London School of Hygiene & Tropical Medicine

Dissecting the mechanistic role of PfACS-ADP in regulating mitochondrial energy metabolism

(London)

We have discovered that PfACS-ADP is a mitochondrial enzyme that diverts energy flux away from the mitochondrion in Plasmodium. Deletion of PfACS-ADP has opposing effects on parasite transmission to the mosquito. It initially enhances gamete formation, but then parasites fail to develop in the mosquito gut. Both point to the importance of tight regulation of mitochondrial activity.

PfACS-ADP contains a predicted GNAT acetylation domain. Protein acetylation is known to regulate mitochondrial activity. We hypothesise that this domain either self-regulates ACS-ADP or acetylates other mitochondrial proteins.

To test this hypothesis, you will (1) generate transgenic parasites lacking GNAT domain or enzymatic domains and study their phenotypes in cell assays; and (2) introduce a bio-ID tag to ACS-ADP and probe for binding partners by mass spectroscopy.  The project will involve cell culture, molecular biology, whole cell assays, and microscopy.

Drug resistance by dormancy: How do trypanosomes sleep and avoid elimination?

(London)

The American trypanosome Trypanosoma cruzi causes Chagas disease, a debilitating infection that leads to cardiac and/or gut pathology, often with fatal outcomes. >6 million people are infected, and treatment is restricted to two toxic drugs with limited efficacy. Treatment failure results when a sub-population of parasites switch to a non-replicating form. We have developed a method for isolating these quiescent trypanosomes.

The project aims are to dissect the signaling pathways that trigger quiescence and to generate parasite lines where the phenotype is inducible. You will use genetic manipulation techniques to identify and characterise genes that drive reduced DNA replication, transcription and metabolic activity. The project is sponsored by the Drugs for Neglected Diseases initiative (DNDi) and the parasite lines generated will be integrated into the drug development pipeline to aid identification of novel chemical entities that can eliminate these “persister” parasites.

Bacterial expression, enzyme kinetics and protein structure of a novel malaria lactate dehydrogenase

(London)

Development of malaria parasites (genus Plasmodium) relies on glycolysis to generate energy, which requires pyruvate to lactate conversion by an essential lactate dehydrogenase enzyme (LDH1). We have recently shown that malaria parasites encode a second putative LDH (LDH2) that is critical for parasite transmission by mosquitoes. LDH2 is distinct from LDH1 and human LDH with respect to its catalytic residues and substrate specificity loop, making it an attractive drug target.

The project aims to determine the enzymatic properties and 3D structure of LDH2 to see how it differs from Plasmodium LDH1 and human LDH to inform the identification of specific inhibitors. The project’s objectives are to: (1) Recombinantly express Plasmodium LDH2 in bacteria; (2) Conduct in vitro enzymatic assays of affinity-purified LDH2 to determine its substrate binding affinities and enzyme kinetics; (3) Carry out 3D structural studies of purified LDH2 through X-ray crystallography or Cryo-EM (time allowing).

Characterisation of novel invasion genes in the malaria parasite Plasmodium knowlesi

(London)

Malaria is a deadly disease caused by Plasmodium parasites. During the asexual blood stage, the parasite invades the red blood cell (RBC), multiplies within and releases daughter cells ready to invade new RBCs. Invasion is critical for parasite survival and invasion proteins are key players in host cell tropism, virulence and as vaccine candidates.

Most cases are caused by Plasmodium falciparum (Pf) and P. vivax (Pv). More research has been done on Pf which is culture adapted, unlike Pv. Recently the zoonotic parasite P. knowlesi (Pk), which is also medically important, was adapted to in vitro culture and closely related to Pv. The aim of this project is to characterise novel invasion genes in the less studied Pk parasite, with a view to discover potential divergent biology between Pf and Pk/Pv. Techniques include CRISPR-Cas9 gene editing, cloning, western blot and microscopy to characterise 1) localisation 2) essentiality and 3) the role of selected invasion protein candidates.

Adaptive variation in parasite sexual commitment for transmission of malaria in The Gambia

(The Gambia)

Malaria parasites in the blood are mostly asexual but must produce sexual forms to infect mosquitoes. There is significant variation in sexual commitment rates of different clinical isolates of the major human parasite Plasmodium falciparum, ranging from less than 3% up to almost 15% per replicative cycle. The adaptive significance and cellular mechanisms of this parasite variation need to be better understood, particularly in populations where levels of malaria transmission have declined over time but elimination remains difficult to achieve. Particular candidate genomic variants in the parasite are shown to have been under natural selection in Gambia over several decades, during which the level of malaria transmission declined.

You will investigate phenotypic variation of cryopreserved parasite isolates collected over a 20 year period, and determine their dependence on genomic and epigenetic markers as well as their relation to asexual multiplication and other phenotypes.

How haptomonad promastigotes influence Leishmania transmission from sand flies

(London)

Leishmania are transmitted from the anterior midgut of sand flies by regurgitation after parasites attach to the stomodeal valve as specialist haptomonad promastigotes and differentiate into infectious metacyclic promastigotes. Previous work from our lab developed a metacyclic-specific RT-qPCR revealing that transmission is more heterogeneous than expected. Using single-cell RNA-seq we have identified candidate genes, improving understanding of how different promastigote stages colonise the valve and influence the transmitted dose.

You will investigate transmission heterogeneity by developing a multiplex RT-qPCR to quantify haptomonad and metacyclic promastigotes in sand fly infections and in parasites transmitted to skin. Using co-infections with red- and green-fluorescent parasites, you will test how variation in transformation rates affects valve colonisation and metacyclogenesis (microscopy), and how this contributes to dose heterogeneity (RT-qPCR).

Decoding rDNA transcriptional regulation in trypanosomatid parasites

(London)

Uniquely amongst studied eukaryotes, Trypanosoma brucei transcribes some of its mRNAs (including its surface coat proteins) with RNA polymerase-I, posing a challenge to rRNA production.  By integrating reporters at rDNA arrays we have shown that not all arrays are equally active in T. brucei and that repressed arrays can be upregulated during differentiation, suggesting that they are tightly regulated.

You will identify and characterise the factors responsible for rDNA array transcriptional repression in T. brucei using our established reporter cell lines, and a combination of forward and reverse genetic approaches. You will gain expertise in a range of molecular and cell biology approaches, and the opportunity to further develop an exciting project exploring a complex research question, with the potential to extend beyond T. brucei.