Amino acid sequences and organization of the <i>Leishmania donovani</i> complex HASPB repeat region.

<p>Panel A. Comparison of peptide repeats motif sequences found in <i>L. infantum</i> and <i>L. donovani</i>. Panel B. Bar code of peptide motif organization for the k26 repeat region of HASPB in <i>Leishmania donovani</i> complex from different geographic regions of the Old World. Peptides A (bright yellow) −14 amino acid peptides found both in <i>L. donovani</i> and/or <i>L. infantum</i>; Peptides a (banana yellow) −14 amino acid peptides found primarily in <i>L. infantum</i> containing the arginine (R) substitution at position 6; Peptides B (blue) −13 amino acid peptides found in <i>L. donovani</i>. Peptide A3 found in both species was chosen as the reference sequence to which the amino acid sequences of all the other peptides are compared: Single amino acid abbreviations in blue indicates a substitution, in red a deletion, and in black conserved; (−) missing amino acid, (.) conserved amino acid. Peptides numbers from 0–19 have amino acid sequences identical to those reported by Maroof et al. <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002031#pntd.0002031-Maroof1" target="_blank">[29]</a>; those with numbers ≥ 20 are new sequences described in this study.</p

Polymorphism in the HASPB Repeat Region of East African <em>Leishmania donovani</em> Strains

<div><h3>Background/Objectives</h3><p>Visceral leishmaniasis (VL) caused by <em>Leishmania donovani</em> is a major health problem in Ethiopia. Parasites in disparate regions are transmitted by different vectors, and cluster in distinctive genotypes. Recently isolated strains from VL and HIV-VL co-infected patients in north and south Ethiopia were characterized as part of a longitudinal study on VL transmission.</p> <h3>Methodology/Principal Findings</h3><p>Sixty-three <em>L. donovani</em> strains were examined by polymerase chain reaction (PCR) targeting three regions: internal transcribed spacer 1 (ITS1), cysteine protease B (cpb), and HASPB (k26). ITS1- and cpb - PCR identified these strains as <em>L. donovani</em>. Interestingly, the k26 - PCR amplicon size varied depending on the patient's geographic origin. Most strains from northwestern Ethiopia (36/40) produced a 290 bp product with a minority (4/40) giving a 410 bp amplicon. All of the latter strains were isolated from patients with HIV-VL co-infections, while the former group contained both VL and HIV-VL co-infected patients. Almost all the strains (20/23) from southwestern Ethiopia produced a 450 bp amplicon with smaller products (290 or 360 bp) only observed for three strains. Sudanese strains produced amplicons identical (290 bp) to those found in northwestern Ethiopia; while Kenyan strains gave larger PCR products (500 and 650 bp). High-resolution melt (HRM) analysis distinguished the different PCR products. Sequence analysis showed that the k26 repeat region in <em>L. donovani</em> is comprised of polymorphic 13 and 14 amino acid motifs. The 13 amino acid peptide motifs, prevalent in <em>L. donovani</em>, are rare in <em>L. infantum</em>. The number and order of the repeats in <em>L. donovani</em> varies between geographic regions.</p> <h3>Conclusions/Significance</h3><p>HASPB repeat region (k26) shows considerable polymorphism among <em>L. donovani</em> strains from different regions in East Africa. This should be taken into account when designing diagnostic assays and vaccines based on this antigen.</p> </div

Origin of Ethiopian <i>Leishmania donovani</i> strains used in this study.

<p>Specific details on all strains are provided in Table S1.</p

Analysis of Ethiopian <i>Leishmania donovani</i> strains by k26 - PCR and agarose gel electrophoresis.

<p>PCR products were separated by electrophoresis in 2% agarose gels and stained with ethidium bromide. Southern Ethiopian (SE): 450 bp amplicon −1 and 360 bp amplicon −2. Northern Ethiopian (NE): 290 bp amplicon −1 and 410 bp amplicon −2. A 100 bp molecular weight marker (Mr) is shown on either side of the gel. DNAs from <i>L. donovani</i> examined by k26 – PCR in order from left to right: DM290, DM317, AM553, DM283, DM291, AM546, DM256, DM257, DM376sp, GR284, DM14, DM297, DM259, DM287, DM299a, DM389.</p

Predicted B-cell epitopes in HASPB repeat regions of <i>Leishmania donovani</i> and <i>L. infantum</i>.

<p>Bar codes, see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002031#pntd-0002031-g005" target="_blank">Figure 5B</a> for legend, showing the A(a) or B motif organization of the repeat region for East African and Indian <i>L. donovani,</i> and for <i>L. infantum</i> cluster 1a. Non-overlapping B-cell epitopes, 16 amino acids long with a threshold ≥ 0.8, were predicted using a recurrent artificial neural network (ABCpred server <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002031#pntd.0002031-Saha1" target="_blank">[36]</a>). The position of each epitope is indicated under the respective k26 – PCR product cluster bar code. The B-cell epitope recognized by infected canine visceral leishmaniasis sera, <u>DGPKEDGRTQKNDGDG</u>, is underlined.</p

Characterization of Ethiopian parasite strains from patients with visceral leishmaniasis by short cpbE/F - PCR.

<p>Amplicons were separated by electrophoresis on 2% agarose gel and staining with ethidium bromide. Reference DNA samples for <i>Leishmania infantum</i> are indicated by Li1 (MCAN/IL/2000/LRC-L792) and Li2 - (MHOM/TN/1980/IPT1), and for <i>L. donovani</i> by Ld (MHOM/SD/1962/1S cl2). Mr −100 bp molecular weight marker. Representative parasite DNA samples examined by short cpbE/F - PCR from left to right Southern Ethiopia (SE): AM546, AM548, AM551, AM552, AM553, AM554, AM560, AM563 and Northern Ethiopia (NE): GR284, GR353, GR356, GR358, GR361, GR378, GR379, GR383.</p

Arginase activity in saliva, PBMCs and plasma.

<p>The activity of arginase was measured by enzymatic assay in (A) Saliva (VL patients: n = 16, VL/HIV patients: n = 13), (B) PBMCs (VL patients: n = 14, VL/HIV patients: n = 13) and (C) plasma (VL patients: n = 14, VL/HIV patients: n = 13). The straight line represents the median. NS = not significant.</p

Liver and kidney functions assays.

<p>GOT = glutamic-oxaloacetic transaminase, GPT = Glutamic-pyruvic transaminase, ALP = Alkaline phosphatase, BUN = blood urea nitrogen.</p

Phenotype of arginase expressing LDGs.

<p>PBMCs were isolated by density gradient from the blood of (A) one VL patient and (B) one VL/HIV patient and the phenotype of arginase-expressing cell was measured by flow cytometry. Data show the results of one representative experiment out of 8 independent experiments for the VL patients and 5 independent experiments for the VL/HIV patients.</p

Haematological data.

<p>Hct = hematocrit; Hb = haemoglobin.</p><p>Normal range: platelets (×10³) = 150–450; white blood cells (×10³) = 4.5–10.5; Hct (%) = 35–60; hb (g/dl) = 11–18.</p><p>The white blood cells were counted in total blood before Ficoll using a COULTER Ac•T diff Hematology Analyzer and are expressed as number of WBC (×10³) per µl of blood.</p