Human African trypanosomiasis, or sleeping sickness, is caused by the protozoan parasite Trypanosoma brucei and induces profound reactivity of glial cells and neuroinflammation when the parasites colonise the central nervous system. However, the transcriptional and functional responses of the brain to chronic T. brucei infection remain poorly understood. By integrating single cell and spatial transcriptomics of the mouse brain, we identify that glial responses triggered by infection are readily detected in the proximity to the circumventricular organs, including the lateral and 3rd ventricle. This coincides with the spatial localisation of both slender and stumpy forms of T. brucei. Furthermore, in silico predictions and functional validations led us to identify a previously unknown crosstalk between homeostatic microglia and Cd138+ plasma cells mediated by IL-10 and B cell activating factor (BAFF) signalling. This study provides important insights and resources to improve understanding of the molecular and cellular responses in the brain during infection with African trypanosomes.

Mutations in the Trypanosoma brucei aquaporin AQP2 are associated with resistance to pentamidine and melarsoprol. We show that TbAQP2 but not TbAQP3 was positively selected for increased pore size from a common ancestor aquaporin. We demonstrate that TbAQP2's unique architecture permits pentamidine permeation through its central pore and show how specific mutations in highly conserved motifs affect drug permeation. Introduction of key TbAQP2 amino acids into TbAQP3 renders the latter permeable to pentamidine. Molecular dynamics demonstrates that permeation by dicationic pentamidine is energetically favourable in TbAQP2, driven by the membrane potential, although aquaporins are normally strictly impermeable for ionic species. We also identify the structural determinants that make pentamidine a permeant although most other diamidine drugs are excluded. Our results have wide-ranging implications for optimising antitrypanosomal drugs and averting cross-resistance. Moreover, these new insights in aquaporin permeation may allow the pharmacological exploitation of other members of this ubiquitous gene family.

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BackgroundThe release of small non-coding RNAs (sRNAs) has been reported in parasitic nematodes, trematodes and cestodes of medical and veterinary importance. However, little is known regarding the diversity and composition of sRNAs released by different lifecycle stages and the portion of sRNAs that persist in host tissues during filarial infection. This information is relevant to understanding potential roles of sRNAs in parasite-to-host communication, as well as to inform on the location within the host and time point at which they can be detected.Methodology and principal findingsWe have used small RNA (sRNA) sequencing analysis to identify sRNAs in replicate samples of the excretory-secretory (ES) products of developmental stages of the filarial nematode Litomosoides sigmodontis in vitro and compare this to the parasite-derived sRNA detected in host tissues. We show that all L. sigmodontis developmental stages release RNAs in vitro, including ribosomal RNA fragments, 5’-derived tRNA fragments (5’-tRFs) and, to a lesser extent, microRNAs (miRNAs). The gravid adult females (gAF) produce the largest diversity and abundance of miRNAs in the ES compared to the adult males or microfilariae. Analysis of sRNAs detected in serum and macrophages from infected animals reveals that parasite miRNAs are preferentially detected in vivo, compared to their low levels in the ES products, and identifies miR-92-3p and miR-71-5p as L. sigmodontis miRNAs that are stably detected in host cells in vivo.ConclusionsOur results suggest that gravid adult female worms secrete the largest diversity of extracellular sRNAs compared to adult males or microfilariae. We further show differences in the parasite sRNA biotype distribution detected in vitro versus in vivo. We identify macrophages as one reservoir for parasite sRNA during infection, and confirm the presence of parasite miRNAs and tRNAs in host serum during patent infection.

