RNAi screening identifies Trypanosoma brucei stress response protein kinases required for survival in the mouse

Protein kinases (PKs) are a class of druggable targets in Trypanosoma brucei, the causative agent of Human African Trypanosomiasis (sleeping sickness), yet little is known about which PKs are essential for survival in mammals. A recent kinome-wide RNAi screen with 176 individual bloodstream form Trypanosoma brucei lines identified PKs required for proliferation in culture. In order to assess which PKs are also potential virulence factors essential in vivo, lines were pooled, inoculated into mice, and screened for loss of fitness after 48 h RNAi. The presence of trypanosomes in the bloodstream was assessed using RNAi target sequencing (RITseq) and compared to growth in culture. We identified 49 PKs with a significant loss of fitness in vivo in two independent experiments, and a strong correlation between in vitro and in vivo loss of fitness for the majority. Nine PKs had a more pronounced growth defect in vivo, than in vitro. Amongst these PKs were several with putative functions related to stress responses mediated through the PI3K/TOR or MAPK signaling cascades, which act to protect the parasite from complement-mediated and osmotic lysis. Identification of these virulence-associated PKs provides new insights into T. brucei-host interaction and reveals novel potential protein kinase drug targets

Genomic and Proteomic Studies on the Mode of Action of Oxaboroles against the African Trypanosome

SCYX-7158, an oxaborole, is currently in Phase I clinical trials for the treatment of human African trypanosomiasis. Here we investigate possible modes of action against Trypanosoma brucei using orthogonal chemo-proteomic and genomic approaches. SILAC-based proteomic studies using an oxaborole analogue immobilised onto a resin was used either in competition with a soluble oxaborole or an immobilised inactive control to identify thirteen proteins common to both strategies. Cell-cycle analysis of cells incubated with sub-lethal concentrations of an oxaborole identified a subtle but significant accumulation of G2 and >G2 cells. Given the possibility of compromised DNA fidelity, we investigated long-term exposure of T. brucei to oxaboroles by generating resistant cell lines in vitro. Resistance proved more difficult to generate than for drugs currently used in the field, and in one of our three cell lines was unstable. Whole-genome sequencing of the resistant cell lines revealed single nucleotide polymorphisms in 66 genes and several large-scale genomic aberrations. The absence of a simple consistent mechanism among resistant cell lines and the diverse list of binding partners from the proteomic studies suggest a degree of polypharmacology that should reduce the risk of resistance to this compound class emerging in the field. The combined genetic and chemical biology approaches have provided lists of candidates to be investigated for more detailed information on the mode of action of this promising new drug clas

Modulation of the surface proteome through multiple ubiquitylation pathways in African Trypanosomes

Recently we identified multiple suramin-sensitivity genes with a genome wide screen in Trypanosoma brucei that includes the invariant surface glycoprotein ISG75, the adaptin-1 (AP-1) complex and two deubiquitylating enzymes (DUBs) orthologous to ScUbp15/HsHAUSP1 and pVHL-interacting DUB1 (type I), designated TbUsp7 and TbVdu1, respectively. Here we have examined the roles of these genes in trafficking of ISG75, which appears key to suramin uptake. We found that, while AP-1 does not influence ISG75 abundance, knockdown of TbUsp7 or TbVdu1 leads to reduced ISG75 abundance. Silencing TbVdu1 also reduced ISG65 abundance. TbVdu1 is a component of an evolutionarily conserved ubiquitylation switch and responsible for rapid receptor modulation, suggesting similar regulation of ISGs in T. brucei. Unexpectedly, TbUsp7 knockdown also blocked endocytosis. To integrate these observations we analysed the impact of TbUsp7 and TbVdu1 knockdown on the global proteome using SILAC. For TbVdu1, ISG65 and ISG75 are the only significantly modulated proteins, but for TbUsp7 a cohort of integral membrane proteins, including the acid phosphatase MBAP1, that is required for endocytosis, and additional ISG-related proteins are down-regulated. Furthermore, we find increased expression of the ESAG6/7 transferrin receptor and ESAG5, likely resulting from decreased endocytic activity. Therefore, multiple ubiquitylation pathways, with a complex interplay with trafficking pathways, control surface proteome expression in trypanosomes

