Multiplex immunoassay antigen screening with serum from benznidazole treated and non-treated <i>T</i>. <i>cruzi</i> infected mice.

(A) Images of multiplex assays screened with serum from a vehicle treated infected BALB/c mouse (BN201-4). (B) Images after screening with serum from an infected mouse (BN206-4) that had been subjected to benznidazole treatment (100 mg kg-1, 5 days), initiated 101 days post-infection (dpi). The dpi are indicated. Antigens were arrayed in duplicate as in Fig 1. For illustrative purposes, specific antigens are framed as follows: IBAG39 (green), IBAG257 (blue), IBAG108 (red), IBAG37 (black), IBAG38 (yellow), IBAG101 (orange), IBAG131 (purple). (C) Longitudinal data showing the proportion of serum samples reacting with selected antigens at a net intensity >10 (minimum set threshold) at each time point (n = 45). Each IBAG is indicated by an arrow. The curves of the two most reactive antigens IBAG257(blue) and IBAG39 (green) overlap.</p

Bioluminescence total flux and IBAG39 intensity of reactivity for each of the 15 mice treated with 100 mg kg^-1 benznidazole (BZ).

The linear correlation coefficient R is given in each graph. The red curve is intensity of IBAG39 and the black curve is log (total flux (p/s)).</p

Average change of IBAG reactivity over time, based on the linear prediction model for mice treated with vehicle, and benznidazole at 30 and 100 mg kg^-1.

The fits correspond to the intercept and slopes in Table 1. The reactivity of (A) IBAG38 and (B) IBAG39 remains stable in mice treated with vehicle and benznidazole at 30 mg kg-1 and sharply decreases in 100 mg kg-1 benznidazole treated mice. (C) IBAG257 declines in reactivity in the three groups. The reactivities of (D) IBAG36, (E) IBAG37 and (F) IBAG101 increase only in vehicle-treated animals. The vertical axis is representative for the real intensity of the IBAGs.</p

<i>Ex vivo</i> imaging of benznidazole-treated mice at the experimental end-point.

At 301 days post infection, mice that had been treated with 100 mg kg-1 benznidazole for 5 days (S1 Fig) were euthanized and subjected to ex vivo imaging (Methods). Bioluminescent foci (examples highlighted by white arrows) were detected in 4 mice, which were designated as non-cured. The organs and tissues are organized as shown in Fig 4. (PPTX)</p

Summary of the linear mixed model (LMM) regression output for each biomarker and in each subgroup.

Both the intercept and slope of the linear regression of each antigen corresponds to an average over the 15 mice in each group. Intercept > 10. Blue: significant p-values (Red: high intensity. We have data on several mice where we have treatment group and biomarker reactivity measured at different time points. Where all mice have the same slope and intercept relating reactivity to time and treatment group, a regular multiple linear regression model can be fitted with time and treatment group as the predictor, and reactivity as the response. Biomarker reactivities < 10 were considered below the positive reactivity threshold and were regarded as non-reactive. Slopes compared between treatment groups were assigned as different based on a 5% significance level.</p

Location of parasites in the stomach and cecum during chronic stage infections.

<p><b>A.</b> Section of stomach tissue from a chronically infected mouse (day 117)—a single slice from a Z-stack through the infected cell (63x scan zoom 0.7). The left-hand panel is a magnified view of the boxed section (63x scan zoom 2.8). The bars represent 5 μm (left hand panel) and 50 μm (right hand panel). <b>B.</b> Section of cecum tissue from a chronically infected mouse (day 117), with a cluster of amastigotes surrounding the nucleus of an infected cell (left hand image, 63x scan zoom 2.5; right hand image, 63x scan zoom 0.7).</p

CRISPR/Cas9 mediated genome editing in the <i>T</i>. <i>cruzi</i> CL-Luc::Neon reporter strain.

<p><b>A.</b> Diagram of the <i>GP72</i> gene indicating sgRNA binding sites (indicated by scissors) and homology arms for integration (blue bars). The lower map shows the configuration of the puromycin (PAC) and blasticidin (BSD) drug resistance cassettes. Grey boxes indicate plasmid derived RNA processing signals; orange box shows the coding sequence for the selectable marker. <b>B.</b> PCR-based confirmation of gene deletion (Methods). Lane 1, parental clone, Lane 2, a null mutant clone. The relative positions of the primers are illustrated on the maps below each panel. The <i>GP72</i> gene is only present in the parental clone and both selectable markers have integrated as expected. <b>C.</b> The <i>GP72</i> null mutant parasites are characterised by detachment of the flagellum from the cell body. Left hand panel, the parental strain (TcCL-LNCas9); right hand panel, the <i>GP72</i> null mutant. Note that the flagellum in the null mutants exits the cell immediately anterior to the kinetoplast, whereas in the parental strain it remains attached along the length of the cell body. <b>D.</b> Strategy for CRISPR-Cas9 mediated replacement of mNeonGreen by mScarlet. The map of the Luc::Neon locus indicates the Cas9/sgRNA mediated cleavage sites. The homologous repair donor is illustrated, with the 30 bp homology arms which correspond to the spacer peptide coding sequence and the 3’-end of the hygromycin resistance gene. The edited locus is shown in which the mScarlet and blasticidin resistance genes have replaced the mNeonGreen and hygromycin resistance genes. <b>E.</b> Images of parasites before (green) and after (red) replacement of the fluorescent reporters. Images taken with 100x objective at 2.3 scan zoom. <b>F.</b> Image of the parasite population under drug selection, 12 days after transfection, taken with 63x objective at 1.3 scan zoom. The majority of parasites now display red fluorescence. In all images, the white bars represent 10μm.</p