34Defining mode of action is vital for both developing new drugs and predicting 35 potential resistance mechanisms. African trypanosome pentamidine and 36 melarsoprol sensitivity is predominantly mediated by aquaglyceroporin 2 (TbAQP2), a 37 channel associated with water/glycerol transport. TbAQP2 is expressed at the flagellar 38 pocket membrane and chimerisation with TbAQP3 renders parasites resistant to both 39 drugs. Two models for how TbAQP2 mediates pentamidine sensitivity have emerged; 40 that TbAQP2 mediates pentamidine translocation or via binding to TbAQP2, with 41 subsequent endocytosis, but trafficking and regulation of TbAQPs is uncharacterised. 42We demonstrate that TbAQP2 is organised as a high order complex, is ubiquitylated 43 and transported to the lysosome. Unexpectedly, mutation of potential ubiquitin 44 conjugation sites, i.e. cytoplasmic lysine residues, reduced folding and tetramerization 45 efficiency and triggered ER retention. Moreover, TbAQP2/TbAQP3 chimerisation also 46 leads to impaired oligomerisation, mislocalisation, and increased turnover. These data 47suggest that TbAQP2 stability is highly sensitive to mutation and contributes towards 48 emergence of drug resistance. 49 50 Human African trypanosomiasis (HAT) is a neglected tropical disease affecting 52 sub-Saharan countries [1-4]. HAT progresses by two stages: a haemolymphatic 53 stage, in which the parasite successfully colonises the bloodstream, lymphatics, skin, 54 adipose tissue and organs and a meningoencephalic stage characterised by the 55 emergence of parasites in the central nervous system (CNS) [2,5]. Several drugs are 56 used to treat HAT; currently suramin and pentamidine are the drugs of choice for 57 treatment of the haemolymphatic stage of T. brucei rhodesiense and T. brucei 58 gambiense infections respectively, whereas melarsoprol, eflornithine or combined 59 nifurtimox-eflornithine (NECT) therapy are recommended for the meningoencephalic 60 stage [6,7]. 61 Two new drugs, fexinidazole and acoziborole, recently completed clinical trials 62 and opened a new front in HAT chemotherapy [8,9]. Drug development, successful 63 public health initiatives and active case-monitoring programs have all contributed to 64 the anticipated eradication of gambiense HAT as a major public health problem in the 65 coming decade [10]. However, vigilance and understanding of drug mechanisms and 66 possible resistance pathways remain essential to maintain this situation, and 67rhodesiense HAT cannot be eliminated in this way as it is highly zoonotic [11]. 68Genome-wide RNAi screens identified a number of genes associated with 69 pentamidine sensitivity that, together with evidence from melarsoprol-pentamidine 70 cross-resistance (MPXR), identified aquaglyceroporin 2 as the primary determinant for 71 drug-uptake [12,13], alongside lesser roles for the TbAT1/P2 aminopurine transporter 72 and the Low Affinity Pentamidine transporter LAPT1 [14]. 73 Aquaglyceroporins (AQPs) are an ancient family of multi-pass membrane 74 proteins, containing b...

Defining mode of action is vital for both developing new drugs and predicting potential resistance mechanisms. Sensitivity of African trypanosomes to pentamidine and melarsoprol is predominantly mediated by aquaglyceroporin 2 (TbAQP2), a channel associated with water/ glycerol transport. TbAQP2 is expressed at the flagellar pocket membrane and chimerisation with TbAQP3 renders parasites resistant to both drugs. Two models for how TbAQP2 mediates pentamidine sensitivity have emerged; that TbAQP2 mediates pentamidine translocation across the plasma membrane or via binding to TbAQP2, with subsequent endocytosis and presumably transport across the endosomal/lysosomal membrane, but as trafficking and regulation of TbAQPs is uncharacterised this remains unresolved. We demonstrate that TbAQP2 is organised as a high order complex, is ubiquitylated and is transported to the lysosome. Unexpectedly, mutation of potential ubiquitin conjugation sites, i.e. cytoplasmicoriented lysine residues, reduced folding and tetramerization efficiency and triggered ER retention. Moreover, TbAQP2/TbAQP3 chimerisation, as observed in pentamidine-resistant parasites, also leads to impaired oligomerisation, mislocalisation and increased turnover. These data suggest that TbAQP2 stability is highly sensitive to mutation and that instability contributes towards the emergence of drug resistance.

African trypanosomes are highly divergent from their metazoan hosts, and as part of adaptation to a parasitic life style have developed a unique endomembrane system. The key virulence mechanism of many pathogens is successful immune evasion, to enable survival within a host, a feature that requires both genetic events and membrane transport mechanisms in African trypanosomes. Intracellular trafficking not only plays a role in immune evasion, but also in homeostasis of intracellular and extracellular compartments and interactions with the environment. Significantly, historical and recent work has unraveled some of the connections between these processes and highlighted how immune evasion mechanisms that are associated with adaptations to membrane trafficking may have, paradoxically, provided specific sensitivity to drugs. Here, we explore these advances in understanding the membrane composition of the trypanosome plasma membrane and organelles and provide a perspective for how transport could be exploited for therapeutic purposes.

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