The <i>N</i>-myristoylome of <i>Trypanosoma cruzi</i>

Protein N-myristoylation is catalysed by N-myristoyltransferase (NMT), an essential and druggable target in Trypanosoma cruzi, the causative agent of Chagas’ disease. Here we have employed whole cell labelling with azidomyristic acid and click chemistry to identify N-myristoylated proteins in different life cycle stages of the parasite. Only minor differences in fluorescent-labelling were observed between the dividing forms (the insect epimastigote and mammalian amastigote stages) and the non-dividing trypomastigote stage. Using a combination of label-free and stable isotope labelling of cells in culture (SILAC) based proteomic strategies in the presence and absence of the NMT inhibitor DDD85646, we identified 56 proteins enriched in at least two out of the three experimental approaches. Of these, 6 were likely to be false positives, with the remaining 50 commencing with amino acids MG at the N-terminus in one or more of the T. cruzi genomes. Most of these are proteins of unknown function (32), with the remainder (18) implicated in a diverse range of critical cellular and metabolic functions such as intracellular transport, cell signalling and protein turnover. In summary, we have established that 0.43–0.46% of the proteome is N-myristoylated in T. cruzi approaching that of other eukaryotic organisms (0.5–1.7%)

Transcriptomic profiling of host-parasite interactions in the microsporidian <i>Trachipleistophora hominis</i>

BACKGROUND: Trachipleistophora hominis was isolated from an HIV/AIDS patient and is a member of a highly successful group of obligate intracellular parasites. METHODS: Here we have investigated the evolution of the parasite and the interplay between host and parasite gene expression using transcriptomics of T. hominis-infected rabbit kidney cells. RESULTS: T. hominis has about 30 % more genes than small-genome microsporidians. Highly expressed genes include those involved in growth, replication, defence against oxidative stress, and a large fraction of uncharacterised genes. Chaperones are also highly expressed and may buffer the deleterious effects of the large number of non-synonymous mutations observed in essential T. hominis genes. Host expression suggests a general cellular shutdown upon infection, but ATP, amino sugar and nucleotide sugar production appear enhanced, potentially providing the parasite with substrates it cannot make itself. Expression divergence of duplicated genes, including transporters used to acquire host metabolites, demonstrates ongoing functional diversification during microsporidian evolution. We identified overlapping transcription at more than 100 loci in the sparse T. hominis genome, demonstrating that this feature is not caused by genome compaction. The detection of additional transposons of insect origin strongly suggests that the natural host for T. hominis is an insect. CONCLUSIONS: Our results reveal that the evolution of contemporary microsporidian genomes is highly dynamic and innovative. Moreover, highly expressed T. hominis genes of unknown function include a cohort that are shared among all microsporidians, indicating that some strongly conserved features of the biology of these enormously successful parasites remain uncharacterised. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12864-015-1989-z) contains supplementary material, which is available to authorized users

Situation analysis for delivering integrated comprehensive sexual and reproductive health services in humanitarian crisis condition for Rohingya refugees in Cox's Bazar, Bangladesh: protocol for a mixed-method study

INTRODUCTION:Rohingya diaspora are one of the most vulnerable groups seeking refuge in camps of Cox's Bazar, Bangladesh, arising an acute humanitarian crisis. More than half of the Rohingya refugees are women and adolescent girls requiring quality sexual and reproductive health (SRH) services. Minimum initial service package of SRH are being rendered in the refugee camps; however, WHO is aiming to provide integrated comprehensive SRH services to meet the unmet needs of this most vulnerable group. For sustainable and successful implementation of such comprehensive SRH service packages, a critical first step is to undertake a situation analysis and understand the current dimensions and capture the lessons learnt on their SRH-specific needs and implementation challenges. This situation analysis is pertinent in current humanitarian condition and will provide an overview of the needs, availability and delivery of SRH services for adolescent girls and women, barriers in accessing and providing those services in Rohingya refugee camps in Cox's Bazar, Bangladesh, and similar humanitarian contexts. METHODS AND ANALYSIS:A concurrent mixed-methods design will be used in this study. A community-based household survey coupled with facility assessments as well as qualitative in-depth interviews, key informant interviews and focus group discussions will be conducted with community people of Rohingya refugee camps and relevant stakeholders providing SRH services to Rohingya population in Cox's Bazar, Bangladesh. Survey data will be analysed using univariate, bivariate and multivariable regression statistics. Descriptive analysis will be done for facility assessment and thematic analysis will be conducted with qualitative data. ETHICS AND DISSEMINATION:Ethical approval from Institutional Review Board of BRAC James P Grant School of Public Health (2018-017-IR) has been obtained. Findings from this research will be disseminated through presentations in local, national and international conferences, workshops, peer-reviewed publications, policy briefs and interactive project report

