Generation of <em>Leishmania</em> Hybrids by Whole Genomic DNA Transformation

<div><p>Genetic exchange is a powerful tool to study gene function in microorganisms. Here, we tested the feasibility of generating <em>Leishmania</em> hybrids by electroporating genomic DNA of donor cells into recipient <em>Leishmania</em> parasites. The donor DNA was marked with a drug resistance marker facilitating the selection of DNA transfer into the recipient cells. The transferred DNA was integrated exclusively at homologous locus and was as large as 45 kb. The independent generation of <em>L. infantum</em> hybrids with <em>L. major</em> sequences was possible for several chromosomal regions. Interfering with the mismatch repair machinery by inactivating the <em>MSH2</em> gene enabled an increased efficiency of recombination between divergent sequences, hence favouring the selection of hybrids between species. Hybrids were shown to acquire the phenotype derived from the donor cells, as demonstrated for the transfer of drug resistance genes from <em>L. major</em> into <em>L. infantum</em>. The described method is a first step allowing the generation of <em>in vitro</em> hybrids for testing gene functions in a natural genomic context in the parasite <em>Leishmania</em>.</p> </div

Targeted replacement of the <i>L. major</i> and <i>L. infantum LRP</i> gene.

<p>Schematic drawing of the <i>LRP</i> locus with <i>Sal</i>I sites of <i>L. major</i> and <i>L. infantum</i> (A, C). (B) Southern blot analysis of <i>L. major</i> Friedlin genomic DNA digested with <i>Sal</i>I and hybridized with a 3′ UTR probe of <i>LRP</i> gene (a ∼500 bp fragment just downstream the stop codon of <i>LRP</i> gene) (upper panel) and then with a LRP PCR amplified gene fragment of ∼500 bp (bottom panel). (D) Southern blot analysis of <i>L. infantum</i> 263 genomic DNA digested with <i>Sal</i>I and hybridized with a 3′ UTR probe of <i>LRP</i> gene (a ∼500 bp fragment just downstream the stop codon of <i>LRP</i> gene) (upper panel) and then with the LRP PCR specific fragment (bottom panel). Molecular weight is indicated on the left. Lane 1, <i>Leishmania</i> WT cells; Lane 2, <i>Leishmania</i> SKO cells (LRP/NEO); 3, <i>Leishmania</i> DKO cells (NEO/NEO) at the <i>LRP</i> locus obtained by loss of heterozygosity. (E, F) SbIII susceptibilities of <i>L. major</i> and <i>L. infantum LRP</i> mutants and hybrid parasites. The EC₅₀ values were determined by culturing promastigotes parasites in the presence of increasing concentrations of SbIII. The average of three independent experiments is shown with a statistical significance observed by Student's t-test (p<0.05) (*).</p

The inactivation of <i>MSH2</i> increases the frequency of generation of cross-species hybrids.

<p>(A) Schematic map of the wild-type and inactivated <i>MSH2</i> alleles of <i>L</i>. <i>infantum</i>. (B) Southern blot analysis of gDNA derived from <i>L. infantum</i> WT (Lane 1), <i>L. infantum MSH2</i> haploid mutant (Lane 2) and <i>L. infantum MSH2</i> null mutant (Lane 3) digested with <i>Sac</i>I and hybridized with a probe specific for the 3′ UTR of <i>MSH2</i> (upper panel). The absence of <i>MSH2</i> gene was confirmed by PCR using a pair of internal primers of the gene (middle panel); and primers for the amplification of <i>LinJ34_V3.0540</i> were used as control (lower panel). Molecular weight is indicated on the left of the blot. (C–F) The number of recombinant clones obtained from 1×10⁸<i>L. infantum</i> 263 WT parasites (black bars), <i>L. infantum</i> Li263Δ<i>MSH2</i>::HYG:BLA (grey bars) and <i>L. infantum</i> Li263Δ<i>MSH2</i>::HYG:BLA complemented <i>in trans</i> for <i>MSH2</i> (white bars) transfected with a linear LinJ28_V3.0330:NEO DNA PCR fragment amplified from <i>L. infantum</i> (C), a linear LmjF30.1250:NEO (PK) DNA PCR fragment amplified from <i>L. major</i> (D), gDNA derived from <i>L. major</i> Friedlin LmΔ<i>LRP</i>::NEO/NEO (E), or gDNA derived from <i>L. major</i> Friedlin LmΔLmjF05.0610::NEO/LmjF05.0610 (F). The average of at least 3 experiments is indicated and the number of colonies is indicated per 10 µg of DNA.</p

Whole genome transformation in <i>Leishmania</i> and cross-species hybrid formation.

