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. 2012;6(12):e1958.
doi: 10.1371/journal.pntd.0001958. Epub 2012 Dec 13.

Cruzipain promotes Trypanosoma cruzi adhesion to Rhodnius prolixus midgut

Affiliations

Cruzipain promotes Trypanosoma cruzi adhesion to Rhodnius prolixus midgut

Lívia Almeida Uehara et al. PLoS Negl Trop Dis. 2012.

Abstract

Background: Trypanosoma cruzi is the etiological agent of Chagas' disease. Cysteine peptidases are relevant to several aspects of the T. cruzi life cycle and are implicated in parasite-mammalian host relationships. However, little is known about the factors that contribute to the parasite-insect host interaction.

Methodology/principal findings: Here, we have investigated whether cruzipain could be involved in the interaction of T. cruzi with the invertebrate host. We analyzed the effect of treatment of T. cruzi epimastigotes with anti-cruzipain antibodies or with a panel of cysteine peptidase inhibitors (cystatin, antipain, E-64, leupeptin, iodocetamide or CA-074-OMe) on parasite adhesion to Rhodnius prolixus posterior midgut ex vivo. All treatments, with the exception of CA074-OMe, significantly decreased parasite adhesion to R. prolixus midgut. Cystatin presented a dose-dependent reduction on the adhesion. Comparison of the adhesion rate among several T. cruzi isolates revealed that the G isolate, which naturally possesses low levels of active cruzipain, adhered to a lesser extent in comparison to Dm28c, Y and CL Brener isolates. Transgenic epimastigotes overexpressing an endogenous cruzipain inhibitor (pCHAG), chagasin, and that have reduced levels of active cruzipain adhered to the insect gut 73% less than the wild-type parasites. The adhesion of pCHAG parasites was partially restored by the addition of exogenous cruzipain. In vivo colonization experiments revealed low levels of pCHAG parasites in comparison to wild-type. Parasites isolated after passage in the insect presented a drastic enhancement in the expression of surface cruzipain.

