The Trypanosoma brucei-Derived Ketoacids, Indole Pyruvate and Hydroxyphenylpyruvate, Induce HO-1 Expression and Suppress Inflammatory Responses in Human Dendritic Cells

The extracellular parasite and causative agent of African sleeping sickness Trypanosoma brucei (T. brucei) has evolved a number of strategies to avoid immune detection in the host. One recently described mechanism involves the conversion of host-derived amino acids to aromatic ketoacids, which are detected at relatively high concentrations in the bloodstream of infected individuals. These ketoacids have been shown to directly suppress inflammatory responses in murine immune cells, as well as acting as potent inducers of the stress response enzyme, heme oxygenase 1 (HO-1), which has proven anti-inflammatory properties. The aim of this study was to investigate the immunomodulatory properties of the T. brucei-derived ketoacids in primary human immune cells and further examine their potential as a therapy for inflammatory diseases. We report that the T. brucei-derived ketoacids, indole pyruvate (IP) and hydroxyphenylpyruvate (HPP), induce HO-1 expression through Nrf2 activation in human dendritic cells (DC). They also limit DC maturation and suppress the production of pro-inflammatory cytokines, which, in turn, leads to a reduced capacity to differentiate adaptive CD4+ T cells. Furthermore, the ketoacids are capable of modulating DC cellular metabolism and suppressing the inflammatory profile of cells isolated from patients with inflammatory bowel disease. This study therefore not only provides further evidence of the immune-evasion mechanisms employed by T. brucei, but also supports further exploration of this new class of HO-1 inducers as potential therapeutics for the treatment of inflammatory conditions.

LncRNAs Are Differentially Expressed between Wildtype and Cell Line Strains of African Trypanosomes

Trypanosoma brucei is a parasitic protist that causes African sleeping sickness. The establishment of T. brucei cell lines has provided a significant advantage for the majority of T. brucei research. However, these cell lines were isolated and maintained in culture for decades, occasionally accumulating changes in gene expression. Since trypanosome strains have been maintained in culture for decades, it is possible that difference may have accumulated in fast-evolving non-coding RNAs between trypanosomes from the wild and those maintained extensively in cultures. To address this, we compared the lncRNA expression profile of trypanosomes maintained as cultured cell lines (CL) to those extracted from human patients, wildtype (WT). We identified lncRNAs from CL and WT from available transcriptomic data and demonstrate that CL and WT have unique sets of lncRNAs expressed. We further demonstrate that the unique and shared lncRNAs are differentially expressed between CL and WT parasites, and that these lncRNAs are more evenly up-regulated and down-regulated than protein-coding genes. We validated the expression of these lncRNAs using qPCR. Taken together, this study demonstrates that lncRNAs are differentially expressed between cell lines and wildtype T. brucei and provides evidence for potential evolution of lncRNAs, specifically in T. brucei maintained in culture.

The Occurrence of Malignancy in Trypanosoma brucei brucei by Rapid Passage in Mice

Pleomorphic Trypanosoma brucei are best known for their tightly controlled cell growth and developmental program, which ensures their transmissibility and host fitness between the mammalian host and insect vector. However, after long-term adaptation in the laboratory or by natural evolution, monomorphic parasites can be derived. The origin of these monomorphic forms is currently unclear. Here, we produced a series of monomorphic trypanosome stocks by artificially syringe-passage in mice, creating snapshots of the transition from pleomorphism to monomorphism. We then compared these artificial monomorphic trypanosomes, alongside several naturally monomorphic T. evansi and T. equiperdum strains, with the pleomorphic T. brucei. In addition to failing to generate stumpy forms in animal bloodstream, we found that monomorphic trypanosomes from laboratory and nature exhibited distinct differentiation patterns, which are reflected by their distinct differentiation potential and transcriptional changes. Lab-adapted monomorphic trypanosomes could still be induced to differentiate, and showed only minor transcriptional differences to that of the pleomorphic slender forms but some accumulated differences were observed as the passages progress. All naturally monomorphic strains completely fail to differentiate, corresponding to their impaired differentiation regulation. We propose that the natural phenomenon of trypanosomal monomorphism is actually a malignant manifestation of protozoal cells. From a disease epidemiological and evolutionary perspective, our results provide evidence for a new way of thinking about the origin of these naturally monomorphic strains, the malignant evolution of trypanosomes may raise some concerns. Additionally, these monomorphic trypanosomes may reflect the quantitative and qualitative changes in the malignant evolution of T. brucei, suggesting that single-celled protozoa may also provide the most primitive model of cellular malignancy, which could be a primitive and inherent biological phenomenon of eukaryotic organisms from protozoans to mammals.

