Phylogeny for 26 <i>Trypanosoma cruzi</i> 350 bp RNA-Binding Protein-19 (<i>RB19</i>) sequences.

<p>Neighbor-Joining tree constructed in MEGA6 with evolutionary distances computed via Maximum Composite Likelihood. The numbers above the nodes represent bootstrap confidence levels for 2,000 replicates. Only values ≥ 50% are shown. The only TcI isolate not obtained in this study is represented by a square. Sequences obtained in this study are indicated by a triangle (Esc = Escondido, SoCal = Southern California, Vall = Vallecito). All other isolates represent published GenBank sequences as listed in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004291#pntd.0004291.s001" target="_blank">S1 Table</a> with their country origin indicated in parentheses. The scale bar indicates the number of nucleotide substitutions per site. Tree is outgroup rooted with <i>T</i>. <i>cruzi marinkellei</i> (TcMark CONTIG 1404).</p

Phylogeny for 35 <i>Trypanosoma cruzi</i> 1288 bp trypanothione reductase sequences.

<p>Depicted is the Neighbor-Joining (NJ) tree constructed in MEGA6 with evolutionary distances computed via the Maximum Composite Likelihood method and the scale bar indicating the number of nucleotide substitutions per site. The numbers above or below the nodes represent the bootstrap confidence levels for 2,000 NJ replicates (1^st value) and 500 Maximum Likelihood replicates (2^nd value presented at nodes with congruent topologies) run under the Kimura 2-parameter model for those values <b>≥</b> 50%. Sequences obtained in this study are indicated by the triangles (Esc = Escondido, SoCal = Southern California, Vall = Vallecito). All other isolates represent published GenBank sequences as listed in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004291#pntd.0004291.s001" target="_blank">S1 Table</a> with their country of origin indicated in parentheses. Tree is outgroup rooted with <i>T</i>. <i>cruzi marinkellei</i> (GenBank #AF359007).</p

PCR assay flowsheet¹ to identify <i>Trypanosoma cruzi</i> discrete typing units (DTUs).

<p>Directional arrows indicate assay order and stop signs denote when sufficient data was gathered to theoretically identify the DTU. The final assay (<i>GPI</i>) is included as a confirmatory step, but is not required for DTU identification. ¹Modified from Lewis et al. [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004291#pntd.0004291.ref037" target="_blank">37</a>] ²The large subunit rDNA assay is also referred to as the 24sα rRNA gene assay. ³An additional band of approximately 125bp may or may not be visible in combination with the 110bp band. ⁴Heat Shock Protein-60 (HSP60) results in an amplicon of 432-462bp, which upon RFLP with <i>Eco</i>V restriction enzyme yields the following patterns: 1 band (432–462), 2 bands (118–148 + 314), or 3 bands (118–148 + 314 + 432–462). ⁵This PCR used a pool of three primers to amplify a portion of the non-transcribed intergenic region of the tandemly repeated mini-exon gene. ⁶Glucose Phosphate Isomerase (GPI) results in an amplicon of approximately 1264bp, which upon RFLP with <i>Hha</i>I restriction enzyme yields the following patterns: 2 bands (447 + 817), 3 bands (253 + 447 + 490), or 4 bands (253 + 447 + 490 + 817). TcIV will display 2 or 3 bands for North American and South American strains, respectively.</p

Phylogeny for 62 <i>Trypanosoma cruzi</i> concatenated 786 bp cytochrome oxidase II-NADH 1 (<i>COII-ND1</i>) sequences.

<p>The TcI clade is condensed in this figure and contains the majority of the sequences obtained in this study (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004291#pntd.0004291.g005" target="_blank">Fig 5</a> for expanded version). Depicted is the Neighbor-Joining (NJ) tree constructed in MEGA6 with evolutionary distances computed via the Maximum Composite Likelihood method and the scale bar indicating the number of nucleotide substitutions per site. The numbers above or below the nodes represent the bootstrap confidence levels for 2,000 NJ replicates (1^st value) and 500 Maximum Likelihood replicates run under the Tamura 3-parameter model (due to slightly incongruent topology, ML bootstrap values are only shown at three nodes) for those values <b>≥</b> 50%. Only one sequence obtained in this study (Esc19 = Escondido 19) was grouped as TcIV based on mitochondrial gene sequences. All other isolates represent published GenBank sequences as listed in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004291#pntd.0004291.s001" target="_blank">S1 Table</a> with their country of origin indicated in parentheses. Tree is outgroup rooted with <i>T</i>. <i>cruzi marinkellei</i> (GenBank #AF359054).</p

TcI subtree represented as TcI on Fig 4 showing four distinct subclades.

<p>This subtree includes 46 <i>COII-ND1</i> concatenated sequences (786 bp). The scale bar indicates the number of nucleotide substitutions per site for the NJ tree. The numbers above or below the nodes represent the bootstrap confidence levels for 2,000 NJ replicates (1^st value) and 500 Maximum Likelihood replicates run under the Tamura 3-parameter model. Only bootstrap values <b>≥</b> 50% are shown. Sequences obtained in this study are indicated by the triangles (Esc = Escondido, SoCal = Southern California, Vall = Vallecito). Esc26 was grouped with TcI in this analysis but was grouped with TcIV in all other analyses. All other isolates represent published GenBank sequences as listed in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004291#pntd.0004291.s001" target="_blank">S1 Table</a> with their country of origin indicated in parentheses.</p

<i>In vitro</i> sensitivity of null mutants to oxidative stress and benznidazole.

