Caracterização proteômica dos Produtos de Excreção-Secreção de Trichuris muris e sua interação in vitro com bactérias da microbiota

Abstract

Trichuriasis is a neglected disease that affects millions of people and is caused by the intestinal nematode Trichuris trichiura. These helminths produce excretory/secretory products (ESPs), which are rich in microRNAs and proteins with various functions, such as immunomodulation. To establish themselves in the host's large intestine, these helminths penetrate the organ wall, promoting epithelial disruption and allowing bacterial translocation. The parasite-microbiota-host interaction has been studied by our group using the T. muris infection model in mice. In this study, our objective was to extract ESPs from T. muris maintained in Swiss mice, either subjected or not to antibiotic therapy, and to verify whether this treatment alters the protein composition of the ESPs. Additionally, we aimed to analyze the antimicrobial activity of these ESPs and assess their potential influence on the interaction between bacteria and intestinal T84 and Caco-2 cells in vitro. First, mice were infected with T. muris and either treated with antibiotics or left untreated (controls). The recovered parasites were maintained in culture in RPMI medium at 37 °C/CO₂ for 24 hours. Subsequently, some of these samples were subjected to mass spectrometry to analyze the proteins, followed by data extraction and analyses of quantification, concentration, KEGG pathways, gene ontology (GO), signal peptide presence, and network analysis. The ESPs were then evaluated for their ability to inhibit bacterial growth and their interaction with Escherichia coli 042, E. coli DH5α, and Salmonella c20 and the T84 and Caco-2 cells. After 6 hours, the material was processed and analyzed using light microscopy and scanning electron microscopy. In our results, we observed differences in the proteins found in each group of treated and untreated mice. The response in the treated group was more complex than in the untreated group, showing more proteins with more diverse functions. Some of the proteins found in both groups have various functions, such as antimicrobial and immunomodulatory activities, according to the literature. However, in the antibiogram, bacterial growth was not inhibited by the ESPs. We did not identify patterns in the bacterial quantity between groups with and without ESPs in any of the cell monolayers. However, we observed differences in bacterial behavior, such as changes in aggregation and invasive potential, particularly in E. coli 042 and E. coli DH5α when in contact with ESPs. We concluded that ESPs are rich in proteins with known biological activities and that their composition varies depending on the environment, potentially interacting with bacteria that comprise the host's microbiota.