Diverse Virulent Pneumophages Infect <i>Streptococcus mitis</i>

<div><p><i>Streptococcus mitis</i> has emerged as one of the leading causes of bacterial endocarditis and is related to <i>Streptococcus pneumoniae</i>. Antibiotic resistance has also increased among strains of <i>S</i>. <i>mitis</i> and <i>S</i>. <i>pneumoniae</i>. Phages are being reinvestigated as alternatives to antibiotics for managing infections. In this study, the two virulent phages Cp-1 (<i>Podoviridae</i>) and Dp-1 (<i>Siphoviridae</i>), previously isolated from <i>S</i>. <i>pneumoniae</i>, were found to also infect <i>S</i>. <i>mitis</i>. Microbiological assays showed that both pneumophages could not only replicate in <i>S</i>. <i>mitis</i> but also produced more visible plaques on this host. However, the burst size and phage adsorption data were lower in <i>S</i>. <i>mitis</i> as compared to <i>S</i>. <i>pneumoniae</i>. A comparison of the genomes of each phage grown on both hosts produced identical nucleotide sequences, confirming that the same phages infect both bacterial species. We also discovered that the genomic sequence of podophage Cp-1 of the Félix d’Hérelle collection is different than the previously reported sequence and thus renamed SOCP.</p></div

Genome alignment of phages SOCP and Cp-1.

<p>The scale above the phage genome SOCP is in base pairs. Each arrow represents a gene, and the numbering for SOCP refers to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118807#pone.0118807.t001" target="_blank">Table 1</a>. Some putative functions of the deduced proteins are indicated above the arrows (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118807#pone.0118807.t001" target="_blank">Table 1</a> for details). Grey arrows indicate that no putative function was attributed to the deduced protein. Arrows with the same color indicate the same general function and have at least 90% identity at the amino acid level. Heterologous regions are shaded gray, and the shaded numbers indicate the number of base pairs between the corresponding gene sequences. Shadows light with colored contour highlight ORFs not found in the genome of phage Cp-1.</p

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

Putative Open Reading Frames deduced from SOCP genome sequences and their predicted functions.

<p>^a Number of amino acids (aa),</p><p>^b IP, isoelectric point and</p><p>^c MM, molecular mass.</p><p>^d RBS, ribosomal binding site. Bases in bold correspond to nucleotides identical to the RBS consensus; lowercase indicates.</p><p>Putative Open Reading Frames deduced from SOCP genome sequences and their predicted functions.</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

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

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