School of BioSciences - Research Publications
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ItemTargeting malaria parasites with novel derivatives of azithromycin(FRONTIERS MEDIA SA, 2022-11-30)INTRODUCTION: The spread of artemisinin resistant Plasmodium falciparum parasites is of global concern and highlights the need to identify new antimalarials for future treatments. Azithromycin, a macrolide antibiotic used clinically against malaria, kills parasites via two mechanisms: 'delayed death' by inhibiting the bacterium-like ribosomes of the apicoplast, and 'quick-killing' that kills rapidly across the entire blood stage development. METHODS: Here, 22 azithromycin analogues were explored for delayed death and quick-killing activities against P. falciparum (the most virulent human malaria) and P. knowlesi (a monkey parasite that frequently infects humans). RESULTS: Seventeen analogues showed improved quick-killing against both Plasmodium species, with up to 38 to 20-fold higher potency over azithromycin after less than 48 or 28 hours of treatment for P. falciparum and P. knowlesi, respectively. Quick-killing analogues maintained activity throughout the blood stage lifecycle, including ring stages of P. falciparum parasites (<12 hrs treatment) and were >5-fold more selective against P. falciparum than human cells. Isopentenyl pyrophosphate supplemented parasites that lacked an apicoplast were equally sensitive to quick-killing analogues, confirming that the quick killing activity of these drugs was not directed at the apicoplast. Further, activity against the related apicoplast containing parasite Toxoplasma gondii and the gram-positive bacterium Streptococcus pneumoniae did not show improvement over azithromycin, highlighting the specific improvement in antimalarial quick-killing activity. Metabolomic profiling of parasites subjected to the most potent compound showed a build-up of non-haemoglobin derived peptides that was similar to chloroquine, while also exhibiting accumulation of haemoglobin-derived peptides that was absent for chloroquine treatment. DISCUSSION: The azithromycin analogues characterised in this study expand the structural diversity over previously reported quick-killing compounds and provide new starting points to develop azithromycin analogues with quick-killing antimalarial activity.
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ItemRoles of the apicoplast across the life cycles of rodent and human malaria parasites(WILEY, 2022-11-12)Malaria parasites are diheteroxenous, requiring two hosts—a vertebrate and a mosquito—to complete their life cycle. Mosquitoes are the definitive host where malaria parasite sex occurs, and vertebrates are the intermediate host, supporting asexual amplification and more significant geographic spread. In this review, we examine the roles of a single malaria parasite compartment, the relict plastid known as the apicoplast, at each life cycle stage. We focus mainly on two malaria parasite species—Plasmodium falciparum and P. berghei— comparing the changing, yet ever crucial, roles of their apicoplasts.
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ItemA genetic screen in rodent malaria parasites identifies five new apicoplast putative membrane transporters, one of which is essential in human malaria parasites(WILEY, 2018-01)The malaria-causing parasite, Plasmodium, contains a unique non-photosynthetic plastid known as the apicoplast. The apicoplast is an essential organelle bound by four membranes. Although membrane transporters are attractive drug targets, only two transporters have been characterised in the malaria parasite apicoplast membranes. We selected 27 candidate apicoplast membrane proteins, 20 of which are annotated as putative membrane transporters, and performed a genetic screen in Plasmodium berghei to determine blood stage essentiality and subcellular localisation. Eight apparently essential blood stage genes were identified, three of which were apicoplast-localised: PbANKA_0614600 (DMT2), PbANKA_0401200 (ABCB4), and PbANKA_0505500. Nineteen candidates could be deleted at the blood stage, four of which were apicoplast-localised. Interestingly, three apicoplast-localised candidates lack a canonical apicoplast targeting signal but do contain conserved N-terminal tyrosines with likely roles in targeting. An inducible knockdown of an essential apicoplast putative membrane transporter, PfDMT2, was only viable when supplemented with isopentenyl diphosphate. Knockdown of PfDMT2 resulted in loss of the apicoplast, identifying PfDMT2 as a crucial apicoplast putative membrane transporter and a candidate for therapeutic intervention.
