The Enhanced Expression of Cruzipain-Like Molecules in the Phytoflagellate Phytomonas serpens Recovered From the Invertebrate and Plant Hosts

Abstract

Phytomonas serpens is a protozoan parasite that alternates its life cycle between two hosts: an invertebrate vector and the tomato fruit. This phytoflagellate is able to synthesize proteins displaying similarity to the cysteine peptidase named cruzipain, an important virulence factor from Trypanosoma cruzi, the etiologic agent of Chagas disease. Herein, the growth of P. serpens in complex medium (BHI) supplemented with natural tomato extract (NTE) resulted in the increased expression of cysteine peptidases, as verified by the hydrolysis of the fluorogenic substrate Z-Phe-Arg-AMC and by gelatin-SDS-PAGE. Phytoflagellates showed no changes in morphology, morphometry and viability, but the proliferation was slightly reduced when cultivated in the presence of NTE. The enhanced proteolytic activity was accompanied by a significant increase in the expression of cruzipain-like molecules, as verified by flow cytometry using anti-cruzipain antibodies. In parallel, parasites incubated under chemically defined conditions (PBS supplemented with glucose) and added of different concentration of NTE revealed an augmentation in the production of cruzipain-like molecules in a typically dose-dependent way. Similarly, P. serpens recovered from the infection of mature tomatoes showed an increase in the expression of molecules homologous to cruzipain; however, cells showed a smaller size compared to parasites grown in BHI medium. Furthermore, phytoflagellates incubated with dissected salivary glands from Oncopeltus fasciatus or recovered from the hemolymph of infected insects also showed a strong enhance in the expression of cruzipain-like molecules that is more relevant in the hemolymph. Collectively, our results showed that cysteine peptidases displaying similarities to cruzipain are more expressed during the life cycle of the phytoflagellate P. serpens both in the invertebrate and plant hosts.

Keywords: Oncopeltus fasciatus; Phytomonas serpens; cruzipain; interaction; invertebrate vector; tomato.

Figure 1

Effects of natural tomato extract (NTE) on Phytomonas serpens morphology and growth after cultivation in BHI medium for 48 h in the absence (BHI) or in the presence of different concentrations of NTE (25 and 50%). pH values were also measured in each culture medium before inoculation (0 h) and after 48 h of growth. Cells were analyzed by flow cytometry in order to measure two morphometric parameters, forward scatter (FSC) and side scatter (SSC). The values expressed represent the mean of fluorescence intensity of three independent experiments. The symbol (*) represents the significant difference (P < 0.05; Student’s t-test) between treated and control groups. The influence of NTE was also evaluated during cell proliferation (initial inoculum of 1 x 10⁶ parasites/mL) by counting cells in a Neubauer chamber after 48 h. In parallel, Giemsa-stained smears were analyzed under an optical microscope. Promastigote forms present a kinetoplast (k), the central nucleus (n), an elongated cell body and a flagellum (f) attached to the parasite cell body. Bars: 10 µM. In scanning electron microscopy analysis of promastigotes, note that no morphological changes were identified after growth of parasites in these different media. Bars: 5 µm.

Figure 2

Effects of natural tomato extract (NTE) on cysteine peptidase activity in promastigotes of P. serpens cultivated in BHI medium for 48 h in the absence or in the presence of different concentrations of NTE (25 and 50%). (A) Cysteine ​​peptidase activity in parasite lysates was assessed by measuring the hydrolysis of Z–Phe–Arg–AMC. Results are expressed as arbitrary fluorescence units (AFU). Data shown are the mean ± standard deviation (SD) of three independent experiments performed in triplicate. Symbols denote statistical differences (P < 0.05; Student’s t-test) between cells grown in BHI medium and cells grown in BHI-NTE medium (25% or 50%). (B) The peptidase profiles in cell extracts were analyzed by means of gelatin-SDS-PAGE; gel strips were incubated at 37°C in sodium phosphate buffer, pH 5.0, supplemented with 2 mM DTT. (C) Gel strips were also incubated in the absence (control) or presence of cysteine peptidase inhibitors: 1 μM cystatin, 1 μM leupeptin, 1 μM antipain, 1 μM iodoacetamide and 1 μM E-64. Molecular masses, expressed in kDa, are represented on the left. The proteolytic class was determined in cell extracts of parasites grown in BHI medium, but samples from BHI-NTE media were tested in parallel, and similar results were detected.

Figure 3

Detection of cruzipain-like molecules in P. serpens cultivated in BHI medium supplemented or not with either 25% or 50% natural tomato extract (NTE). Promastigotes were cultured for 48 h and then fixed and processed for flow cytometry analysis using anti-cruzipain antibody. The histogram expresses the mean of fluorescence intensity (MFI) levels. Values represented in the table express the percentage of fluorescent cells (%FC). Each experiment was performed at least three independent times. The symbols indicate the experimental systems considered statistically significant from the BHI medium (*, P < 0.05 and **, P < 0.01; Student’s t-test).

Figure 4

Detection of cruzipain-like molecules in P. serpens cultivated in PBS-glucose supplemented or not with 0.1%, 1% or 10% natural tomato extract (NTE). Promastigotes were cultured for 3 h and then fixed and processed for flow cytometry analysis using anti-cruzipain antibody. The histogram expresses the mean of fluorescence intensity (MFI) levels. Values represented in the table express the percentage of fluorescent cells (%FC). Each experiment was performed at least three independent times. The symbols indicate experimental systems considered statistically significant from the PBS-glucose medium (*, P < 0.05 and **, P < 0.01; Student’s t-test).

Figure 5

Detection of cruzipain-like molecules in P. serpens after infection of tomatoes (S. lycopersicum). Promastigotes recovered from infected tomatoes or cultured in BHI medium were washed and analyzed by flow cytometry. (A) Morphometric parameters analysis, forward scatter (FSC) and side scatter (SSC); (B) Analysis of the expression of cruzipain-like molecules by flow cytometry using anti-cruzipain antibody. The histogram expresses the mean of fluorescence intensity (MFI) levels. Values represented in the table express the percentage of fluorescent cells (%FC). Each experiment was performed at least three independent times. The symbols indicate experimental systems considered statistically significant from the BHI medium (**, P < 0.01; Student’s t-test).

Figure 6

Detection of cruzipain-like molecules in P. serpens after infection of Oncopeltus fasciatus. Parasites recovered after parasite interaction with explanted salivary glands of O. fasciatus and recovered from the hemolymph of infected insects were washed, resuspended in PBS and processed for flow cytometry analysis. The histogram expresses the mean of fluorescence intensity (MFI) levels using anti-cruzipain antibody. Values represented in the table express the percentage of fluorescent cells (%FC). Each experiment was performed at least three independent times. The symbols indicate the experimental systems considered statistically significant from the BHI medium (*, P < 0.05 and **, P < 0.01; Student’s t-test).