Conclusions/significance: These data highlight, for the first time, that cruzipain contributes to the interaction of T. cruzi with the insect host.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Cysteine peptidase inhibitors reduce the interaction between T. cruzi and R. prolixus midgut.
The parasites (×ばつ107 cells) were treated for 60 min at room temperature with chicken egg white cystatin (1 μg/mL), antipain, E-64, leupeptin, iodocetamide or CA-074-OMe, at 10 μM. The viability of the parasites was not affected by the treatments used in this set of experiments. Following interaction for 15 min with the insect gut, the number of adhered parasites/insect gut epithelial cells was estimated by randomly counting at least 100 epithelial cells in quadruplicate. The results are shown as the mean ± SEM of two independent experiments. The symbol indicates systems significantly different from untreated (control) cells by means of Students' t test (formula image, P<0.001).
Figure 2
Figure 2. Dose dependent effect of cystatin on the interaction between T. cruzi and R. prolixus midgut.
Epimastigotes (×ばつ107 cells) were treated for 60 min at room temperature with different concentrations of cystatin from chicken egg white (0.1, 1, 2.5 or 10 μg/mL). The viability of the parasites was not affected by the treatments used in this set of experiments. Following interaction for 15 min with the insect gut, the number of parasites/insect gut epithelial cells was estimated by randomly counting at least 100 epithelial cells in quadruplicate. The results are shown as the mean ± SEM of two independent experiments. Symbols indicate systems significantly different from untreated (control) cells by means of Students' t test (formula image, P<0.001).
Figure 3
Figure 3. Antibodies to cruzipain affect the interaction between T. cruzi and R. prolixus midgut.
The epimastigotes (×ばつ107 cells) were treated for 60 min at room temperature with anti-cruzipain antibodies at 1∶1000 or 1∶2500 dilution, or pre-immune serum at 1∶1000. The viability of the parasites was not affected by the treatment used in this set of experiments. Following interaction for 15 min with the insect gut, the number of adhered parasites/insect gut epithelial cells was estimated by randomly counting at least 100 epithelial cells in quadruplicate. The results are shown as the mean ± SEM of two independent experiments. Symbols indicate systems significantly different from untreated (control) cells by means of Students' t test (formula image, P = 0.000802; formula image formula image, P = 0.004351).
Figure 4
Figure 4. T. cruzi chagasin overexpressing transfectants present reduced interaction rate with R. prolixus midguts.
Epimastigotes (×ばつ107 cells) wild-type, or transfected with empty vector (pTEX) or with the vector containing the chagasin gene (pCHAG) were incubated with the dissected guts of the triatomine for 15 min at room temperature. The number of adhered parasites/insect gut epithelial cells was estimated by randomly counting at least 100 epithelial cells in quadruplicate. Results are shown as the mean ± SEM of two independent experiments. Parasites pCHAG showed adhesion rate significantly different from the wild-type and of control pTEX parasites by means of Students' t test (formula image, P<0.001) (A). The insets show phase-contrast photomicrography of T. cruzi wild-type (B) or pCHAG (C) incubated ex vivo with dissected posterior midgut epithelial cells of R. prolixus, 10 days after the blood meal. ×ばつ· The arrows in (B) and (C) indicate the parasites attached to the epithelial cells.
Figure 5
Figure 5. Exogenous cruzipain restores the interaction of chagasin overexpressing transfectants with R. prolixus midguts.
Exogenous cruzipain was supplemented in the interaction media at 1.875, 3.75 and 7.5 ng/μL. Following interaction for 15 min with the insect gut, the number of parasites/insect gut epithelial cells was estimated by randomly counting at least 100 epithelial cells in quadruplicate. The results are shown as the mean ± SEM of two independent experiments. Parasites pCHAG supplemented with exogenous cruzipain had an adhesion rate significantly different from the parasites pCHAG using Students' t test (formula image, P<0.001).
Figure 6
Figure 6. Interaction rate of different T. cruzi isolates with R. prolixus midguts.
Epimastigote forms (×ばつ107 cells) of Dm28c, CL-Brener, Y and G strains were incubated with the dissected guts of the triatomine for 15 minutes at room temperature. Following, the number of parasites/insect gut epithelial cells was estimated by randomly counting at least 100 epithelial cells in quadruplicate. The results are shown as the mean ± SEM of two independent experiments. G strain had an adhesion rate significantly different from the others T. cruzi isolates by means of ANOVA variance (P≤0.05).
Figure 7
Figure 7. In vivo infection of R. prolixus by T. cruzi is reduced upon chagasin overexpression.
The insects were fed with defibrinated rabbit blood containing ×ばつ106 parasites/mL (Wild-type, pTEX, pCHAG) ad libitum. Twenty days later, intact pools of 4 midguts or 8 recta were excised from the insects, and processed as described in Methods section. The parasites were quantified by the following methods: (A) microscopic counts of the infection levels of T. cruzi in the rectum of R. prolixus, (B, C) quantitative PCR (qPCR) for estimating the infection levels of T. cruzi in the midgut or rectum of R. prolixus. The results are shown as the mean ± SEM of three independent experiments. Parasites pCHAG showed a rate of adhesion to the cells not statistically different from control and pTEX by means of Students' t test, due to intrinsic biological triatomine variance.
Figure 8
Figure 8. Cruzipain expression in T. cruzi cells is enhanced after passage in R. prolixus.
Cells re-isolated after the colonization (3) or cells obtained from cultivation in axenic medium (BHI) (2) were incubated in the presence or absence (1) of anti-cruzipain antibodies at 1∶2000 dilution and then analyzed by flow cytometry. Representative data of the analysis of 10,000 cells from one of three experiments are shown. The curve 3 represents 10,000 events positive for cruzipain.

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