Generation and expression in <i>T</i>. <i>cruzi</i> of a chimeric bioluminescent–fluorescent fusion protein.

<p><b>A.</b> Amplification of the mNeonGreen ORF and flanking targeting sequences, together with the coding sequence for an 8 amino-acid spacer peptide (purple line) (Methods) (PCR1). The product was used as the downstream primer to amplify the Luc::Neon fusion fragment (PCR2). The purple box indicates the 8 amino acid spacer peptide. <b>B.</b> The PCR2 product was inserted into plasmid pTRIX2-RE9-Hyg [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006388#pntd.0006388.ref015" target="_blank">15</a>] to create pTRIX2-Luc::Neon-Hyg. <b>C.</b> SbfI/AscI linearised pTRIX2-Luc::Neon-Hyg was transfected into <i>T</i>. <i>cruzi</i> CL-Luc such that integration replaces the original luciferase sequence with the fusion gene, with the G418 resistance gene (Neo) exchanged for the gene encoding hygromycin resistance (Hyg). <b>D.</b> Western blot probed with a polyclonal anti-luciferase antibody (Promega). Lane 1, <i>T</i>. <i>cruzi</i> CL-Luc, the original bioluminescent strain; Lane 2, <i>T</i>. <i>cruzi</i> CL-Luc::Neon parasites expressing the 88 kDa fusion protein. <b>E.</b> Epimastigote luciferase activity in parasite lysates (Methods). Extracts were assayed in quadruplicate, data show mean activity, and error bars indicate standard deviation. <b>F.</b> Epimastigotes fixed in 2% paraformaldehyde and imaged on a Zeiss LSM 510 confocal microscope. The bar indicates 10μm. Only cells carrying the fusion protein exhibit green fluorescence. DNA is stained with DAPI (coloured red on images).</p

Expression of the dual reporter does not impinge on the infection profile.

<p><b>A.</b> Bioluminescence imaging of representative infected mice (ventral and dorsal images shown) following i.p. infection with 1x10³ bloodstream trypomastigotes; upper panel, infection with the original <i>T</i>. <i>cruzi</i> CL-Luc clone; lower panel, infection with the <i>T</i>. <i>cruzi</i> CL-Luc::Neon clone. DPI; days post-infection. <b>B.</b> The total body flux for each group in photons/second (p/s). Data points represent the sum of the total flux from both dorsal and ventral views. Red line, group infected with <i>T</i>. <i>cruzi</i> CL-Luc; Blue lines, two individual groups infected with <i>T</i>. <i>cruzi</i> CL-Luc::Neon (n = 3 per group). Error bars represent SEM. <b>C.</b> <i>Post mortem</i> imaging of organs from mice in the acute phase of infection (day 13). <b>D.</b> Organs imaged in the chronic phase of infection (day 117). The image in the lower panel (left) indicates the arrangement of tissues. The image inset in the lower panel (right) shows organs from the chronic phase of infection (day 117) from a mouse immunosuppressed with cyclophosphamide (Methods), that was infected with <i>T</i>. <i>cruzi</i> CL-Luc::Neon. All images in D are calibrated to the same scale as indicated by the side bar.</p

Incorporation of CRISPR/Cas9 functionality into the <i>T</i>. <i>cruzi</i> CL Luc::Neon reporter line.

<p><b>A.</b> Map of pLEW13-Cas9 showing the construct and the integration of linearised DNA into the <i>T</i>. <i>cruzi</i> genome via the tubulin locus (Methods). <b>B.</b> Western blots showing expression of the <i>Cas9</i> gene. Wild type <i>T</i>. <i>cruzi</i> CL-Brener before (lane 1) and after (lane 2) transfection with pLEW13-Cas9. Right hand panel; <i>T</i>. <i>cruzi</i> CL-Luc::Neon reporter strain before (lane 3) and after (lane 4) transfection. The blot was probed with anti-Cas9 monoclonal 7A9 (Merck). <b>C.</b> Cas9 has no effect on growth when expressed from the tubulin array. Growth curves comparing both parental lines with their Cas9 expressing counterparts. Growth assays were performed in triplicate; the error bars represent standard deviation.</p