <i>Amastin</i> gene expression used as markers for promastigotes (both pro- and metacyclics), metacyclics, and amastigotes, respectively. Data are from four independent biological replicates. The error bars indicate standard deviations. <i>LmxM</i>.<i>07</i>.<i>0510</i> was used for normalization. T-test statistical values are symbolized by asterisks: * (≤ 0.05), ** (≤ 0.01), or N.S. (not statistically significant).</p

Macrophage infection <i>in vitro</i>.

<p>The percentage of infected macrophages and the average number of parasite per infected cell were calculated for WT and KO <i>L</i>. <i>mexicana</i> infecting non-stimulated (A) or LPS/IFN-γ stimulated (B) primary murine macrophages. Data are summarized from four independent biological replicates (400 cells per condition). The error bars indicate standard deviations. T-test statistical values are symbolized by asterisks: * (≤ 0.05), ** (≤ 0.01), *** (≤ 0.001), or N.S. (not statistically significant). N/A, not available.</p