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The role of the gut in experimental visceral leishmaniasis and Chagas disease

Final Report Summary - GUTTRYP (The role of the gut in experimental visceral leishmaniasis and Chagas disease)

This project was designed to investigate the importance of the gut and its commensal microbiota as factors in diseases caused by two major human pathogens, Leishmania donovani and Trypanosoma cruzi. These are protozoan parasite species that cause the neglected infectious diseases visceral leishmaniasis (VL) and Chagas disease (CD) respectively. People normally have a sufficiently strong immune response to control the initial wave of infection but, for largely unknown reasons, the parasites are not completely eliminated. Consequently, VL and CD are chronic conditions that develop gradually over time and often progress to life-threatening clinical syndromes. One way L. donovani and T. cruzi evade host immunity and maintain chronicity is by persisting in only a limited set of favoured cell and tissue types. Previous work had shown that the gut is one of the most important reservoirs of chronic T. cruzi infection and that L. donovani may also target the gut. Barrier sites like the digestive tract are constantly exposed to the external environment, including diverse commensal microbial species. Inflammatory immune responses against commensal microbe, food and environmental antigens are usually unwarranted and potentially dangerous. Therefore, a valid hypothesis is that a hyporesponsive immune environment in the gut is a key factor underpinning the host’s failure to completely clear T. cruzi and L. donovani infections. Parasite persistence in the gut may also compromise microbiota containment, contributing to VL/CD pathology or leading to secondary syndromes, as is seen in chronic HIV infection.

In the outgoing phase of this project I spent two years at the US National Institute for Allergy and Infectious Disease. I was engaged in a training programme that helped me develop a broad range of new technical and conceptual competencies to investigate general and gut-specific immunity and host-commensal interactions, as well as enhanced transferable skills. The overall aim of the research was to test the hypothesis that the gut and its microbiota are important factors in visceral leishmaniasis. I used two animal species, the mouse and hamster, which are models of mild and severe VL respectively. I trained in a variety of experimental techniques and used them to compare tissue-specific parasite loads, immune responses and pathology in these animal models. I generated genetically engineered parasites that express a model antigen, 2W1S, that allows helper T cells involved in the parasite-specific immune response to be identified and analysed. In order to study the relevance of the gut microbiota, animals were treated with broad-spectrum antibiotics to remove most of the normal commensal bacteria and compared with untreated control animals in the context of L. donovani infection. The data indicated that transgenic parasites engineered to express the 2W1S antigen could be a useful experimental tool to study immune responses in mice. The technology is not transferable to the hamster model due to genetic differences compared with mice and a lack of suitable reagents. The data showed that L. donovani parasitized the tissues of the GI tract in symptomatic hamsters but not in asymptomatic mice. Hamsters in the antibiotic treatment groups had delayed onset and progression symptoms. Hamsters that were infected with L. donovani and also treated with antibiotics survived for significantly longer than hamsters that did not receive antibiotics. Antibiotic-treated hamsters also had significantly less tissue pathology than untreated controls even though infection intensities were not significantly different. There were no significant differences in disease presentation in mice with antibiotic-induced dysbiosis. These results may have clinical significance because secondary bacterial infections could be an under-appreciated aspect of human VL progression. This is an interesting avenue for future research.

In the return phase of this project I spent one year at the London School of Hygiene and Tropical Medicine. I engaged in a training programme that was designed to help me re-integrate, transfer new knowledge and skills acquired during the overseas phase to the return host institution and implement them through research and career development activities. The primary research objective was to define the key features of host-parasite interaction in chronic T. cruzi gut infection. A system was developed to localise infected host cells based on transgenic parasites co-expressing bioluminescent and fluorescent reporter genes. A series of experiments generated tissue samples necessary to characterise tissue-specific host responses at the protein level. Approaches developed during the outgoing phase for inducing GI dysbiosis and studying barrier function were applied to the T. cruzi infection model to evaluate the role of the host microbiota in Chagas disease and the impact of chronic infection on GI tissue structure and function. The gut microbiota was found not to be a critical factor mediating permissiveness to T. cruzi infection. T. cruzi was found to parasitize the muscle layers tissues of the GI tract in chronically infected mice and cause major pathological alterations of relevance to human Chagas disease. Preliminary findings indicated that host tissue biomarker profiles differ and significant differences in peristalsis occur according to the genotypes of both the host and the parasite.

This project has enabled me to gain considerable experience in terms of both research and complementary skills, which have helped me to advance towards a position of scientific independence. The research led to significant new insights into the biology of T. cruzi and L. donovani and their interactions with mammalian hosts. My findings provide a clearer idea of when, how and why parasitic protozoa can persistently infect the gastrointestinal tract, and how this impacts on the development and severity of chronic disease. In addition, they provide a better understanding of the impact of the host’s microbiota on the outcome of infection with both L. donovani and T. cruzi. Although some final elements of analysis are currently ongoing, the research findings will be published in at least two peer-reviewed publications in 2018. The results have the potential for impact on efforts to develop new, more effective treatments against two major neglected infectious diseases.