Altered immune responses in mice infected with Tm-sAChE.

<p>(A) Specific antibody responses (end-point titres) to <i>T</i>. <i>musculi</i> at d14 post-infection. (B) Cytokine responses from splenocytes at d14 post-infection. Data are shown as the mean ±SEM (n = 5) and are representative of three independent experiments. **p<0.01, ***p<0.001. White bar = uninfected mice; Light grey bar = mice infected with Tm-luc; Dark grey bar = mice infected with Tm-sAChE.</p

Expression of <i>N</i>. <i>brasiliensis</i> AChE B in <i>T</i>. <i>musculi</i>.

<p>(A) Detection by western blot. NbSP: Secreted products from <i>N</i>. <i>brasiliensis</i>; TmE: <i>T</i>. <i>musculi</i> extracts; TmSP: <i>T</i>. <i>musculi</i> secreted products. WT: Wild type trypanosomes; AChE; <i>T</i>. <i>musculi</i> expressing cytosolic AChE; sAChE: <i>T</i>. <i>musculi</i> expressing secreted AChE. (B) Tm-sAChE is glycosylated. Extracts and secreted products as in A), either with (+) or without (-) PNGase F treatment. Molecular mass markers are shown in kDa. (C) Tm-sAChE stained with antibody to <i>N</i>. <i>brasiliensis</i> AChE B and DAPI and viewed by indirect immunofluorescence. (D) Visualisation of AChE activity after non-denaturing gel electrophoresis, abbreviations as in panel A. (E) AChE activity measured by Ellman assay, abbreviations as in panel A. TmE: <i>T</i>. <i>musculi</i> extracts from 5 x 10⁵ trypanosomes; TmSP: <i>T</i>. <i>musculi</i> secreted products from 5 x 10⁴ trypanosomes cultured for 24 hrs. Data are shown as the mean ±SEM, assayed in triplicate.</p

Cellularity of spleens from mice infected with transgenic <i>T</i>. <i>musculi</i>.

<p>Phenotyping was performed by flow cytometry as described in Materials and Methods. Data are shown as the mean ± 1SEM (n = 5) and are representative of two independent experiments with 5 mice in each group. *p<0.05, **p<0.01. White bar = uninfected mice; Light grey bar = mice infected with Tm-luc; Dark grey bar = mice infected with Tm-sAChE.</p

Generation of transgenic <i>T</i>. <i>musculi</i> and expression of GFP.

<p>(A) Schematic depiction of expression vector pSSUGFP, utilising 5´ and 3´ regions to integrate into the 18S SSU rRNA gene locus, and both PFR and β-α tubulin intergenic regions to effect RNA processing of eGFP and blasticidin resistance genes. (B) Detection of eGFP expression by western blot. Lane 1: Wild type <i>T</i>. <i>musculi</i>; Lane 2: Parasites transformed with expression cassette in which eGFP was incorporated into the tubulin gene array (pTubGFP); Lane 3: Expression cassette with eGFP incorporated into the SSU rRNA locus (pSSUT7GFP) in a cell line with T7 RNA polymerase incorporated into the tubulin array (pT7polyNeo); Lane 4: Expression cassette with eGFP incorporated into the SSU rRNA locus alone (pSSUGFP). Molecular mass markers are shown in kDa. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005998#ppat.1005998.s002" target="_blank">S2 Fig</a> for different constructs. (C) Detection of eGFP expression by fluorescence microscopy: eGFP incorporated into the SSU rRNA locus.</p

Growth and survival of transgenic <i>T</i>. <i>musculi</i> in vitro and in vivo.

<p>(A) Growth <i>in vitro</i> of wild type <i>T</i>. <i>musculi</i> (WT), those engineered to express secreted AChE (Tm-sAChE) and expressing cytosolic luciferase (Tm-luc). (B) Survival of Tm-sAChE compared to Tm-luc <i>in vivo</i>. Parasitaemia in peripheral blood monitored over the course of infection of female BALB/c mice. Data are shown as the mean ±SEM (n = 5) and are representative of three independent experiments. *p<0.05, **p<0.01, ***p<0.001. (C). Detection of AChE in serum of mice 8 days post-infection with Tm-luc and Tm-sAChE by activity-based gel assay [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005998#ppat.1005998.ref023" target="_blank">23</a>]. Samples of serum (5 μl) from individual mice were loaded in each lane.</p