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

Simone S C Oliveira,Camila G R Elias,Marta H Branquinha,Felipe A Dias,Claudia M D'Avila-Levy,André L S Santos,Angela H Lopes

doi:10.3389/fcimb.2021.819133

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.

Highlights

The genus Phytomonas (Kinetoplastea: Trypanosomatidae) was first mentioned in the early 1900s when proposed a classification for flagellate trypanosomatids found in plants (Camargo, 1999; Jaskowska et al, 2015)
Analysis by flow cytometry showed that parasites grown in brain–heart infusion (BHI)-natural tomato extract (NTE) 25% and BHI-NTE 50% for 48 h presented similar cell size and granularity when compared to parasites grown in the standard BHI medium, as revealed by both forward scatter (FSC) and side scatter (SSC) flow cytometry parameters (Figure 1)
The presence of NTE in the culture medium was able to reduce the proliferation rate of P. serpens in a dosedependent manner: the cultivation in BHI-NTE 25% promoted a reduction of approximately 17%, while the cultivation in BHI-NTE 50% of around 30% (Figure 1)

Summary

Introduction

The genus Phytomonas (Kinetoplastea: Trypanosomatidae) was first mentioned in the early 1900s when proposed a classification for flagellate trypanosomatids found in plants (Camargo, 1999; Jaskowska et al, 2015). There is no precise available information about the pathogenicity of trypanosomatids in edible fruits such as tomatoes, oranges and grapes, flagellates usually remain limited around the point of inoculation. When it comes to P. serpens, the presence of these trypanosomatids provoke only yellowish spots on the fruit surface that are not ascertained to be real injuries to tomato; a loss in nutritional quality and, above all, a loss in economic value added to the product are undoubtedly documented (Camargo, 1999)

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Conclusion