Reliable, scalable functional genetics in bloodstream-form Trypanosoma congolense in vitro and in vivo.

Animal African trypanosomiasis (AAT) is a severe, wasting disease of domestic livestock and diverse wildlife species. The disease in cattle kills millions of animals each year and inflicts a major economic cost on agriculture in sub-Saharan Africa. Cattle AAT is caused predominantly by the protozoan parasites Trypanosoma congolense and T. vivax, but laboratory research on the pathogenic stages of these organisms is severely inhibited by difficulties in making even minor genetic modifications. As a result, many of the important basic questions about the biology of these parasites cannot be addressed. Here we demonstrate that an in vitro culture of the T. congolense genomic reference strain can be modified directly in the bloodstream form reliably and at high efficiency. We describe a parental single marker line that expresses T. congolense-optimized T7 RNA polymerase and Tet repressor and show that minichromosome loci can be used as sites for stable, regulatable transgene expression with low background in non-induced cells. Using these tools, we describe organism-specific constructs for inducible RNA-interference (RNAi) and demonstrate knockdown of multiple essential and non-essential genes. We also show that a minichromosomal site can be exploited to create a stable bloodstream-form line that robustly provides >40,000 independent stable clones per transfection-enabling the production of high-complexity libraries of genome-scale. Finally, we show that modified forms of T. congolense are still infectious, create stable high-bioluminescence lines that can be used in models of AAT, and follow the course of infections in mice by in vivo imaging. These experiments establish a base set of tools to change T. congolense from a technically challenging organism to a routine model for functional genetics and allow us to begin to address some of the fundamental questions about the biology of this important parasite

Clostridium perfringens beta-toxin induces necrostatin-inhibitable, calpain-dependent necrosis in primary porcine endothelial cells

Clostridium perfringens β-toxin (CPB) is a β-barrel pore-forming toxin and an essential virulence factor of C. perfringens type C strains, which cause fatal hemorrhagic enteritis in animals and humans. We have previously shown that CPB is bound to endothelial cells within the intestine of affected pigs and humans, and that CPB is highly toxic to primary porcine endothelial cells (pEC) in vitro. The objective of the present study was to investigate the type of cell death induced by CPB in these cells, and to study potential host cell mechanisms involved in this process. CPB rapidly induced lactate dehydrogenase (LDH) release, propidium iodide uptake, ATP depletion, potassium efflux, a marked rise in intracellular calcium [Ca(2+)]i, release of high-mobility group protein B1 (HMGB1), and caused ultrastructural changes characteristic of necrotic cell death. Despite a certain level of caspase-3 activation, no appreciable DNA fragmentation was detected. CPB-induced LDH release and propidium iodide uptake were inhibited by necrostatin-1 and the two dissimilar calpain inhibitors PD150606 and calpeptin. Likewise, inhibition of potassium efflux, chelation of intracellular calcium and treatment of pEC with cyclosporin A also significantly inhibited CPB-induced LDH release. Our results demonstrate that rCPB primarily induces necrotic cell death in pEC, and that necrotic cell death is not merely a passive event caused by toxin-induced membrane disruption, but is propagated by host cell-dependent biochemical pathways activated by the rise in intracellular calcium and inhibitable by necrostatin-1, consistent with the emerging concept of programmed necrosis ("necroptosis")

Clostridium perfringens Beta-Toxin Induces Necrostatin-Inhibitable, Calpain-Dependent Necrosis in Primary Porcine Endothelial Cells

International audienceClostridium perfringens β-toxin (CPB) is a β-barrel pore-forming toxin and an essential virulence factor of C. perfringens type C strains, which cause fatal hemorrhagic enteritis in animals and humans. We have previously shown that CPB is bound to endothelial cells within the intestine of affected pigs and humans, and that CPB is highly toxic to primary porcine endothelial cells (pEC) in vitro. The objective of the present study was to investigate the type of cell death induced by CPB in these cells, and to study potential host cell mechanisms involved in this process. CPB rapidly induced lactate dehydrogenase (LDH) release, propidium iodide uptake, ATP depletion, potassium efflux, a marked rise in intracellular calcium [Ca(2+)]i, release of high-mobility group protein B1 (HMGB1), and caused ultrastructural changes characteristic of necrotic cell death. Despite a certain level of caspase-3 activation, no appreciable DNA fragmentation was detected. CPB-induced LDH release and propidium iodide uptake were inhibited by necrostatin-1 and the two dissimilar calpain inhibitors PD150606 and calpeptin. Likewise, inhibition of potassium efflux, chelation of intracellular calcium and treatment of pEC with cyclosporin A also significantly inhibited CPB-induced LDH release. Our results demonstrate that rCPB primarily induces necrotic cell death in pEC, and that necrotic cell death is not merely a passive event caused by toxin-induced membrane disruption, but is propagated by host cell-dependent biochemical pathways activated by the rise in intracellular calcium and inhibitable by necrostatin-1, consistent with the emerging concept of programmed necrosis ("necroptosis")

pEC exposed to rCPB do not exhibit apoptotic but typical features of necrotic cell death.

