PhD Students

Structural Elucidation of the Interaction of Apicoplast-Resident Ferredoxin with its Interacting Proteins

Ferredoxin (Fd) and its reductase (FNR) are an essential redox system in the apicoplast organelle in both T. gondii  and Plasmodium sp. Fd is presumably an important electron donor for the apicoplast-resident synthesis pathways of fatty acids and isoprenoid precursors. However, the precise role of this redox system is ill-defined. In particular, how Fd is capable of selectively passing on electrons via protein-protein interactions to the three different enzymes (LipA, GcpE & LytB) involved in these essential pathways is unknown. 

Metabolic Characterisation and Chemotherapeutic Targeting of Persistent T. gondii and P. falciparum Parasites

Phenotypic variation in protozoan parasites is an important strategy to evade the immune responses and antimicrobial treatments. The apicomplexans Toxoplasma gondii and Plasmodium falciparum employ this strategy through the formation of tissue cysts and dormant blood stages, respectively. These stages are marked by attenuated growth and drug resistance. The ability to persist facilitates transmission to the next host. In order to successfully target these evasive stages we need to understand how resistance to antimicrobials is achieved. To this end we aim to characterize the metabolism of dormant parasites by using untargeted metabolomics and test their susceptibility to novel inhibitors.

Determinants of Semi-Immunity and Disease Tolerance in Malaria

The outcome of malaria in individual patients critically depends on their history of previous infections. Patients who have repeatedly been exposed to Plasmodium tolerate the presence of blood-stage parasites far better than those with first-time infections, e.g. with respect to organ damage. Populations of highly malaria-endemic areas even develop a state of complete absence of symptoms, despite ongoing parasitemia. Hence, tolerance is a major determinant of what is frequently referred to as “semi-immunity”. Acquired tolerance can also be disadvantageous, particularly with respect to vaccination. For instance, whole sporozoite vaccines (PfSPZ) elicit weaker immune responses and consequently less protection in vaccines in endemic regions than in malaria-naïve individuals.

Novel adjuvants for pre-erythrocytic stage vaccines

The project will combine self-devised immunoglobulin transgenic mice and lentiviral knockdown libraries for the evidence-based development of adjuvants that permit superior B cell responses against inactivated sporozoites, which are notoriously weak in eliciting lasting immunity. Hence, this project could remove a major roadblock in development of whole sporozoite vaccines for use in endemic countries.

Cholesterol as the molecular key to antiparasitic drug delivery

The collective fight against malaria has been an ongoing battle for many thousands of years. From early naturally derived compounds, to modern synthetic drug therapies; it is a molecular siege of epic proportions which continues to take hundreds of thousands of lives each year. As the malarial parasite continues to evolve, so must our approach towards finding new strategies to combat this brutal disease. Cholesterol-drug conjugation presents itself as a new tactic to overcome poor selectivity and enhance the potency of current and emerging antimalarial drugs. Cholesterol is imported by the malarial parasite from host sources in large quantities. The mechanism by which this import occurs can by hijacked by cholesterol linked conjugates to deliver drugs to parasite targets. Understanding the molecular nature of this delivery is vital to the development of improved drug conjugates. Towards this end, a series of drug linked cholesterol analogues with a wide range of varied structural and chemical features have been synthesized to probe this transport using medicinal chemistry. Furthermore, the development of a metabolically activated fluorescent-dye conjugate has been achieved in an attempt to visualize import and metabolism of cholesterol linked constructs using microscopy. Future studies seeking to further elucidate this unlocking and entry of cholesterol into T. gondii will involve metabolomic profiling of drug-treated parasites using targeted and untargeted metabolomic studies to understand the phenotype of drug-treated parasites and the fate of drug and sterol components.

Modeling ion homeostasis in Plasmodium falciparum using non-equilibrium thermodynamics

In this project we model different aspects of interactions between Plasmodium falciparum and the infected red blood cell with a focus on lipid metabolism and ion homeostasis of the erythrocyte before and after parasite infection. Interference with lipid metabolism is known to affect viability of the malaria parasite. Consequently, the identification of new ways to intervene with the parasite's lipid metabolism could lead to new potential drug targets. After parasite infection, ion homeostasis of the red blood cell changes drastically, mainly due to novel permeability pathways emerging in the erythrocyte's membrane. Analyzing the interplay between host- and parasite-derived ion transport ways will provide new insights into regulatory mechanisms of ion transport inhibitors that have recently emerged as potential antimalarial drugs.

Tubulin - A Novel Lead to Antimalarial Drug Discovery

The highly dynamic microtubule cytoskeleton plays an essential role in the structural integrity of malarial parasites. Consistent with its important role, microtubule-disruptive drugs have great potential as anti-malarial agents. The current therapeutic options of microtubule-disruptive drugs, however, are limited by their high toxicity to mammalian cells. The aim of this PhD project is to characterise Plasmodium tubulin biochemically and biophysically in order to improve its druggability and to advance the potential of Plasmodium tubulin as anti-malarial.

