Development of a trypanosome-based system to investigate the function of helminth secreted proteins in vivo

Abstract

Parasitic helminths are able to secrete molecules which modulate their host’s immune system. Evidence from epidemiological data and lab models suggest that these molecules could prevent and treat immunopathologies, such as allergy and autoimmune disorders. Although some parasitic helminth-derived proteins have been shown to be immunomodulatory, the role of many secreted proteins have not been elucidated. Unfortunately reverse genetic approaches such as RNAi and transgenic gene knockdown have been difficult in many species of parasitic nematode. Here, it is proposed that a gain of function approach using the murine protozoan parasite Trypanosoma musculi could be used to investigate immunomodulatory molecules in vivo. This parasite is extracellular, so can deliver secreted proteins in a similar fashion to worms, and it has a distinct, short pattern of infection, which allows for quick detection of immunomodulatory effects. Necessary steps to generate a T. musculi heterologous expression system were performed, including optimising in vitro culture of this species, isolation of genomic sequences for the design and construction of plasmids for the expression and secretion of exogenous gene products and testing a proof of principle molecule in vivo. A T. musculi line was generated that secreted an active acetylcholinesterase (AChE) from the nematode Nippostrongylus brasiliensis, and this caused a truncated infection in mice compared to a control line. Immunological assays suggest that this was due to changes in macrophage polarisation, with increased expression of M1 markers and nitric oxide when AChE is secreted by T. musculi. This was also reflected in cytokine production, with higher levels of IFNγ and TNFα and lower levels of IL-4. M1 macrophages are involved in the killing of protozoa and bacteria, while M2 macrophages are important for immune responses against helminths. Skewing of macrophage polarisation to a more M1 phenotype should increase the survival of parasitic worms. Not only does this suggest a novel role for parasitic helminth secreted AChEs, but it also shows that this trypanosome-based system could be used to investigate secreted proteins with unknown function.