<p>Epimastigotes seeded at 5 x 10⁵ ml^-1 were exposed to various concentrations of <b>A</b> hydrogen peroxide and <b>B</b> benznidazole. The number of viable cells after 9 days was measured using resazurin fluorescence. TcΔAPx1comp refers to TcΔAPx1 cells retransformed with pTEX-APx to complement the null phenotype. Data were analysed by sigmoidal curve fitting using GraphPad Prism. The table below shows the EC₅₀ values (μM) for each compound against the various cell lines +/- standard deviation. Significance of differences in the EC₅₀ for H₂O₂ was measured using the F-test. (ND—not done).</p

Numbers of single and co-infected sand flies examined by fluorescence microscopy.

<p>PBM, post bloodmeal;</p><p>Dis, number of dissected females;</p><p>Inf, number of females infected with at least one <i>Leishmania</i> strain;</p><p>B, number of females infected with both <i>Leishmania</i> strains;</p><p>H, number of females with hybrid promastigotes seen and photographed under fluorescence microscope;</p><p>-, not done.</p

Phenotypic assessment of null mutants <i>in vitro</i>.

<p><b>A</b> Growth rate of <i>T</i>. <i>cruzi</i> epimastigotes for wild type and null mutant (TcΔAPx1 and 2) clones. Triplicate cultures were followed for 10 days. There was no significant difference in growth rate. <b>B</b> Parasites lacking TcAPx can differentiate to amastigotes (AM) and trypomastigotes (TR). Examples shown are Giemsa stained wild type and null mutant (TcΔAPx2) cells. <b>C</b><i>In vitro</i> infectivity for L6 rat myoblast cells. Metacyclic trypomastigotes were used to infect L6 cells at a ratio of 5 trypanosomes per cell and left for 48 hours (Methods). Cells were Giemsa stained and the number of infected cells counted. Infections were carried out with seven replicates per parasite line. TcΔAPx1comp refers to TcΔAPx1 cells retransformed with pTEX-APx to complement the null phenotype. Data presented as mean <u>+</u> SD. Significance of difference between each pair was assessed by Student’s t-test, (**) corresponds to <i>P</i> = 0.007, (***) <i>P</i> = 0.0006. <i>P</i> values for wild type:TcΔAPx1 indicated by short horizontal line, wild type:TcΔAPx2 indicated by long horizontal line. The difference between the wild type and complemented lines was not significant. <b>D</b><i>In vitro</i> infectivity for Vero epithelial cells. Metacyclic trypomastigotes were used to infect Vero cells at a ratio of 5 trypanosomes per cell and left for 48 hours (Methods). Cells were Giemsa stained and the number of infected cells counted. Infections were carried out with seven replicates per parasite line. Data presented as mean <u>+</u> SD. Significance of difference was assessed by Student’s t-test, (***) corresponds to <i>P</i> = 0.0006. <i>P</i> values for wild type:TcΔAPx1 indicated by short horizontal line, wild type:TcΔAPx2 indicated by long horizontal line.</p

Course of infection in a murine model monitored by bioluminescence imaging.

<p><b>A</b> Female BALB/c mice were infected with 2 x 10⁵ culture-derived bloodstream trypomastigotes modified to express a red-shifted luciferase gene (Methods, [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003707#pntd.0003707.ref019" target="_blank">19</a>]). Mice were imaged at the time points shown using an IVIS Illumina II system (Caliper Life Sciences). Images were analysed using the same signal intensity scale for radiance (right) where purple indicates low signal intensity and red indicates a high signal. The maximum (5x10⁷) and minimum (6.5 x10³) signals are indicated at the top and bottom of the scale bar respectively. Three representative mice are shown from each group of animals (<i>n</i> = 6 per group). DPI: days post infection, time 0 represents image taken one hour after infection. <b>B</b> Graph showing the mean total body flux measured in each group of animals throughout the experiment. The grey line indicates the wild type infection, blue is TcΔAPx1 and red is TcΔAPx2. Data are plotted as mean values, error bars indicate standard deviation. The black lines indicate the mean (solid line), and mean +2SD (dotted line) of background luminescence of control uninfected mice. All data were acquired and analysed using Living Image software (Caliper Life Sciences).</p

Development of GFP/RFP transfected <i>Leishmania</i> strains in sand flies.

<p>Infection rates and intensities of co-infections in three combinations of <i>L. donovani</i> strains in <i>L. longipalpis</i> (LO) and <i>P. perniciosus</i> (PE). A, LEM3804 plus LV9: in late stage infections (days 7 and 10) LEM3804 mostly over-grew LV9, in some females LV9 disappeared. B, Gebre-1 plus LV9: Gebre-1 grew better than LV9, in a high proportion of females LV9 was lost during late stage infections. C, Gebre-1 plus LEM4265: in late stage infections Gebre-1 mostly over-grew LEM4265, in a high proportion of females LEM4265 disappeared.</p