Doomed drones? Using passage experiments and mathematical modelling to determine Deformed wing virus population dynamics in male honey bees

Funding: This research was funded by the BBSRC (Biotechnology and Biological Sciences Research Council), grant numbers: BB/M010996/1 and BB/R00305X/1. P.C.S was supported by the BBSRC, grant number: BB/S00243X/1.Varroa destructor is an ectoparasitic mite of honeybees which vectors a range of pathogenic viruses, the most notable being Deformed wing virus (DWV). Mites parasitise bees during pupal development and male honeybees, drones, have a longer development cycle than female workers (24 versus 21 days), allow for more progeny mites to develop per foundress (1.6–2.5 compared to 0.7–1.45). How this longer exposure time influences evolution of the transmitted virus population is unknown. Using uniquely tagged viruses recovered from cDNA we investigated the replication, competition and morbidity of DWV genotypes in drones. Assays examining virus replication and morbidity revealed drones are highly susceptible to both predominant genotypes of DWV. In virus passage studies using an equimolar inocula of major DNA genotypes and their recombinants, the recombinant form dominated but did not reach 100% of the virus population within 10 passages. Using an in-silico model of the virus–mite–bee system we examined bottlenecks during virus acquisition by the mite and subsequent injection of viruses into the host, which may play a significant role in shaping virus diversity. This study furthers our understanding of the variables influencing DWV diversity changes and provides insight into areas of future research in the mite–virus–bee system.Publisher PDFPeer reviewe

Benzoxaborole treatment perturbs S-adenosyl-L-methionine metabolism in Trypanosoma brucei

The parasitic protozoan Trypanosoma brucei causes Human African Trypanosomiasis and Nagana in other mammals. These diseases present a major socio-economic burden to large areas of sub-Saharan Africa. Current therapies involve complex and toxic regimens, which can lead to fatal side-effects. In addition, there is emerging evidence for drug resistance. AN5568 (SCYX-7158) is a novel benzoxaborole class compound that has been selected as a lead compound for the treatment of HAT, and has demonstrated effective clearance of both early and late stage trypanosomiasis in vivo. The compound is currently awaiting phase III clinical trials and could lead to a novel oral therapeutic for the treatment of HAT. However, the mode of action of AN5568 in T. brucei is unknown. This study aimed to investigate the mode of action of AN5568 against T. brucei, using a combination of molecular and metabolomics-based approaches.Treatment of blood-stage trypanosomes with AN5568 led to significant perturbations in parasite metabolism. In particular, elevated levels of metabolites involved in the metabolism of S-adenosyl-L-methionine, an essential methyl group donor, were found. Further comparative metabolomic analyses using an S-adenosyl-L-methionine-dependent methyltransferase inhibitor, sinefungin, showed the presence of several striking metabolic phenotypes common to both treatments. Furthermore, several metabolic changes in AN5568 treated parasites resemble those invoked in cells treated with a strong reducing agent, dithiothreitol, suggesting redox imbalances could be involved in the killing mechanism

Reliable, scalable functional genetics in bloodstream-form Trypanosoma congolense in vitro and in vivo.

Animal African trypanosomiasis (AAT) is a severe, wasting disease of domestic livestock and diverse wildlife species. The disease in cattle kills millions of animals each year and inflicts a major economic cost on agriculture in sub-Saharan Africa. Cattle AAT is caused predominantly by the protozoan parasites Trypanosoma congolense and T. vivax, but laboratory research on the pathogenic stages of these organisms is severely inhibited by difficulties in making even minor genetic modifications. As a result, many of the important basic questions about the biology of these parasites cannot be addressed. Here we demonstrate that an in vitro culture of the T. congolense genomic reference strain can be modified directly in the bloodstream form reliably and at high efficiency. We describe a parental single marker line that expresses T. congolense-optimized T7 RNA polymerase and Tet repressor and show that minichromosome loci can be used as sites for stable, regulatable transgene expression with low background in non-induced cells. Using these tools, we describe organism-specific constructs for inducible RNA-interference (RNAi) and demonstrate knockdown of multiple essential and non-essential genes. We also show that a minichromosomal site can be exploited to create a stable bloodstream-form line that robustly provides >40,000 independent stable clones per transfection-enabling the production of high-complexity libraries of genome-scale. Finally, we show that modified forms of T. congolense are still infectious, create stable high-bioluminescence lines that can be used in models of AAT, and follow the course of infections in mice by in vivo imaging. These experiments establish a base set of tools to change T. congolense from a technically challenging organism to a routine model for functional genetics and allow us to begin to address some of the fundamental questions about the biology of this important parasite

Diminazene resistance in Trypanosoma congolense is not caused by reduced transport capacity but associated with reduced mitochondrial membrane potential

