Characterisation and diagnostic potential of extracellular small RNAs in filarial nematodes

Abstract

Filarial infections (lymphatic filariasis and onchocerciasis) are amongst the major neglected tropical diseases, and together account for more than 120 million infections in tropical and subtropical regions. The gold-standard technique for the diagnosis of filariases relies on the detection of microfilariae (mf) either in blood smears (lymphatic filariasis) or in skin biopsies (onchocerciasis). The secretion of extracellular RNAs (exRNAs) by parasitic nematodes has opened new avenues for the development of novel biomarkers for helminthiases, including filariasis. However, rather little is known about the origin and regulation of these RNAs inside the nematodes. One outstanding question is whether the secretion of small RNAs is distinct across the developmental stages of parasitic nematodes. Similarly, it is not clear whether the secretion of miRNAs is affected by treatment with anthelminthic chemotherapy or their potential as biomarkers for infection. Litomosoides sigmodontis is a murine filarial nematode closely related to filarial nematodes of medical and veterinary importance, including Onchocerca spp. and Brugia spp. L. sigmodontis has been extensively used to decipher multiple aspects of filarial biology, including parasite development, vaccine, and host-pathogen interactions. Therefore, we decided to use this model to address fundamental questions regarding the secretion of small RNAs and their biomarker potential. Our in vitro studies demonstrate that some extracellular miRNAs are enriched in a sexand stage-specific manner in the Excretion/Secretion (ES) products from early larval and adult stages from the rodent filarial nematode Litomosoides sigmodontis. Moreover, our data demonstrates that the gravid adult female worms secrete a plethora of miRNAs enriched in the secretome of this developmental stage when compared to adult males or mf. Further characterization studies show that the miRNAs are likely to be secreted in association with extracellular vesicles (EVs), as previously reported for other parasitic nematodes, including the human pathogen Brugia malayi. Interestingly, Ivermectin, which is typically used to treat filarial infections, does not have consistent effects on the secretion of miRNAs by gravid adult female worms in vitro, requiring further in vivo experiments to determine the effect of IVM on detection of extracellular parasite-derived miRNAs. In vivo experiments, using murine models of infection with L. sigmodontis (gerbils and BALB/c mice), as well as human samples from patients infected with Onchocerca volvulus and cattle infected with Onchocerca ochengi, demonstrated the presence of filarial-derived miRNAs, including female-specific miRNA markers, in biofluids from infected hosts. Further statistical analysis showed that two parasite-derived miRNAs, miR-71 and miR-100d, can significantly discriminate infected animals from naïve controls with high sensitivity/specificity (>80%/100%). The results presented in this PhD thesis provide an initial framework to understand the secretion of small RNAs throughout nematode development, the potential interactions between anthelminthic chemotherapy and small RNA trafficking and secretion, as well as the use of parasite-derived miRNAs for the development of a new generation of biomarkers for filarial infections.