Investigating the pathogenesis of African trypanosome infection via the skin

African trypanosomes (Trypanosoma brucei sp.) are single-celled extracellular protozoan parasites that are transmitted via the tsetse fly vector across sub- Saharan Africa. T. brucei subspecies cause trypanosomiasis in both humans and animals, inflicting substantial disease and economic strains in affected regions. Mammalian infection begins when the tsetse fly takes a blood meal and injects trypanosomes into the dermal layer of skin. The parasites then invade the circulatory and lymphatic systems, reaching the draining lymph nodes and disseminate systemically. Little is understood about the host-pathogen interactions which influence the establishment of host infection at the initial bite site in the skin. Most experimental transmissions of African trypanosomiasis have studied the intraperitoneal or intravenous routes of exposure. However, these by-pass the natural route of infection via the skin. Therefore the aim of this thesis is to investigate the pathogenesis of African trypanosome infection via the skin. Chemokines play important roles in attracting leukocytes towards the lymphatics and lymph nodes. To investigate how trypanosomes migrate from the bite site to the draining lymph nodes, the hypothesis that chemokines may act as chemoattractants for trypanosomes was tested. Chemokines can also possess antimicrobial properties, including against the related protozoan parasite Leishmania mexicana, therefore their potential cytotoxic effects against T. brucei were tested. Data presented in this thesis shows that these chemokines do not induce the chemotaxis of T. brucei. The motility characteristics of the parasites were also not affected by chemokine exposure. Nor did these chemokines exert any trypanostatic effects on trypanosomes. These data suggest trypanosomes use alternative cues to reach the lymphatics post-infection. The skin is an overlooked area of research for African trypanosome infections. Therefore work in this thesis sought to investigate the hypothesis that the infection kinetics would be different in a host infected by the natural intradermal route when compared with the routinely-researched intraperitoneal route. Experiments in this thesis revealed clear differences in the infection kinetics and disease progression in mice infected intradermally when compared with those infected by the intraperitoneal route. These data imply that further infection models should utilise intradermal injections and investigate the overlooked skin stage of disease which occurs naturally in the wild. Upon deposition in the skin the trypanosomes home towards the lymphatic system before migrating systemically. Lymphotoxin-β-receptor signalling (LTβR) is essential for lymphoid organogenesis and the maintenance of secondary lymphoid tissue microarchitecture. For example, LTβ-/- mice lack most lymph nodes and have grossly disturbed splenic microarchitecture. As a consequence of these disturbances LTβ-/- mice have impaired antibody isotype class-switching. Experiments in this thesis were performed to test the hypothesis that deficiencies in lymph node development and antibody isotype class-switching would influence disease pathogenesis. These data show that disease susceptibility and pathogenesis were exacerbated in LTβ-/- mice, which lacked class-switched antibody isotypes in their sera. This disease profile was then reversed in LTβ-/- mice which received wild-type bone-marrow transfers after their haematopoietic system was ablated through lethal irradiation. These data could identify the importance of the class-switching capability of the adaptive immune system to combat trypanosome infection. Little is known of the early host-parasite interactions following injection of T. brucei into the dermis of the skin. Macrophages are key players in the innate immune response against African trypanosome infection, and manipulating these cells during infection may help our understanding of the disease pathogenesis. To address their potential role in disease susceptibility, experiments were designed to manipulate the density and inflammatory status of the macrophages in the skin prior to infection with T. brucei. These data show, that manipulation of the inflammatory status of the skin reduced susceptibility to infection with T. brucei via the skin. A greater understanding of the macrophage-parasite interactions which occur during the early stages of African trypanosome infection is important for understanding how the immune system responds to infection and how we can boost immunity to combat infection. A thorough identification of the mechanisms involved in establishing African trypanosome infections in the skin and their systemic dissemination will aid the development of novel approaches to block disease transmission

To the skin and beyond: the immune response to African trypanosomes as they enter and exit the vertebrate host

African trypanosomes are single-celled extracellular protozoan parasites transmitted by tsetse fly vectors across sub-Saharan Africa, causing serious disease in both humans and animals. Mammalian infections begin when the tsetse fly penetrates the skin in order to take a blood meal, depositing trypanosomes into the dermal layer. Similarly, onward transmission occurs when differentiated and insect pre-adapted forms are ingested by the fly during a blood meal. Between these transmission steps, trypanosomes access the systemic circulation of the vertebrate host via the skin-draining lymph nodes, disseminating into multiple tissues and organs, and establishing chronic, and long-lasting infections. However, most studies of the immunobiology of African trypanosomes have been conducted under experimental conditions that bypass the skin as a route for systemic dissemination (typically via intraperitoneal or intravenous routes). Therefore, the importance of these initial interactions between trypanosomes and the skin at the site of initial infection, and the implications for these processes in infection establishment, have largely been overlooked. Recent studies have also demonstrated active and complex interactions between the mammalian host and trypanosomes in the skin during initial infection and revealed the skin as an overlooked anatomical reservoir for transmission. This highlights the importance of this organ when investigating the biology of trypanosome infections and the associated immune responses at the initial site of infection. Here, we review the mechanisms involved in establishing African trypanosome infections and potential of the skin as a reservoir, the role of innate immune cells in the skin during initial infection, and the subsequent immune interactions as the parasites migrate from the skin. We suggest that a thorough identification of the mechanisms involved in establishing African trypanosome infections in the skin and their progression through the host is essential for the development of novel approaches to interrupt disease transmission and control these important diseases