Trypanosoma congolense is a principal agent causing livestock trypanosomiasis in Africa, costing developing economies billions of dollars and undermining food security. Only the diamidine diminazene and the phenanthridine isometamidium are regularly used, and resistance is widespread but poorly understood. We induced stable diminazene resistance in T. congolense strain IL3000 in vitro. There was no cross‐resistance with the phenanthridine drugs, melaminophenyl arsenicals, oxaborole trypanocides, or with diamidine trypanocides, except the close analogues DB829 and DB75. Fluorescence microscopy showed that accumulation of DB75 was inhibited by folate. Uptake of [3H]‐diminazene was slow, low affinity and partly but reciprocally inhibited by folate and by competing diamidines. Expression of T. congolense folate transporters in diminazene‐resistant T. b. brucei significantly sensitized the cells to diminazene and DB829, but not to oxaborole AN7973. However, [3H]‐diminazene transport studies, whole genome sequencing and RNA‐seq found no major changes in diminazene uptake, folate transporter sequence or expression. Instead, all resistant clones displayed a moderate reduction in the mitochondrial membrane potential. We conclude that diminazene uptake in T. congolense proceed via multiple low affinity mechanisms including folate transporters; while resistance is associated with a reduction in Ψm it is unclear whether this is the primary cause of the resistance

The TgsGP gene is essential for resistance to human serum in Trypanosoma brucei gambiense

Trypanosoma brucei gambiense causes 97% of all cases of African sleeping sickness, a fatal disease of sub-Saharan Africa. Most species of trypanosome, such as T. b. brucei, are unable to infect humans due to the trypanolytic serum protein apolipoprotein-L1 (APOL1) delivered via two trypanosome lytic factors (TLF-1 and TLF-2). Understanding how T. b. gambiense overcomes these factors and infects humans is of major importance in the fight against this disease. Previous work indicated that a failure to take up TLF-1 in T. b. gambiense contributes to resistance to TLF-1, although another mechanism is required to overcome TLF-2. Here, we have examined a T. b. gambiense specific gene, TgsGP, which had previously been suggested, but not shown, to be involved in serum resistance. We show that TgsGP is essential for resistance to lysis as deletion of TgsGP in T. b. gambiense renders the parasites sensitive to human serum and recombinant APOL1. Deletion of TgsGP in T. b. gambiense modified to uptake TLF-1 showed sensitivity to TLF-1, APOL1 and human serum. Reintroducing TgsGP into knockout parasite lines restored resistance. We conclude that TgsGP is essential for human serum resistance in T. b. gambiense

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

ExploreASL: an image processing pipeline for multi-center ASL perfusion MRI studies

Arterial spin labeling (ASL) has undergone significant development since its inception, with a focus on improving standardization and reproducibility of its acquisition and quantification. In a community-wide effort towards robust and reproducible clinical ASL image processing, we developed the software package ExploreASL, allowing standardized analyses across centers and scanners. The procedures used in ExploreASL capitalize on published image processing advancements and address the challenges of multi-center datasets with scanner-specific processing and artifact reduction to limit patient exclusion. ExploreASL is self-contained, written in MATLAB and based on Statistical Parameter Mapping (SPM) and runs on multiple operating systems. To facilitate collaboration and data-exchange, the toolbox follows several standards and recommendations for data structure, provenance, and best analysis practice. ExploreASL was iteratively refined and tested in the analysis of >10,000 ASL scans using different pulse-sequences in a variety of clinical populations, resulting in four processing modules: Import, Structural, ASL, and Population that perform tasks, respectively, for data curation, structural and ASL image processing and quality control, and finally preparing the results for statistical analyses on both single-subject and group level. We illustrate ExploreASL processing results from three cohorts: perinatally HIV-infected children, healthy adults, and elderly at risk for neurodegenerative disease. We show the reproducibility for each cohort when processed at different centers with different operating systems and MATLAB versions, and its effects on the quantification of gray matter cerebral blood flow. ExploreASL facilitates the standardization of image processing and quality control, allowing the pooling of cohorts which may increase statistical power and discover between-group perfusion differences. Ultimately, this workflow may advance ASL for wider adoption in clinical studies, trials, and practice

Pathogenicity and virulence of African trypanosomes: From laboratory models to clinically relevant hosts

ABSTRACT African trypanosomes are vector-borne protozoa, which cause significant human and animal disease across sub-Saharan Africa, and animal disease across Asia and South America. In humans, infection is caused by variants of Trypanosoma brucei, and is characterized by varying rate of progression to neurological disease, caused by parasites exiting the vasculature and entering the brain. Animal disease is caused by multiple species of trypanosome, primarily T. congolense, T. vivax, and T. brucei. These trypanosomes also infect multiple species of mammalian host, and this complexity of trypanosome and host diversity is reflected in the spectrum of severity of disease in animal trypanosomiasis, ranging from hyperacute infections associated with mortality to long-term chronic infections, and is also a main reason why designing interventions for animal trypanosomiasis is so challenging. In this review, we will provide an overview of the current understanding of trypanosome determinants of infection progression and severity, covering laboratory models of disease, as well as human and livestock disease. We will also highlight gaps in knowledge and capabilities, which represent opportunities to both further our fundamental understanding of how trypanosomes cause disease, as well as facilitating the development of the novel interventions that are so badly needed to reduce the burden of disease caused by these important pathogens