<p>Genomic DNAs derived from LRP haploid mutants of <i>L. infantum</i> 263 (Li263Δ<i>LRP</i>::NEO/LRP) or <i>L. major</i> Friedlin (LmΔ<i>LRP</i>::NEO/LRP) were used to transfect <i>L. infantum</i> 263 or <i>L. infantum</i> JPCM5 (WT) parasites. (A) Chromosomes were separated by CHEF, transferred, and hybridized with probes specific for the <i>NEO</i> gene or for LmjF34.0540 gene located just upstream of the <i>LRP</i> gene on chromosome 34. The 2,000 kb chromosome 34 is indicated by an arrow on the right. Lane 1, <i>L. infantum</i> 263 WT; Lanes 2–4, <i>L. infantum</i> 263 transfected with Li263Δ<i>LRP</i>::NEO/LRP gDNA (2); LmΔ<i>LRP</i>::NEO/LRP gDNA (3–4); Lane 5, <i>L. infantum</i> JPCM5 WT; Lanes 6–7, <i>L. infantum</i> JPCM5 transfected with Li263Δ<i>LRP</i>::NEO/LRP gDNA or with LmΔ<i>LRP</i>::NEO/LRP gDNA, respectively. (B) Schematic map of the genomic region encompassing the <i>LRP</i> gene of <i>L. infantum</i> JPCM5 (black boxes) and <i>L. major</i> Friedlin (white boxes). Hybrids with one allele of <i>L. major</i> and one allele of <i>L. infantum</i> are shown in gray. The genomic regions exchanged in the hybrids were mapped by sequencing individual amplified genes using primers amplifying both <i>L. major</i> and <i>L. infantum</i> genes (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001817#pntd.0001817.s005" target="_blank">Tables S3</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001817#pntd.0001817.s006" target="_blank">S4</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001817#pntd.0001817.s007" target="_blank">S5</a>) located in the vicinity of the integrated <i>NEO</i> marker. The sequence of the hybrid JPCM5 (1) was also obtained by next generation sequencing. The extent of the genomic DNA exchanged is indicated after sequence analysis. In the JPCM5 clone 1 hybrid, both <i>L. infantum</i> alleles were replaced by <i>L. major</i>.</p

Generation of <i>L. infantum</i> hybrids.

<p>Genomic DNAs derived from <i>L. major</i> Friedlin <i>NEO</i> recombinants were electroporated into <i>L. infantum</i>. Chromosomes of putative hybrids were separated by pulsed-field gel electrophoresis. (A) <i>L. infantum</i> 263 and JPCM5 wild-type cells (lanes 1, 3) were transformed with gDNA derived from LmΔ<i>PK</i>::NEO/PK (LmjF30.1250) (lanes 2, 4). The hybridization of chromosome sized blots with probes derived from the <i>NEO</i> (left panel) or LmjF30.1255 (right panel) genes confirmed the proper integration of <i>L. major</i> DNA in <i>L. infantum PK</i> locus. (B) <i>L. infantum</i> JPCM5 and 263 WT cells (lanes 1, 3) were transfected with LmΔLmjF<i>01.0315</i>::NEO/LmjF01.0315 gDNA (lanes 2, 4). The hybridization of chromosome sized blots with probes derived from the <i>NEO</i> (left panel) or LmjF01.0310 (right panel) genes confirmed the proper integration of <i>L. major</i> DNA in <i>L. infantum.</i> (C) Generation of hybrids into <i>L. infantum</i> 263 <i>MSH2</i> null mutants. The hybridization of chromosome sized blots with probes specific for the <i>NEO</i> gene (upper panel) confirmed the proper integration of <i>L. major</i>-derived DNA at the level of targeted chromosomes in <i>L. infantum</i>. Hybridizing the same blots with a probe specific for <i>MSH2</i> (middle panel) confirmed the <i>MSH2</i>-null mutant status of the <i>L. infantum</i> recipients while <i>PTR1</i> hybridization served as a loading control. <i>L. infantum</i> 263 wild-type parasites transfected with gDNA derived from <i>L. major</i> LmΔ<i>LRP</i>::NEO/NEO (lane 1); <i>L. infantum</i> Li263Δ<i>MSH2</i>::HYG/BLA transfected with gDNA derived from <i>L. major</i> LmΔ<i>LRP</i>::NEO/NEO (lanes 2 and 3) or <i>L. major</i> LmΔLmjF05.0610::NEO/LmjF05.0610 (lane 4). Schematic map of the genomic region encompassing the <i>PK</i> (D), the LmjF01.0315/LinJ01_V3.0315 (E), and <i>LRP</i> (F) genes of <i>L. infantum</i> JPCM5 (black boxes) or <i>L. major</i> Friedlin (white boxes) and hybrid regions with one allele derived from <i>L. major</i> and one allele from <i>L. infantum</i> (grey boxes) as determined by multilocus PCR sequencing (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001817#pntd.0001817.s008" target="_blank">Tables S6</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001817#pntd.0001817.s009" target="_blank">S7</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001817#pntd.0001817.s010" target="_blank">S8</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001817#pntd.0001817.s011" target="_blank">S9</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001817#pntd.0001817.s012" target="_blank">S10</a>).</p

Cross species functional phenotypic transfer.

