Heterologous expression in Caenorhabditis elegans as an alternative approach to functional studies in Schistosoma mansoni.

Sandra G Gava,Ã‚Ngela Volpini,Riva De Paula Oliveira,Guilherme Oliveira,Larissa L S Scholte

doi:10.3389/fgene.2014.00120

Abstract

The lack of an accurate diagnosis has been a serious obstacle to the advancement of the anti-Trypanosoma cruzi chemotherapy and long-term infection can result in different health risks to human. PCRs are alternative methods, more sensitive than conventional parasitological techniques, which due to their low sensitivities are considered unsuitable for these purposes. The aim of this study was to investigate a sensitive diagnostic strategy to quantify blood and cardiac tissues parasites based on real-time PCR tools during acute and chronic phases of murine Chagas disease, as well as to monitor the evolution of infection in those mice under specific treatment. In parallel, fresh blood examination, immunological analysis and quantification of cardiac inflammation were also performed to confront and improve real-time PCR data. Similar profiles of parasitemia curves were observed in both quantification techniques during the acute phase of the infection. In contrast, parasites could be quantified only by real-time PCR at 60 and 120 days of infection. In cardiac tissue, real-time PCR detected T. cruzi DNA in 100% of infected mice, and using this tool a significant Pearson correlation between parasite load in peripheral blood and in cardiac tissue during acute and chronic phases was observed. Levels of serum CCL2, CCL5 and nitric oxide were coincident with parasite load but focal and diffuse mononuclear infiltrates was observed, even with significant (p < 0.05) reduction of parasitism after 60 days of infection. Later, this methodology was used to monitor the evolution of infection in animals treated with itraconazole (Itz). Itz-treatment induced a reduction of parasite load in both blood and cardiac muscle at the treatment period, but after the end of chemotherapy an increase of parasitism was detected. Interestingly, inflammatory mediators levels and heart inflammation intensity had similar evolution to the parasite load, in the group of animals treated. Taken together, our data show that real-time PCR strategy used was suitable for studies of murine T. cruzi infection and may prove useful in investigations involving experimental chemotherapy of the disease and the benefits of treatment in relation to parasitism and inflammatory respons.

Highlights

The use of heterologous expression systems and expression vectors provide a powerful tool for studying the cellular functions of specific genes in their natural cellular environment or in specialized host organisms (Porro et al, 2005)
Genetic manipulation it is difficult to be achieved in parasitic helminthes, significant advances have been reported towards development of transgenic schistosomes as gene silencing by RNA interference (RNAi) and transient and stable transfection including transgenesis mediated by genome integration using competent vectors or retrovirus (Beckmann and Grevelding, 2012; Mann et al, 2014)
We described for the first time the use of C. elegans as an alternative heterologous host to functional studies in S. mansoni

Summary

Introduction

The use of heterologous expression systems and expression vectors provide a powerful tool for studying the cellular functions of specific genes in their natural cellular environment or in specialized host organisms (Porro et al, 2005). In order to advance our knowledge concerning SmJNK gene function we explored the nematode Caenorhabditis elegans as a heterologous platform to investigate whether S. mansoni JNK represents a physiologically functional protein. We tested whether the overexpression of SmJNK in C. elegans would result in similar phenotypes, enabling the demonstration that the schistosome enzyme is active and capable of complementing the function of the original gene.

Results

Conclusion