Sphingolipid Degradation in <em>Leishmania (Leishmania) amazonensis</em>

<div><h3>Background</h3><p>Human leishmaniasis is caused by more than 20 <em>Leishmania</em> species and has a wide range of symptoms. Our recent studies have demonstrated the essential role of sphingolipid degradation in the virulence of <em>Leishmania (Leishmania) major</em>, a species responsible for localized cutaneous leishmaniasis in the Old World. In this study, we investigated the function of sphingolipid degradation in <em>Leishmania (Leishmania) amazonensis</em>, an etiological agent of localized and diffuse cutaneous leishmaniasis in South America.</p> <h3>Methodology/Principal Findings</h3><p>First, we identified the enzyme LaISCL which is responsible for sphingolipid degradation in <em>L. amazonensis</em>. Primarily localized in the mitochondrion, LaISCL shows increased expression as promastigotes progress from replicative log phase to non-replicative stationary phase. To study its function, null mutants of LaISCL (<em>Laiscl⁻</em>) were generated by targeted gene deletion and complemented through episomal gene add-back. In culture, loss of LaISCL leads to hypersensitivity to acidic pH and poor survival in murine macrophages. In animals, <em>Laiscl⁻</em> mutants exhibit severely attenuated virulence towards C57BL6 mice but are fully infective towards BALB/c mice. This is drastically different from wild type <em>L. amazonensis</em> which cause severe pathology in both BALB/c and C57BL 6 mice.</p> <h3>Conclusions/Significance</h3><p>A single enzyme LaISCL is responsible for the turnover of sphingolipids in <em>L. amazonensis</em>. LaISCL exhibits similar expression profile and biochemical property as its ortholog in <em>L. major</em>. Deletion of LaISCL reduces the virulence of <em>L. amazonensis</em> and the outcome of <em>Laiscl⁻</em>-infection is highly dependent on the host's genetic background. Therefore, compared to <em>L. major</em>, the role of sphingolipid degradation in virulence is substantially different in <em>L. amazonensis</em>. Future studies may reveal whether sphingolipid degradation is required for <em>L. amazonensis</em> to cause diffuse cutaneous infections in humans.</p> </div

Functional analysis of ISCL by mutagenesis.

<p>(<b>A</b>) Schematic diagram of ISCL open reading frame and the mutations introduced in this study. Asterisks represent the three aspartic acids (D116, D200, and D383) that were mutated; grey bars represent the region recognized by the anti-ISCL peptide antibody; and black bars represent the transmembrane helices. (<b>B</b>) Western-blot to confirm the expression of mutated ISCL. Whole cell lysates from WT, <i>iscl⁻</i>, <i>iscl⁻</i>/+ISCL, <i>iscl⁻</i>/+ISCL D116G, <i>iscl⁻</i>/+ISCL D200G, <i>iscl⁻</i>/+ISCL D383G, <i>iscl⁻</i>/+ISCLΔ and <i>iscl⁻</i>/+pXG (empty vector) promastigotes were probed with either anti-ISCL (top) or anti-α-tubulin (bottom) antibody. Each lane contained material from 6×10⁵ cells. (<b>C–D</b>) The SMase and IPCase activities in log phase promastigotes were determined as described in <b><i>Materials and Method</i></b>. Error bars represent standard deviations from 3 independent experiments.</p

Cytokine production in <i>Laiscl</i>⁻-infected mice.

<p>C57BL6 mice or BALB/c mice were infected in the footpads and sacrificed after 10 weeks. Lymphocytes (dLNs) were isolated and plated on 24-well dishes. After SLA stimulation for 3 days, culture supernatants were collected to measure the level of IFN-γ (<b>A</b>), IL-4 (<b>B</b>), and IL-10 (<b>C</b>). Ratios of SLA-stimulated/un-stimulated were calculated for each cytokine (<b>A</b>–<b>C</b>). Ratios of IL-4/IFN-γ and IL-10/IFN-γ (both from SLA-treated samples) were also calculated (<b>D</b>–<b>E</b>). Error bars represent standard deviations from 3 replicates (*: <i>p</i><0.05).</p

Increased expression of LaISCL in the infective stages of <i>L. amazonensis</i>.