Plasmodium falciparum lipid metabolism as a target for malaria intervention strategies

The survival of P. falciparum parasites relies on the metabolism of lipids. The aim of this project is to explore methods of interfering with this metabolism to decrease either the survival or virulence of these parasites. I will investigate drugs and processes known to affect metabolism, and identify other key lipid pathways by utilising both biochemical and theoretical modelling approaches. This research provides new insights into malaria treatment and prevention strategies.

RNA Processing and Turnover in Apicomplexan Organelles

Apicoplasts and mitochondria contain their own genetic information. Gene expression in these organelles is essential for parasite survival. Antibiotics targeting gene expression in the apicoplast and mitochondrion serve as potent antimalarials. Very little is known about the organellar gene expression machinery in Apicomplexans. We recently identified a family of RNA binding proteins named heptatricopeptide repeat proteins (HPR proteins). Members of this family were demonstrated to reside in RNA granules in human mitochondria and play a role in rRNA and mRNA processing. Initial gene tagging and knock-out approaches demonstrated that these proteins reside in mitochondria and are essential for parasite survival. Their exact functions in mitochondrial RNA metabolism remains enigmatic. In general, RNA turnover and its regulation is hardly addressed in Apicomplexans, neither in organelles nor in the nucleo-cytoplasmic compartment. This project aims at filling this gap in our knowledge.

Riboflavin requirements and metabolism by the intraerythrocytic stage of Plasmodium falciparum.

The intraerythrocytic stage of the human malaria parasite Plasmodium falciparum has unique vitamin requirements. Whilst progress has been made in our understanding of the parasite’s requirement for certain vitamins (e.g. pantothenate and thiamine), very little is known about the parasite’s requirement for other vitamins and the enzyme cofactors they are metabolised into. This project will investigate the parasite’s requirement for riboflavin (vitamin B2) with a view of determining whether riboflavin metabolism can serve as an antimalarial drug target.

Dissecting the distinct metabolic roles of the ferredoxin redox system

The overall aim of this project is to analyze the role of the small apicoplast-resident protein ferredoxin (Fd) in the different metabolic pathways it has been implicated in. Fd provides electrons to several important enzymes (LipA, LytB, GcpE) via protein-protein interactions (PPI), which we want to disrupt by cyclic peptides (dissociators). Identifying these dissociators by a genetic screen in E. coli and validating their specificity by biophysical binding studies and in situ expression in the apicoplast will be one aim of this project. In addition, an inducible Fd gene knockout will be attempted in Plasmodium falciparum using a conditional chemical rescue scheme. The resulting phenotypes in these transgenic parasite lines will be determined with a focus on parasite replication and growth, morphology, and biochemical profiles (e.g. fatty acid or isoprenoid content). Since a previous report implied a role of Fd in artemisinin resistance this can be validate by complementation of the Fd knockout with a set of Fd mutants.

Conserved and Lineage-specific Functions of Plasmodium Membrane Transport Proteins

An intracellular life style and population expansion strictly depends on efficient uptake of ions, nutrients, building blocks and membrane lipids. Mammalian Plasmodium parasites have very tight host species barriers and co-evolved with their respective hosts. Work in two Plasmodium parasite models, the murine malaria model P. berghei and P. falciparum blood cultures, combines phenotyping of the entire parasite life cycle and parasite/host cross-talk during infection with in-depth analysis of blood infection by the human human pathogen. These studies are expected to uncover druggable targets and generate virulence attenuated parasite lines for preclinical testing of malaria vaccine strategies.

TBA

Lipids – together with proteins and nucleid acids – are the major building blocks of cells. We have recently determined the lipid composition of the different life cycle stages of Plasmodium falciparum and Plasmodium berghei. Based on this data we will now determine key molecules that are important for lipid import and metabolism and explore their role and potential as drug targets. A bioinformatic overview obtained in silico will guide us to identify rate limiting steps in the lipid metabolism. These predictions will be verified using transgenic approaches using CRISPR/Cas9 and various tagging strategies. Our approaches will also determine the localization, function and importance of these enzymes and transporters for the survival of the malaria parasites.

Characterising Lipid droplets (LD) in various life cycle stages of Plasmodium sp.

Many stages across the Plasmodium cycle have changing demands in storage and mobilisation of lipids. We have identified that the profile of lipid groups, including neutral lipids, changes significantly between blood stage Plasmodium falciparum parasites (1). 

Furthermore, the ATP-binding cassette transporter gABCG2, which has been suggested to transport neutral lipids into a lipid storage compartment (2), is primarily transcribed in sexual stages and accumulates in a single lipid-rich dot in female gametocytes, reminiscent of a large lipid droplet (LD), organelles known to accumulate neutral lipids. Our gABCG2 ko cell line produces more gametocytes of both sexes than wild type parasites and neutral lipids are significantly reduced in gABCG2-knockout gametocytes indicating that the transporter plays a crucial role in regulation of gametocyte numbers and the accumulation of neutral lipids in gametocytes, which are likely important for parasite development of insect stages (2). Here, we set out to study the biogenesis, morphology and composition of Plasmodium LDs, which have not been explored in detail. Identifying proteins involved in lipid metabolism, stress responses and cell signalling and functional studies of these regulatory molecules might reveal new drug targets to combat malaria.