Oxidative Phosphorylation Is Required for Powering Motility and Development of the Sleeping Sickness Parasite Trypanosoma brucei in the Tsetse Fly Vector

African trypanosomes cause disease in humans and their livestock and are transmitted by tsetse flies. The insect ingests these parasites with its blood meal, but to be transmitted to another mammal, the trypanosome must undergo complex development within the tsetse fly and migrate from the insect's gut to its salivary glands.

Genomic occupancy of the bromodomain protein Bdf3 is dynamic during differentiation of African trypanosomes from bloodstream to procyclic forms

Trypanosoma brucei , the causative agent of Human and Animal African trypanosomiasis, cycles between a mammalian host and a tsetse fly vector. The parasite undergoes huge changes in morphology and metabolism as it adapts to each host environment. These changes are reflected in the differing transcriptomes of parasites living in each host. While changes in the transcriptome have been well catalogued for parasites differentiating from the mammalian bloodstream to the insect stage, it remains unclear whether chromatin interacting proteins mediate transcriptomic changes during life cycle adaptation. We and others have shown that chromatin interacting bromodomain proteins localize to transcription start sites in bloodstream parasites, but whether the localization of bromodomain proteins changes as parasites differentiate from bloodstream to insect stage parasites remains unknown. To address this question, we performed Cleavage Under Target and Release Using Nuclease (CUT&RUN) timecourse experiments using a tagged version of Bromodomain Protein 3 (Bdf3) in parasites differentiating from bloodstream to insect stage forms. We found that Bdf3 occupancy at most loci increased at 3 hours following onset of differentiation and decreased thereafter. A number of sites with increased bromodomain protein occupancy lie proximal to genes known to have altered transcript levels during differentiation, such as procyclins, procyclin associated genes, and invariant surface glycoproteins. While most Bdf3 occupied sites are observed throughout differentiation, a very small number appear de novo as differentiation progresses. Notably, one such site lies proximal to the procyclin gene locus, which contains genes essential for remodeling surface proteins following transition to the insect stage. Overall, these studies indicate that occupancy of chromatin interacting proteins is dynamic during life cycle stage transitions, and provides the groundwork for future studies aimed at uncovering whether changes in bromodomain protein occupancy affect transcript levels of neighboring genes. Additionally, the optimization of CUT&RUN for use in Trypanosoma brucei may prove helpful for other researchers as an alternative to Chromatin Immunoprecipitation (ChIP).

Trypanin Disruption Affects the Motility and Infectivity of the Protozoan Trypanosoma cruzi

The flagellum of Trypanosomatids is an organelle that contributes to multiple functions, including motility, cell division, and host–pathogen interaction. Trypanin was first described in Trypanosoma brucei and is part of the dynein regulatory complex. TbTrypanin knockdown parasites showed motility defects in procyclic forms; however, silencing in bloodstream forms was lethal. Since TbTrypanin mutants show drastic phenotypic changes in mammalian stages, we decided to evaluate if the Trypanosoma cruzi ortholog plays a similar role by using the CRISPR-Cas9 system to generate null mutants. A ribonucleoprotein complex of SaCas9 and sgRNA plus donor oligonucleotide were used to edit both alleles of TcTrypanin without any selectable marker. TcTrypanin −/− epimastigotes showed a lower growth rate, partially detached flagella, normal numbers of nuclei and kinetoplasts, and motility defects such as reduced displacement and speed and increased tumbling propensity. The epimastigote mutant also showed decreased efficiency of in-vitro metacyclogenesis. Mutant parasites were able to complete the entire life cycle in vitro; however, they showed a reduction in their infection capacity compared with WT and addback cultures. Our data show that T. cruzi life cycle stages have differing sensitivities to TcTrypanin deletion. In conclusion, additional work is needed to dissect the motility components of T. cruzi and to identify essential molecules for mammalian stages.

