Molecular Recognition of Surface Trans-Sialidases in Extracellular Vesicles of the Parasite Trypanosoma cruzi Using Atomic Force Microscopy (AFM)

Abstract

Trans-sialidases (TS) are important constitutive macromolecules of the secretome present on the surface of Trypanosoma cruzi (T. cruzi) that play a central role as a virulence factor in Chagas disease. These enzymes have been related to infectivity, escape from immune surveillance and pathogenesis exhibited by this protozoan parasite. In this work, atomic force microscopy (AFM)-based single molecule-force spectroscopy is implemented as a suitable technique for the detection and location of functional TS on the surface of extracellular vesicles (EVs) released by tissue-culture cell-derived trypomastigotes (Ex-TcT). For that purpose, AFM cantilevers with functionalized tips bearing the anti-TS monoclonal antibody mAb 39 as a sense biomolecule are engineered using a covalent chemical ligation based on vinyl sulfonate click chemistry; a reliable, simple and efficient methodology for the molecular recognition of TS using the antibody-antigen interaction. Measurements of the breakdown forces between anti-TS mAb 39 antibodies and EVs performed to elucidate adhesion and forces involved in the recognition events demonstrate that EVs isolated from tissue-culture cell-derived trypomastigotes of T. cruzi are enriched in TS. Additionally, a mapping of the TS binding sites with submicrometer-scale resolution is provided. This work represents the first AFM-based molecular recognition study of Ex-TcT using an antibody-tethered AFM probe.

Integrated morphological and transcriptome profiles reveal a highly-developed extrusome system associated to virulence in the notorious fish parasite, Ichthyophthirius multifiliis

“…We hypothesized that the theront releases these enzymes to induce necrosis of fish epithelial cells. Similar mechanisms have been reported in other parasites, including Taxoplasma [ 35 ], Plasmodium [ 36 ], and Trypanosoma [ 37 ]. In addition, we found 14 proteins in the leishmanolysin family specifically expressed in the theront and trophont stages (Table S1).…”

Guidelines for the purification and characterization of extracellular vesicles of parasites

“…Thus, the characterization and functional assays (Cronemberger‐Andrade et al., 2020 ; Gutierrez et al., 2022 ; Nogueira et al., 2015 ) and the detection of biomarkers (Madeira et al., 2021 , 2022 ) can be performed. Osuna group (Prescilla‐Ledezma et al., 2022 ) suggested that EVs isolated from tissue‐culture derived trypomastigotes can be characterized using AFM‐based single molecule‐force spectroscopy as a suitable technique for the detection and location of functional components on the surface of EVs. To detect trans‐sialidase (TS) and other specific genes in EVs, it is possible to use specific monoclonal antibodies.…”

“…To detect trans‐sialidase (TS) and other specific genes in EVs, it is possible to use specific monoclonal antibodies. For example, it has been shown the presence of TS using coupled antibodies to sensors (Prescilla‐Ledezma et al., 2022 ). The components present in T. cruzi EVs obtained with different methodologies, their characterization, and the biological function are summarized in Table 1 .…”

The End Justifies the Means: Chagas Disease from a Perspective of the Host–Trypanosoma cruzi Interaction

“…Extracellular vesicles are lipid bilayer nanoparticles that mainly comprise exosomes and microvesicles. EVs released by T. cruzi contain several virulence factors, such as trans-sialidases, peptidyl-prolyl cis-trans isomerase, oligopeptidases and proteases [130,[132][133][134]. In addition to EVs secreted by the parasite, EVs coming from infected host cells or cells in contact with the parasite also participate in communication (Figure 4).…”