Characterizing Plasmodium Protein Function at the Parasite-Host Interface During both Liver and Blood Infection Stages

While protein export and host cell membrane remodeling are processes conserved across Plasmodium species, their influence on the ability of parasites to thrive in their hosts has been primarily studied in Plasmodium falciparum. While P. falciparum is the deadliest malaria species, it is evolutionarily distinct from the other human-infecting species, such as P. vivax and P. knowlesi. We have been studying P. berghei proteins that are conserved in most human- or rodent-infecting species, but absent from P. falciparum. These proteins are expressed in both the liver and blood stages of infection and are exported into the host red blood cell where they localize to membranes formed in the host cytoplasm and influence the ability of infected red blood cells to sequester in the microvasculature.

How innate immune recognition shapes plasmodium-specific T-cell responses

There is common consensus that successful eliminating of malaria will require the implementation of an efficacious vaccine against the disease. Thus far, most vaccine candidates provide only modest and rather short-lived protection in the field. Live P. falciparum sporozoites (PfSPZ) have shown promise in healthy volunteers, yet appear to provide short-lived protection in highly endemic areas.

Cross-species association of genotype and transcriptome response as driver of susceptibility and virulence

Understanding host-pathogen interactions is key to understanding infection mechanisms.  In malaria, little is known about genes and proteins involved in these interactions. I am studying the effects of perturbations in gene regulatory networks and of correlated gene expression on infection mechanisms, first, using online databases and second, using gene expression data of mouse models.

Susceptibility to and Manifestation of Malaria: Role of Iron Deficiency and Polymorphisms of Iron Regulation

Iron deficiency (ID) inhibits Plasmodium growth, increases parasite phagocytosis, and reduces malaria risk. Upregulation of the iron master-regulator hepcidin due to malaria or inflammation reduces hepatic parasite survival. Several host polymorphisms influence iron status whereas hepcidin is regulated by inflammation. We have previously shown that ID as well as a common ferroportin polymorphism associate with relative resistance to malaria. 

Role of host lipids in the establishment of the human malaria parasite in the mosquito 

Establishment of Plasmodium falciparum in the mosquito is essential for malaria transmission. However, the host and mosquito factors that regulate the first steps of this critical process are not well understood. P. falciparum transmission stages represent an interesting evolutionary model of plasticity as they require a rapid parasite adaptation to fundamentally different environments (e.g. temperature, metabolites, immune system). The biological mechanisms that underlie parasite plasticity that deals with changing environment, however, remain unknown. 

Dissecting innate immune responses of dendritic cells to blood-stage and pre-blood-stage Plasmodium falciparum

High-throughput CRISPR/Cas9-genome engineering will be used to identify and characterize DC-Plasmodium sensing pathways, which will then be validated together with our Australian partners from the Cockburn lab. New insights may play a role in informed vaccine or adjuvant development against malaria.

Analysis of heptatricopeptide repeat proteins in Toxoplasma gondii

In the course of the project a novel family of apicomplexan organellar RNA binding proteins will be studied. Initial evidence suggests that they function in RNA stabilization, processing and ribosome assembly. As a genetically tractable model for Plasmodium biology, the role of these proteins in organellar RNA metabolism will be investigated in Toxoplasma gondii using a combination of RNA biochemistry and experimental genetics.

Conserved and lineage-specific functions of Plasmodium membrane transport proteins

Membrane transport proteins (MTPs) transport nutrients, metabolic products and inorganic ions across biological membranes. Although they are essential for the cell physiology of Plasmodium parasites, the actual functions of many transport proteins are still insufficiently clarified. Thus, functional analyses of MTPs are of great importance, not only due to their influence on the development of drug resistances, but also as possible targets in the development of active pharmaceutical ingredients. The aim of this work is to analyse a selection of MTPs in Plasmodium and to compare their importance, conservation, occurrence and function in the life cycle of Apicomplexan species.

Tubulin – A novel lead to antimalarial drug discovery

The highly dynamic microtubulecytoskeleton plays an essential  role in the structural integrity of malarial parasites. Consistent with its important role, microtubule-disruptive drugs have great potential as anti-malarial agents. The current therapeutic options

of microtubule-disruptive drugs, however, are limited by their high toxicity to mammalian cells. The aim of this PhD project is to characterize Plasmodium tubulin biochemically and biophysically in order to improve its druggability and to advance the potential of Plasmdium tubulin as anti-malarial.

Characterizing protein function at the parasite-host interface during both liver and blood infection stages.

Plasmodium parasites remodel their host cells to create an environment conducive to their development. This project investigates Plasmodium proteins present in the remodeled membranes at the interface between the parasite and host in both liver and blood cells. Understanding these membrane structures and the functions of these proteins will elucidate key mechanisms by which parasites influence their host environments. The project will use genetics, biochemistry and advanced microscopy to investigate the function of membrane structures generated by Plasmodium in their liver and erythrocyte host cells.