Trypanosoma cruzi interaction with host tissues modulate the composition of large extracellular vesicles

Abstract

Trypanosoma cruzi is the protozoan that causes Chagas disease (CD), an endemic parasitosis in Latin America distributed around the globe. If CD is not treated in acute phase, the parasite remains silent for years in the host's tissues in a chronic form, which may progress to cardiac, digestive or neurological manifestations. Recently, studies indicated that the gastrointestinal tract represents an important reservoir for T. cruzi in the chronic phase. During interaction T. cruzi and host cells release extracellular vesicles (EVs) that modulates the immune system and infection, but the dynamics of secretion of host and parasite molecules through these EVs is not understood. Now, we used two cell lines: mouse myoblast cell line C2C12, and human intestinal epithelial cell line Caco-2to simulate the environments found by the parasite in the host. We isolated large EVs (LEVs) from the interaction of T. cruzi CL Brener and Dm28c/C2C12 and Caco-2 cells upon 2 and 24 h of infection. Our data showed that at two hours there is a strong cellular response mediated by EVs, both in the number, variety and enrichment/targeting of proteins found in LEVs for diverse functions. Qualitative and quantitative analysis showed that proteins exported in LEVs of C2C12 and Caco-2 have different patterns. We found a predominance of host proteins at early infection. The parasite-host cell interaction induces a switch in the functionality of proteins carried by LEVs and a heterogeneous response depending on the tissues analyzed. Protein-protein interaction analysis showed that cytoplasmic and mitochondrial homologues of the same parasite protein, tryparedoxin peroxidase, were differentially packaged in LEVs, also impacting the interacting molecule of this protein in the host. These data provide new evidence that the interaction with T. cruzi leads to a rapid tissue response through the release of LEVs, reflecting the enrichment of some proteins that could modulate the infection environment.

Keywords: Trypanosoma cruzi; Chagas disease; Extracellular vesicles; Mass spectrometry; Microvesicles.

Figure 1

Workflow of the experimental design to obtain 2 and 24 h post infection (h.p.i.) LEVs. Myoblasts (C2C12) and intestinal epithelium (Caco-2) cells were seeded and infected with T. cruzi TCTs belonging to two distinct strains (CL Brener, DTU Tc VI and Dm28c DTU Tc I). LEVs were collected in the first 2 h and after 24 h of infection. LEVs derived from isolated C2C12, Caco-2 and T. cruzi TCTs were used as controls. LEVs were lysed, their proteins were separated and digested in-gel with trypsin and the peptides were analyzed by LC–MS/MS for protein identification and quantification.

Figure 2

(A) Number of proteins identified in the 2 h and 24 h LEVs of myoblasts (C2C12) and epithelial cells (Caco-2) cells with or without contact with T. cruzi. (B) Proportion of host (mouse or human) and T. cruzi proteins identified in LEVs. (C) Subcellular localization of host proteins present in LEVs 2 h. (D) Subcellular localization of host proteins present in LEVs 24 h.

Figure 3

Venn diagrams of proteins present in LEVs. (A) 2 h LEVs of myoblasts (C2C12). (B) 2 h LEVs of epithelial cells (Caco-2). (C) 24 h LEVs of myoblasts (C2C12). (D) 24 h LEVs of epithelial cells (Caco-2).

Figure 4

Hierarchical clustering of T. cruzi LEVs proteins. Heatmaps showing the expression level of LEV proteins from T. cruzi strains CL Brener and Dm28c isolated and during interaction for 2 h and 24 h with myoblasts (C2C12) (A) and epithelial cells (Caco-2) (B). Each condition is represented by a color and numbered according to the replicate. CL Bre Ctl and Dm28c Ctl correspond to LEV proteins coming from the isolated parasite (without contact with host cells) for 2 h; the other samples refer to the LEVs proteins coming from the contact of each strain with the host cell for 2 h or 24 h.

Figure 5

Hierarchical clustering of host cell LEV proteins. Heatmaps showing the expression level of LEV proteins from myoblasts (C2C12) (A) and epithelial cells (Caco-2) (B) isolated and under interaction for 2 h and 24 h with T. cruzi CL Brener and Dm28c strains. Each condition is represented by a color and numbered according to the replicate. C2C12 Ctl and Caco-2 Ctl correspond to LEV proteins coming from the isolated host cells (without contact with T. cruzi) for 2 h; the other samples refer to the LEV proteins coming from the contact of host cell with each T. cruzi strain with the for 2 h or 24 h.

Figure 6

Differentially abundant proteins in LEVs. Volcano plots representing statistically significant proteins identified in LEVs of interaction between T. cruzi with C2C12 and Caco-2 during 2 h.p.i (A) and 24 h.p.i. (B). Statistically significant proteins are in red (host proteins) and blue (T. cruzi proteins).

Figure 7

Biological processes enriched in C2C12 (A) and Caco-2 (B) LEV proteins during 2 h.p.i. with each T. cruzi strain. Colors indicate the value of -log₁₀(p-value). Terms indicated in gray were not significantly enriched in the condition.

Figure 8

Interactome of LEV proteins of T. cruzi CL Brener (A) and Dm28c (B) strains with epithelial cells (Caco-2). Circles: Caco-2 proteins, diamonds: T. cruzi proteins.

Figure 9

Interactome of LEV proteins of T. cruzi CL Brener (A) and Dm28c (B) strains with myoblast (C2C12). Circles: C2C12 proteins, diamonds: T. cruzi proteins.