<p>(<b>A</b>) Promastigote lysates from <i>La</i> WT (Log: log phase; S1–S4: day 1–4 in stationary phase; Meta: metacyclics), <i>Laiscl⁻</i> (log phase), and <i>Laiscl⁻/+LaISCL</i> (log phase) were analyzed by western blot using the anti-LmISCL antibody (top) or anti-α-tubulin antibody (bottom). (<b>B</b>) Immunoblot of cell lysates from log phase <i>La</i> WT promastigotes (Pro) and lesion-derived amastigotes (Ama). Relative intensities of ISCL and tubulin bands were determined using a FluoroChem E imager and shown below the blots. Each lane contained material from 5×10⁵ cells.</p

Introduction of a sole SMase or sole IPCase into <i>iscl⁻</i>.

<p>Whole cell lysates from late log phase promastigotes of WT, <i>iscl⁻</i>, <i>iscl⁻</i>/+HA-ISCL, <i>iscl⁻</i>/+HA-BcSMase, <i>iscl⁻</i>/+5′HASPb-BcSMase-HA, and <i>iscl⁻</i>/+CnISC1 parasites were incubated with NBD-labeled SM (<b>A</b> and <b>C</b>) or NBD-labeled IPC (<b>B</b> and <b>D</b>) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031059#s4" target="_blank">Materials and Methods</a>. Each reaction contained ∼40 µg of total protein corresponding to 4×10⁶ cells. (<b>A–B</b>) After incubation, lipids were extracted and separated by thin layer chromatography (TLC). Ceramide (Cer), a product of SMase and IPCase, migrates faster than sphingomyelin (SM in <b>A</b>) or IPC (<b>B</b>). O: origin of migration. Positive control (+): 0.1 unit of purified <i>Bc</i>SMase (<b>A</b>) or 0.1 unit of purified <i>Bc</i>PI-PLC (<b>B</b>). Negative control (−): boiled WT lysate. (<b>C–D</b>) Activity of SMase (<b>C</b>) or IPCase (<b>D</b>) in <i>Leishmania</i> cell lysates was quantified after TLC analysis based on the amount ceramide produced and the amount of protein in each reaction. Error bars represent standard deviations from 3 independent experiments.</p

The SMase activity alone was sufficient to restore virulence in <i>iscl⁻</i>.

<p>(<b>A–B</b>) Bone marrow-derived Mφs from BALB/c mice were infected by stationary phase promastigotes (•: WT, ○: <i>iscl⁻</i>, ▾: <i>iscl⁻</i>/+HA-ISCL, ▪: <i>iscl⁻</i>/+CnISC1, □: <i>iscl⁻</i>/+HA-BcSMase, ⧫: <i>iscl⁻</i>/+5′HASPb-BcSMase-HA, ▵: WT+activated Mφs) at a ratio of 15 parasites per Mφ. Percentages of infected Mφs (<b>A</b>) and the number of parasites in 100 Mφs (<b>B</b>) were recorded. (<b>C–D</b>) BALB/c mice were infected in the footpads with day 3 stationary promastigotes (•: WT, ○: <i>iscl⁻</i>, ▾: <i>iscl⁻</i>/+HA-ISCL, ▵: <i>iscl⁻</i>/+HA-BcSMase, ▪: <i>iscl⁻</i>/+5′HASPb-BcSMase-HA, □: <i>iscl⁻</i>/+CnISC1) at 1×10⁶ parasites per mouse (5 mice per group). Lesion sizes were measured with a caliper (<b>C</b>) and parasite numbers in infected footpads were determined by the limiting dilution assay (<b>D</b>). Error bars represent standard deviations from 2 independent experiments.</p

Replacement of <i>LaISCL</i> alleles by antibiotic resistance marker genes.