<p>(A) Presence of cleaved caspase 3 was assessed by Western blotting at different time points after exposure to 30 ng/ml rCPB or staurosporine. Tubulin was used as loading control. Representative results of 3 independent experiments. (B) Internucleosomal DNA fragmentation was assessed after 16 h of exposure to rCPB. Staurosporine was used as a control for apoptosis, while incubation with 5 mM of H₂O₂ or freeze thawing served as controls for necrosis. Representative results of 3 independent experiments. (C) Intracellular ATP levels were determined at the indicated time points. Results represent mean ± SEM of 3 independent experiments. Statistical difference to untreated control cells was assessed by 2-way ANOVA and Bonferroni multiple comparisons test. *P<0.05, **P<0.01, †P<0.001, ††P<0.0001. (D) Cytoplasmic translocation of HMGB-1 (red) was detectable by immunofluorescence in pEC already 30 min after exposure to 30 ng/ml rCPB. Control cells incubated with toxin-free medium exhibit typical nuclear localization. Nuclear counterstain with Hoechst 33258 (blue). (E) Electron microscopic image of pEC 6 h after incubation with 30 ng/ml rCPB. Cells exhibit small irregular clumps of chromatin abutting to the nuclear membrane (asterisk), swelling of cell organelles with disappearance of the elongated mitochondria with cristae (arrows), and plasma membrane discontinuities (arrowhead).</p

Attenuation of rCPB-induced necrosis by high extracellular K⁺, chelation of intracellular Ca²⁺, and calpain inhibition.

<p>(A) Suppression of potassium efflux by high K⁺ medium (140 mM) significantly inhibited rCPB-induced LDH release from pEC at 4 h, and still reduced LDH release at 16 h, when cells were exposed to 30 ng/ml rCPB. (B) Chelation of intracellular Ca²⁺ with BAPTA-AM (2 or 20 µM) or treatment with cyclosporin A (200 nM) markedly inhibited rCPB induced LDH release at 4 h, and to a lesser extend at 16 h post-exposure. LDH release of rCPB-treated cells in absence or presence of increasing concentrations of PD150606 (C) or calpeptin (D) was measured at 4 and 16 h. Bar graphs show summary of results from 2 to 5 independent experiments, which are expressed as percentage ± SEM relative to rCPB-treated cells in the absence of inhibitor or presence of low K⁺ medium ( = control). Statistical difference to control was assessed by 1-way ANOVA and Dunnnet post-hoc test. *P<0.05, **P<0.01. u = untreated cells, t = treated cells.</p

rCPB-induced efflux of K⁺ and accumulation of intracellular Ca²⁺.

<p>(A) Incubation of confluent pEC with 30 ng/ml rCPB resulted in rapid and significant reduction of intracellular K⁺. rCPB preincubated with mAb-CPB did not cause K⁺ efflux. Values represent means ± SEM from 3 independent experiments. Statistical difference to cells treated with neutralized toxin ( = control) was assessed by 2-way ANOVA and Bonferroni multiple comparisons test. *P<0.05, **P<0.01, ****P<0.0001. (B) Intracellular calcium ([Ca²⁺]_i) levels were measured by Fluo-4 fluorescence in the presence of 3 mM extracellular CaCl₂. Results are expressed as mean ± SEM from 3 independent experiments.</p

Characterization of rCBP-induced cell death by FACS analysis.

<p>pEC were either left untreated or exposed to 30 or 250 ng/ml of rCPB, or staurosporine, detached from the culture dishes by gentle trypsinization, and analyzed by FACS for PI uptake and GFP-annexin V staining at 2, 4 and 16 h of incubation. (A) Representative cytograms and bar graphs (B) in which cells in the lower left quadrant (PI/annexin V double-negative) were considered to be viable (white), cells in the upper left quadrant (PI single-positive) to be necrotic (gray), cells in the lower right quadrant (annexin V single-positive) to be apoptotic (light gray), and cells in the upper right quadrant (PI/annexin double-positive) to be either necrotic or secondary necrotic after apoptosis (dark gray). Bar graphs show quantitative summary of FACS analysis from 3 to 6 independent experiments. Results are expressed as mean ± SD. Statistical difference to control cells was assessed by non-parametric Kruskal-Wallis and Dunn’s post-hoc test. *P<0.05, **P<0.01, †P<0.001.</p

Effect of QVD and nectrostatin-1 on CPB-induced cell death.

<p>(A) Representative cytograms of cells exposed to 30 ng/ml of rCPB in the absence or presence of QVD (50 µM) (at 16 h) or necrostatin-1 (Nec-1, 50 µM) (at 4 h) (B) Bar graphs show quantitative summary of FACS analysis from 4 to 6 independent experiments. Results are expressed as mean ± SD. Same key as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064644#pone-0064644-g002" target="_blank">Figure 2B</a>. Statistical difference to control cells was assessed by non-parametric Kruskal-Wallis and Dunn’s post-hoc test. *P<0.05, **P<0.01, †P<0.001. (C) Bar graphs show % inhibition of LDH release of cells treated with QVD or Nec-1 compared to cells treated with toxin only (control) and represent results from 3 to 5 independent experiments. Statistical difference to control was assessed by 1-way ANOVA and Dunnnet post-hoc test, †P<0.001.</p

Morphological changes and LDH release induced by rCPB in pEC.

<p>(A) pEC were either left untreated (white) or exposed to rCPB or staurosporine (200 nM) for 4 h. Cell morphology was visualized using phase contrast microscopy. (B) Changes in total cell number (determined using automated cell counter) of pEC after 2, 4 and 16 h incubation with control medium, rCPB at indicated concentrations, neutralized rCPB using monoclonal anti-CPB antibodies, or staurosporine. Bar graphs represent the mean ± SEM of n = 3–6 independent experiments. Statistical difference to control cells was assessed by 1-way ANOVA and Dunnnet post-hoc test. *P<0.05, **P<0.01, †P<0.001. (C) The supernatants of pEC cultures from B were analyzed for LDH activity to determine LDH release after different times of exposure. Bar graph shows summary of results from 3 to 6 independent experiments expressed as percentage of activity compared to lysed control cells ( = 100%). Statistical difference to non-treated control cells was assessed by 1-way ANOVA and Dunnnet post-hoc test. *P<0.05, **P<0.01, †P<0.001.</p