Cell Cycle Inhibition To Treat Sleeping Sickness

African trypanosomiasis is caused by infection with the protozoan parasite Trypanosoma brucei. During infection, this pathogen divides rapidly to high density in the bloodstream of its mammalian host in a manner similar to that of leukemia. Like all eukaryotes, T. brucei has a cell cycle involving the de novo synthesis of DNA regulated by ribonucleotide reductase (RNR), which catalyzes the conversion of ribonucleotides into their deoxy form. As an essential enzyme for the cell cycle, RNR is a common target for cancer chemotherapy. We hypothesized that inhibition of RNR by genetic or pharmacological means would impair parasite growth in vitro and prolong the survival of infected animals. Our results demonstrate that RNR inhibition is highly effective in suppressing parasite growth both in vitro and in vivo. These results support drug discovery efforts targeting the cell cycle, not only for African trypanosomiasis but possibly also for other infections by eukaryotic pathogens

Endothelial transmigration by Trypanosoma cruzi.

Chagas heart disease, the leading cause of heart failure in Latin America, results from infection with the parasite Trypanosoma cruzi. Although T. cruzi disseminates intravascularly, how the parasite contends with the endothelial barrier to escape the bloodstream and infect tissues has not been described. Understanding the interaction between T. cruzi and the vascular endothelium, likely a key step in parasite dissemination, could inform future therapies to interrupt disease pathogenesis. We adapted systems useful in the study of leukocyte transmigration to investigate both the occurrence of parasite transmigration and its determinants in vitro. Here we provide the first evidence that T. cruzi can rapidly migrate across endothelial cells by a mechanism that is distinct from productive infection and does not disrupt monolayer integrity or alter permeability. Our results show that this process is facilitated by a known modulator of cellular infection and vascular permeability, bradykinin, and can be augmented by the chemokine CCL2. These represent novel findings in our understanding of parasite dissemination, and may help identify new therapeutic strategies to limit the dissemination of the parasite

CCL2 augments <i>T. cruzi</i> TEM.

<p>(A) To assess the effect of the chemokine CCL2 on TEM, EC monolayers were pre-incubated with CCL2 for 1 hour before the start of the standard TEM assay. Samples were washed briefly before the start of TEM, leaving a gradient of CCL2 in the collagen matrix. Other reagents were also pre-incubated with parasites and EC (in addition to CCL2) for 30 min. at the indicated concentrations before the start of the assay. After adding the parasites to the monolayers, TEM was allowed to proceed for 3 hours. Samples were incubated in the continuous presence of the reagents, except CCL2, which was only present during pre-incubation. Samples were then washed, fixed and scored as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081187#s2" target="_blank">Methods</a>. Data were collected from three experiments with 2–3 replicates per experiment. To correct for variations between parasite preparations and aid in visualization, data are shown as the fold change relative to control. * denotes p<0.05 relative to the control. (B) Permeability was quantified in samples that were identical to those in panel (A) except that 100 µg/mL FITC-dextran was included in the media during the incubation. After washing and fixation, the amount of FITC-dextran that had crossed the monolayer into the collagen matrix was quantified using fluorescence spectrophotometry. Data represent the average and standard deviation of six independent replicates. *n.s. - None of the indicated samples were statistically significant (p <0.05) relative to each other, and CCL2 treatment (200 nM) under these conditions did not significantly alter monolayer permeability in the presence or absence of <i>T. cruzi</i> (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081187#pone-0081187-g006" target="_blank">Figure 6B</a> compared to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081187#pone-0081187-g002" target="_blank">Figure 2</a>).</p

Elucidating determinants for <i>T. cruzi</i> TEM.

<p>ECs and <i>T. cruzi</i> were pre-incubated with the indicated reagents for 30 min prior to infection. Non-specific mouse IgG (ns-mIgG), mouse anti-human PECAM, and mouse anti-human CD99 antibodies were used at 20 µg/mL. Thapsigargin and wortmannin were used at 10 mM and 100 nM, respectively. To disrupt cellular energy production, samples were treated with 10 mM sodium azide and 6 mM deoxyglucose. After adding the parasites to the ECs, samples were then incubated for 3 hours in the continuous presence of the reagents before being washed, fixed and scored for transmigration as described. Data shown are the mean and standard error of the mean for three separate experiments with 2–3 replicates for each condition per experiment. * denotes p<0.05 relative to the control.</p

Comparative TEM among the <i>Kinetoplastidae</i>.

<p>(A) TEM was assayed for <i>T. cruzi</i>, <i>T. brucei</i>, and <i>Leishmania major</i> using our standard TEM method with 1×10⁵ parasites/well. Parasites were prepared as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081187#s2" target="_blank">Methods</a>. Where indicated, samples were treated (both during a 30 minute pre-incubation and during infection/TEM) with HK or HOE 140 at 20 ng/mL and 200 nM, respectively. Data shown represent the total TEM events per high power field to highlight the large differences observed with <i>T. brucei</i> and <i>L. major</i>. Data were collected from three independent experiments with 2–3 replicates per sample per experiment. * denotes significant difference relative to <i>T. cruzi</i> non-treated controls, p <0.05. (B) To confirm that <i>T. brucei</i> TEM was responsive to manipulations of the bradykinin signaling pathway, we treated samples with the indicated reagents (both pre-incubation and during TEM) and assayed TEM using the standard assay<b>.</b> To correct for variations between parasite preparations and aid in visualization, data are shown as the fold change relative to control (no treatment). * denotes p<0.05 relative to the control.</p

Permeability is not altered during <i>T. cruzi</i> infection and TEM.

<p>EC monolayers were incubated with <i>T. cruzi</i> identical to the standard TEM reaction except that 100 µg/mL FITC-dextran was included in the media to assay monolayer permeability. After a three hour incubation, samples were washed extensively and the amount of FITC-dextran that had crossed into the collagen matrix was quantified using a fluorescence spectrophotometer. Data shown represent the mean and standard deviation of six individual replicates. * denotes p<0.05 for the indicated comparisons.</p

Bradykinin augments <i>T. cruzi</i> TEM.

<p>Agonists and inhibitors of the bradykinin pathway were pre-incubated with parasites and EC for 30 min at the indicated concentrations before combining the two for the standard TEM assay. TEM was allowed to proceed for 3 hours in continuous presence of the reagents. Samples were then washed, fixed and scored as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081187#s2" target="_blank">Methods</a>. Data were collected from three experiments with 2–3 replicates per experiment. To correct for variations between parasite preparations and aid in visualization, data are shown as the fold change relative to control (no treatment). * denotes p<0.05 relative to the control.</p