<p>Genomic DNAs from <i>La</i> WT, <i>LaISCL+/−</i> (<i>▵LaISCL::PAC/LaISCL</i>), and <i>Laiscl⁻</i> (<i>▵LaISCL::PAC/▵LaISCL::BSD</i>; clone #1 and #2) were digested and probed with a radioactive probe that recognized a 550-bp downstream region of <i>LaISCL</i>.</p

Localization of LaISCL protein in promastigotes.

<p>Day 1 stationary phase promastigotes of <i>La</i> WT (left column), <i>Laiscl</i>⁻ (middle column), and <i>Laiscl</i>⁻/+<i>LaISCL</i> (right column) were analyzed by immunofluorescence microscopy. (<b>A</b>): differential interference contrast images; (<b>B</b>): DNA staining using Hoechst 33242; (<b>C</b>): immuno-staining with rabbit anti-LmISCL antibody, followed by goat-anti-rabbit IgG-FITC; (<b>D</b>): labeling with Mitotracker Red 580; (<b>E</b>): merge of <b>C</b> and <b>D</b>.</p

Temporal and spatial expression of ISCL in <i>L. major</i>.

<p>(<b>A</b>) Elevated ISCL transcript level in stationary phase promastigotes. <i>L. major</i> WT promastigotes were cultured in M199 medium at a starting density of 1.0×10⁵ cells/ml and total RNA was extracted daily. The relative abundance of ISCL mRNA was determined by quantative RT-PCR using the constitutively expressed rRNA45 gene as an internal control. EL: early log phase culture (1–2×10⁶ cells/ml); LL: late log phase culture (6–10×10⁶ cells/ml); S1–S4: day 1–4 stationary phase culture (2.0–3.0×10⁷ cells/ml). *: <i>p</i><0.05, **: <i>p</i><0.01. Error bars represent standard deviations from two independent experiments. (<b>B</b>) Increased expression of ISCL protein in stationary phase promastigotes and lesion amastigotes. Left: whole cell lysates from WT late log phase promastigotes (LL), day 1–4 stationary phase promastigotes (S1–S4), and late log phase promastigotes of <i>iscl⁻</i> and <i>iscl⁻/+ISCL</i> were subjected to immunoblot analysis using the affinity purified anti-ISCL antibody (top) or an anti-α-tubulin antibody as loading control (bottom). Right: immunoblot of cell lysates from WT day 3 stationary phase promastigotes (S3 pro.) and lesion amastigotes (ama.). Each lane contained material from 1×10⁶ cells. (<b>C–J</b>) Localization of endogenous ISCL in <i>L. major</i>. WT promastigotes (<b>C–F</b>) and amastigotes (<b>G–J</b>) were fixed and permeabilized with ethanol as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031059#s4" target="_blank"><b><i>Materials and Methods</i></b></a>. Cells were then labeled with rabbit anti-ISCL antibody, followed by staining with goat-anti-rabbit IgG-FITC (<b>D</b>, <b>H</b>) and Mitotracker Red 580 (<b>E</b>, <b>I</b>). (<b>F</b>) Overlay of <b>D</b> and <b>E</b>. (<b>J</b>) Overlay of <b>H</b> and <b>I</b>. Control cells that were labeled with secondary antibody only did not show any green fluorescence (data not shown). Experiments in <b>C–F</b> were performed three times and results from one representative set are shown here.</p

LaISCL is required for the hydrolysis of sphingomyelin and IPC in <i>L. amazonensis</i>.

<p>Promastigote lysates were incubated with TX100-based micelles containing either NBD-SM (<b>A–B</b>) or NBD-IPC (<b>C–D</b>) as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001944#s2" target="_blank">Methods</a>. Lipids were then extracted and separated on TLC plates (<b>A</b> and <b>C</b>). The activity of SMase (<b>B</b>) or IPCase (<b>D</b>) was calculated based on the amount of ceramide produced and the amount of protein in each sample. 0.1 unit of <i>B. cereus</i> SMase (<b>A</b>) and <i>B. cereus</i> PI-PLC (<b>C</b>) were used as positive controls. Boiled <i>La</i> WT cell lysate was used as negative controls (-).</p