Sesquiterpene Lactones with Dual Inhibitory Activity against the Trypanosoma brucei Pteridine Reductase 1 and Dihydrofolate Reductase

The parasite Trypanosoma brucei (T. brucei) is responsible for human African trypanosomiasis (HAT) and the cattle disease “Nagana” which to this day cause severe medical and socio-economic issues for the affected areas in Africa. So far, most of the available treatment options are accompanied by harmful side effects and are constantly challenged by newly emerging drug resistances. Since trypanosomatids are auxotrophic for folate, their pteridine metabolism provides a promising target for an innovative chemotherapeutic treatment. They are equipped with a unique corresponding enzyme system consisting of the bifunctional dihydrofolate reductase-thymidylate synthase (TbDHFR-TS) and the pteridine reductase 1 (TbPTR1). Previously, gene knockout experiments with PTR1 null mutants have underlined the importance of these enzymes for parasite survival. In a search for new chemical entities with a dual inhibitory activity against the TbPTR1 and TbDHFR, a multi-step in silico procedure was employed to pre-select promising candidates against the targeted enzymes from a natural product database. Among others, the sesquiterpene lactones (STLs) cynaropicrin and cnicin were identified as in silico hits. Consequently, an in-house database of 118 STLs was submitted to an in silico screening yielding 29 further virtual hits. Ten STLs were subsequently tested against the target enzymes in vitro in a spectrophotometric inhibition assay. Five compounds displayed an inhibition over 50% against TbPTR1 as well as three compounds against TbDHFR. Cynaropicrin turned out to be the most interesting hit since it inhibited both TbPTR1 and TbDHFR, reaching IC50 values of 12.4 µM and 7.1 µM, respectively.

Free-ranging pigs identified as a multi-reservoir of Trypanosoma brucei and Trypanosoma congolense in the Vavoua area, a historical sleeping sickness focus of Côte d’Ivoire

Background The existence of an animal reservoir of Trypanosoma brucei gambiense (T. b. gambiense), the agent of human African trypanosomiasis (HAT), may compromise the interruption of transmission targeted by World Health Organization. The aim of this study was to investigate the presence of trypanosomes in pigs and people in the Vavoua HAT historical focus where cases were still diagnosed in the early 2010’s. Methods For the human survey, we used the CATT, mini-anion exchange centrifugation technique and immune trypanolysis tests. For the animal survey, the buffy coat technique was also used as well as the PCR using Trypanosoma species specific, including the T. b. gambiense TgsGP detection using single round and nested PCRs, performed from animal blood samples and from strains isolated from subjects positive for parasitological investigations. Results No HAT cases were detected among 345 people tested. A total of 167 pigs were investigated. Free-ranging pigs appeared significantly more infected than pigs in pen. Over 70% of free-ranging pigs were positive for CATT and parasitological investigations and 27–43% were positive to trypanolysis depending on the antigen used. T. brucei was the most prevalent species (57%) followed by T. congolense (24%). Blood sample extracted DNA of T. brucei positive subjects were negative to single round TgsGP PCR. However, 1/22 and 6/22 isolated strains were positive with single round and nested TgsGP PCRs, respectively. Discussion Free-ranging pigs were identified as a multi-reservoir of T. brucei and/or T. congolense with mixed infections of different strains. This trypanosome diversity hinders the easy and direct detection of T. b. gambiense. We highlight the lack of tools to prove or exclude with certainty the presence of T. b. gambiense. This study once more highlights the need of technical improvements to explore the role of animals in the epidemiology of HAT.