A Target-Based High Throughput Screen Yields Trypanosoma brucei Hexokinase Small Molecule Inhibitors with Antiparasitic Activity

African sleeping sickness is a disease found in sub-Saharan Africa that is caused by the single-celled parasite Trypanosoma brucei. The drugs used widely now to treat infections are 50 years old and notable for their toxicity, emphasizing the need for development of new therapeutics. In the search for potential drug targets, researchers typically focus on enzymes or proteins that are essential to the survival of the infectious agent while being distinct enough from the host to avoid accidental targeting of the host enzyme. This work describes our research on one such trypanosome enzyme, hexokinase, which is a protein that the parasite requires to make energy. Here we describe the results of our search for inhibitors of the parasite enzyme. By screening 220,223 compounds for anti-hexokinase activity, we have identified new inhibitors of the parasite enzyme. Some of these are toxic to trypanosomes while having no effect on mammalian cells, suggesting that they may hold promise for the development of new anti-parasitic compounds

Growth Arrest of BCR-ABL Positive Cells with a Sequence-Specific Polyamide-Chlorambucil Conjugate

Chronic myeloid leukemia (CML) is characterized by the presence of a constitutively active Abl kinase, which is the product of a chimeric BCR-ABL gene, caused by the genetic translocation known as the Philadelphia chromosome. Imatinib, a selective inhibitor of the Bcr-Abl tyrosine kinase, has significantly improved the clinical outcome of patients with CML. However, subsets of patients lose their response to treatment through the emergence of imatinib-resistant cells, and imatinib treatment is less durable for patients with late stage CML. Although alternative Bcr-Abl tyrosine kinase inhibitors have been developed to overcome drug resistance, a cocktail therapy of different kinase inhibitors and additional chemotherapeutics may be needed for complete remission of CML in some cases. Chlorambucil has been used for treatment of B cell chronic lymphocytic leukemia, non-Hodgkin's and Hodgkin's disease. Here we report that a DNA sequence-specific pyrrole-imidazole polyamide-chlorambucil conjugate, 1R-Chl, causes growth arrest of cells harboring both unmutated BCR-ABL and three imatinib resistant strains. 1R-Chl also displays selective toxicities against activated lymphocytes and a high dose tolerance in a murine model

Discovery of Inhibitors of Leishmania β-1,2-Mannosyltransferases Using a Click-Chemistry-Derived Guanosine Monophosphate Library

Leishmania spp. are a medically important group of protozoan parasites that synthesize a novel intracellular carbohydrate reserve polymer termed mannogen. Mannogen is a soluble homopolymer of β-1,2-linked mannose residues that accumulates in the major pathogenic stages in the sandfly vector and mammalian host. While several steps in mannogen biosynthesis have been defined, none of the enzymes have been isolated or characterized. We report the development of a simple assay for the GDP-mannose–dependent β-1,2-mannosyltransferases involved in mannogen synthesis. This assay utilizes octyl α-d-mannopyranoside to prime the formation of short mannogen oligomers up to 5 mannose residues. This assay was used to screen a focussed library of 44 GMP-triazole adducts for inhibitors. Several compounds provided effective inhibition of mannogen β-1,2-mannosyltransferases in a cell-free membrane preparation. This assay and inhibitor compounds will be useful for dissecting the role of different mannosyltransferases in regulating de novo biosynthesis and elongation reactions in mannogen metabolism