<p>(A) <i>L. infantum</i> 263 WT parasites were transfected with total gDNA derived from <i>L. major</i> Friedlin LmMF80.3ΔLmjF<i>13.1540</i>::NEO/LmjF13.1540 (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001817#pntd.0001817.s001" target="_blank">Figure S1C</a>). Chromosome sized blots were hybridized with probes specific for the <i>LmjF13.1520</i> gene (left panel), located just upstream of the <i>MT</i> gene on chromosome 13, or for the <i>NEO</i> marker (right panel). Lane 1, <i>L. infantum</i> 263 WT; Lane 2, <i>L. infantum</i> hybrid for <i>L. major</i> LmMF80.3 gDNA at its <i>MT</i> locus. (B) Schematic map of the genomic region encompassing the <i>MT</i> gene of <i>L. infantum</i> JPCM5 (black boxes), <i>L. major</i> Friedlin MF80.3 (white boxes), and the hybrid region (grey boxes). The genomic regions exchanged in the <i>L. infantum</i> 263 hybrid were mapped by sequencing the genes located in the vicinity of the integrated <i>NEO</i> marker (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001817#pntd.0001817.s013" target="_blank">Table S11</a>) and by whole genome short reads next generation sequencing. (C) Sequence of the <i>MT</i> genes of <i>L. major</i> Friedlin (LmjF13.1530), <i>L. major</i> Friedlin MF80.3 <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001817#pntd.0001817-Coelho1" target="_blank">[19]</a> and <i>L. infantum</i> JPCM5 WT (LinJ13_V3.1590) respectively. The <i>MT</i> gene from <i>L. infantum</i> hybrids was amplified and cloned into the pGEM-T-easy vector. The analysis 10 independent <i>E. coli</i> clones identified <i>L. infantum</i> WT and <i>L. major</i> MF80.3 alleles in similar proportions indicating that the <i>L. infantum</i> hybrid is heterozygous at its <i>MT</i> locus. The asterisk (*) indicates the deletion of three nucleotides (M547del) present in <i>L. major</i> Friedlin MF80.3 <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001817#pntd.0001817-Coelho1" target="_blank">[19]</a> and sequence in red are polymorphisms between <i>L. major</i> and <i>L. infantum</i>. (D) <i>L. infantum</i> 263 WT parasites (Δ) and <i>L. infantum</i> 263 hybrid for LmMF80.3Δ<i>LmjF13.1540</i>::NEO/<i>LmjF13.1540</i> at their <i>MT</i> locus (▴) were grown in increasing concentrations of miltefosine and their EC₅₀ values determined. The mean of three independent experiments is indicated. A statistical significance was observed by Student's t-test (p<0.05).</p

Summary of generation of <i>Leishmania</i> hybrids using different gDNA.

<p>Transfection efficiency was calculated by dividing the number of colonies by the number of transfected cells. The relative abundance of stable and episomal transformants were determined on clones by CHEF, followed by Southern blot as described in the <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001817#s2" target="_blank">Methods</a> using the respective drug resistance marker as probe.</p

Additional file 3: Figure S3. of A genomic approach to understand interactions between Streptococcus pneumoniae and its bacteriophages

Gene expression profiling in S. pneumoniae R6Δspr0058. Gene expression was compared between S. pneumoniae R6 WT and R6Δspr0058 by RNA-seq. The dots represent the 2043 genes from the S. pneumoniae R6 genome and their level of expression in R6 WT and R6Δspr0058 (represented in terms of fragments per kilobase of transcript per million fragments mapped) is indicated on the x- and y-axis, respectively. (TIFF 151 kb

Boletín de Segovia: Número 137 - 1880 noviembre 12

Copia digital. Madrid : Ministerio de Cultura. Subdirección General de Coordinación Bibliotecaria, 200

Phylogenetic Analysis of <i>Leishmania</i> Isolates Responsible for ACL by Comparing the Sequences of <i>PTR1</i>

<p>The <i>PTR1</i> gene was sequenced in 31 Iranian isolates and control L. major cells (LV39; Friedlin), L. donovani 1S2D, L. infantum (MHOM/67/ITMAP-263), L. tropica ATCC50129, and L. tarentolae TarII. Strain 848 is identical, as determined by RAPD-PCR, to L. tropica MHOM/IR/99, and strain 749 is identical to L. major MHOM/IR/75/ER. Strains 444 and 454 were <i>Leishmania</i> parasites isolated from dogs in Iran. The sequences were compared and clustered using the Molecular Evolutionary Genetics Analysis (MEGA3) software. </p