Moraes, C. B. | bioRxiv Science

bioRxiv2025

Ultra-high throughput in vitro translation assay for identification of Trypanosoma cruzi-specific protein synthesis inhibitors

Chagas disease, a global health threat exacerbated by the migration of infected individuals, raises significant concern due to its restricted treatment options, resulting in around 12,000 fatalities annually. The disease is caused by Trypanosoma cruzi, a protozoan parasite. T. cruzi possesses a divergent translation apparatus, characterized by enlarged ribosomes and a unique mRNA cap appended to the 5 end of a 39-nucleotide spliced leader inserted upstream of all its mRNAs. Given that many anti-microbial agents are protein synthesis inhibitors and the divergence of T. cruzi protein synthetic machinery compared to its mammalian host, it is likely that T. cruzi-specific protein synthesis inhibitors can be employed for the treatment of Chagas disease. Such inhibitors can be identified by screening chemical libraries in an in vitro translation assay. However, lysates of T. cruzi cannot reinitiate translation of exogenous mRNAs. Here, we demonstrate that T. cruzi extracts derived from a strain deficient for hemin accumulation efficiently translate a capped and polyadenylated reporter mRNA. We optimized this assays conditions and adapted it to a 384-well format. In a proof of principle pilot study, we identified quinazoline compounds that inhibit translation of reporter mRNAs by T. cruzi extracts. These compounds inhibited protein synthesis in live wild-type parasites. We further miniaturized this assay to a 5 {micro}L volume, demonstrating its suitability for ultra-high throughput screening in a 1536-well format with appropriate automation. This first-of-its-kind T. cruzi ultra-high-throughput in vitro translation assay will enable screening of very large chemical libraries while hit compounds identified in the pilot study can be developed into lead compounds by synthesis and/or assembly of focused libraries to develop therapeutics for Chagas disease.

biochemistry↗

bioRxiv2023

ST8Sia2 polysialyltransferase protects against infection by Trypanosoma cruzi

Glycosylation is one of the most structurally and functionally diverse co- and post-translational modifications in a cell. Addition and removal of glycans, especially to proteins and lipids, characterize this process which have important implications in several biological processes. In mammals, the repeated enzymatic addition of a sialic acid unit to underlying sialic acids (Sia) by polysialyltransferases, including ST8Sia2, leads to the formation of a sugar polymer called polysialic acid (polySia). The functional relevance of polySia has been extensively demonstrated in the nervous system. However, the role of polysialylation in infection is still poorly explored. Previous reports have shown that Trypanosoma cruzi (T. cruzi), a flagellated parasite that causes Chagas disease (CD), changes host sialylation of glycoproteins. To understand the role of host polySia during T. cruzi infection, we used a combination of in silico and experimental tools. We observed that T. cruzi reduces both the expression of the ST8Sia2 and the polysialylation of target substrates. We also found that chemical and genetic inhibition of host ST8Sia2 increased the parasite load in mammalian cells. These findings suggest a novel approach to interfere with parasite infections through modulation of host polysialylation. AUTHOR SUMMARYGlycosylation is a co- and/or post-translational modification regulated by the addition and removal of glycans. This process shapes the cellular glycome, which in turn, holds significant implications in various biological processes. Trypanosoma cruzi (T. cruzi), the etiological agent of Chagas disease, a globally concerning neglected tropical disease affecting 6 to 8 million individuals worldwide, exerts a profound influence on host glycoprotein sialylation. Remarkably, T. cruzi is incapable of synthesizing sialic acid (Sia) and relies on acquiring it from host glycoconjugates. In mammals, the formation of polysialic acid (polySia) is mediated by polysialyltransferases, such as ST8Sia2. The functional relevance of polySia has been extensively documented in the nervous system. Nevertheless, its role within the context of infectious processes remains largely unexplored. Herein, we demonstrate that in T. cruzi-infected host cells, the expression of the ST8Sia2 enzyme is downregulated, resulting in diminished levels of polysialylation. Furthermore, a reduction in the levels of NCAM1 and SCN5A was observed, which can be attributed to the decreased host polysialylation. Moreover, enzymatic removal of polySia, along with chemical inhibition and genetic silencing of ST8Sia2, led to a marked increase in the number of intracellular parasites. We posit that ST8Sia2 inhibition favors T. cruzi infection, thereby elucidating novel avenues for understanding the mechanisms associated with Chagas disease pathogenesis, prominently featuring the pivotal role of host polysialylation.

microbiology↗