Effects of host-derived chemokines on the motility and viability of Trypanosoma brucei

African trypanosomes (Trypanosoma brucei spp.) are extracellular, hemoflagellate, protozoan parasites. Mammalian infection begins when the tsetse fly vector injects trypanosomes into the skin during blood feeding. The trypanosomes then reach the draining lymph nodes before disseminating systemically. Intravital imaging of the skin post-tsetse fly bite revealed that trypanosomes were observed both extravascularly and intravascularly in the lymphatic vessels. Whether host-derived cues play a role in the attraction of the trypanosomes towards the lymphatic vessels to aid their dis-semination from the site of infection is not known. Since chemokines can mediate the attraction of leucocytes towards the lymphatics, in vitro chemotaxis assays were used to determine whether chemokines might also act as chemoattractants for tryp-anosomes. Although microarray data suggested that the chemokines CCL8, CCL19, CCL21, CCL27 and CXCL12 were highly expressed in mouse skin, they did not stimu-late the chemotaxis of T brucei. Certain chemokines also possess potent antimicrobial properties. However, none of the chemokines tested exerted any parasiticidal ef-fects on T brucei. Thus, our data suggest that host-derived chemokines do not act as chemoattractants for T brucei. Identification of the mechanisms used by trypano-somes to establish host infection will aid the development of novel approaches to block disease transmission

Divergent metabolism between Trypanosoma congolense and Trypanosoma brucei results in differential sensitivity to metabolic inhibition

Animal African Trypanosomiasis (AAT) is a debilitating livestock disease prevalent across sub-Saharan Africa, a main cause of which is the protozoan parasite Trypanosoma congolense. In comparison to the well-studied T. brucei, there is a major paucity of knowledge regarding the biology of T. congolense. Here, we use a combination of omics technologies and novel genetic tools to characterise core metabolism in T. congolense mammalian-infective bloodstream-form parasites, and test whether metabolic differences compared to T. brucei impact upon sensitivity to metabolic inhibition. Like the bloodstream stage of T. brucei, glycolysis plays a major part in T. congolense energy metabolism. However, the rate of glucose uptake is significantly lower in bloodstream stage T. congolense, with cells remaining viable when cultured in concentrations as low as 2 mM. Instead of pyruvate, the primary glycolytic endpoints are succinate, malate and acetate. Transcriptomics analysis showed higher levels of transcripts associated with the mitochondrial pyruvate dehydrogenase complex, acetate generation, and the glycosomal succinate shunt in T. congolense, compared to T. brucei. Stable-isotope labelling of glucose enabled the comparison of carbon usage between T. brucei and T. congolense, highlighting differences in nucleotide and saturated fatty acid metabolism. To validate the metabolic similarities and differences, both species were treated with metabolic inhibitors, confirming that electron transport chain activity is not essential in T. congolense. However, the parasite exhibits increased sensitivity to inhibition of mitochondrial pyruvate import, compared to T. brucei. Strikingly, T. congolense exhibited significant resistance to inhibitors of fatty acid synthesis, including a 780-fold higher EC50 for the lipase and fatty acid synthase inhibitor Orlistat, compared to T. brucei. These data highlight that bloodstream form T. congolense diverges from T. brucei in key areas of metabolism, with several features that are intermediate between bloodstream- and insect-stage T. brucei. These results have implications for drug development, mechanisms of drug resistance and host-pathogen interactions

Divergent metabolism between Trypanosoma congolense and Trypanosoma brucei results in differential sensitivity to metabolic inhibition

Animal African Trypanosomiasis (AAT) is a debilitating livestock disease prevalent across sub-Saharan Africa, a main cause of which is the protozoan parasite Trypanosoma congolense. In comparison to the well-studied T. brucei, there is a major paucity of knowledge regarding the biology of T. congolense. Here, we use a combination of omics technologies and novel genetic tools to characterise core metabolism in T. congolense mammalian-infective bloodstream-form parasites, and test whether metabolic differences compared to T. brucei impact upon sensitivity to metabolic inhibition. Like the bloodstream stage of T. brucei, glycolysis plays a major part in T. congolense energy metabolism. However, the rate of glucose uptake is significantly lower in bloodstream stage T. congolense, with cells remaining viable when cultured in concentrations as low as 2 mM. Instead of pyruvate, the primary glycolytic endpoints are succinate, malate and acetate. Transcriptomics analysis showed higher levels of transcripts associated with the mitochondrial pyruvate dehydrogenase complex, acetate generation, and the glycosomal succinate shunt in T. congolense, compared to T. brucei. Stable-isotope labelling of glucose enabled the comparison of carbon usage between T. brucei and T. congolense, highlighting differences in nucleotide and saturated fatty acid metabolism. To validate the metabolic similarities and differences, both species were treated with metabolic inhibitors, confirming that electron transport chain activity is not essential in T. congolense. However, the parasite exhibits increased sensitivity to inhibition of mitochondrial pyruvate import, compared to T. brucei. Strikingly, T. congolense exhibited significant resistance to inhibitors of fatty acid synthesis, including a 780-fold higher EC(50) for the lipase and fatty acid synthase inhibitor Orlistat, compared to T. brucei. These data highlight that bloodstream form T. congolense diverges from T. brucei in key areas of metabolism, with several features that are intermediate between bloodstream- and insect-stage T. brucei. These results have implications for drug development, mechanisms of drug resistance and host-pathogen interactions