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ItemApicoplast isoprenoid precursor synthesis and the molecular basis of fosmidomycin resistance in Toxoplasma gondii(ROCKEFELLER UNIV PRESS, 2011-07-04)Apicomplexa are important pathogens that include the causative agents of malaria, toxoplasmosis, and cryptosporidiosis. Apicomplexan parasites contain a relict chloroplast, the apicoplast. The apicoplast is indispensable and an attractive drug target. The apicoplast is home to a 1-deoxy-D-xylulose-5-phosphate (DOXP) pathway for the synthesis of isoprenoid precursors. This pathway is believed to be the most conserved function of the apicoplast, and fosmidomycin, a specific inhibitor of the pathway, is an effective antimalarial. Surprisingly, fosmidomycin has no effect on most other apicomplexans. Using Toxoplasma gondii, we establish that the pathway is essential in parasites that are highly fosmidomycin resistant. We define the molecular basis of resistance and susceptibility, experimentally testing various host and parasite contributions in T. gondii and Plasmodium. We demonstrate that in T. gondii the parasite plasma membrane is a critical barrier to drug uptake. In strong support of this hypothesis, we engineer de novo drug-sensitive T. gondii parasites by heterologous expression of a bacterial transporter protein. Mice infected with these transgenic parasites can now be cured from a lethal challenge with fosmidomycin. We propose that the varied extent of metabolite exchange between host and parasite is a crucial determinator of drug susceptibility and a predictor of future resistance.
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ItemCharacterization of Two Malaria Parasite Organelle Translation Elongation Factor G Proteins: The Likely Targets of the Anti-Malarial Fusidic Acid(PUBLIC LIBRARY SCIENCE, 2011-06-10)Malaria parasites harbour two organelles with bacteria-like metabolic processes that are the targets of many anti-bacterial drugs. One such drug is fusidic acid, which inhibits the translation component elongation factor G. The response of P. falciparum to fusidic acid was characterised using extended SYBR-Green based drug trials. This revealed that fusidic acid kills in vitro cultured P. falciparum parasites by immediately blocking parasite development. Two bacterial-type protein translation elongation factor G genes are identified as likely targets of fusidic acid. Sequence analysis suggests that these proteins function in the mitochondria and apicoplast and both should be sensitive to fusidic acid. Microscopic examination of protein-reporter fusions confirm the prediction that one elongation factor G is a component of parasite mitochondria whereas the second is a component of the relict plastid or apicoplast. The presence of two putative targets for a single inhibitory compound emphasizes the potential of elongation factor G as a drug target in malaria.
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ItemYcf93 (Orf105), a Small Apicoplast-Encoded Membrane Protein in the Relict Plastid of the Malaria Parasite Plasmodium falciparum That Is Conserved in Apicomplexa(PUBLIC LIBRARY SCIENCE, 2014-04-04)Malaria parasites retain a relict plastid (apicoplast) from a photosynthetic ancestor shared with dinoflagellate algae. The apicoplast is a useful drug target; blocking housekeeping pathways such as genome replication and translation in the organelle kills parasites and protects against malaria. The apicoplast of Plasmodium falciparum encodes 30 proteins and a suite of rRNAs and tRNAs that facilitate their expression. orf105 is a hypothetical apicoplast gene that would encode a small protein (PfOrf105) with a predicted C-terminal transmembrane domain. We produced antisera to a predicted peptide within PfOrf105. Western blot analysis confirmed expression of orf105 and immunofluorescence localised the gene product to the apicoplast. Pforf105 encodes a membrane protein that has an apparent mass of 17.5 kDa and undergoes substantial turnover during the 48-hour asexual life cycle of the parasite in blood stages. The effect of actinonin, an antimalarial with a putative impact on post-translational modification of apicoplast proteins like PfOrf105, was examined. Unlike other drugs perturbing apicoplast housekeeping that induce delayed death, actinonin kills parasites immediately and has an identical drug exposure phenotype to the isopentenyl diphosphate synthesis blocker fosmidomycin. Open reading frames of similar size to PfOrf105, which also have predicted C-terminal trans membrane domains, occur in syntenic positions in all sequenced apicoplast genomes from Phylum Apicomplexa. We therefore propose to name these genes ycf93 (hypothetical chloroplast reading frame 93) according to plastid gene nomenclature convention for conserved proteins of unknown function.
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ItemMacrolides rapidly inhibit red blood cell invasion by the human malaria parasite, Plasmodium falciparum(BMC, 2015-07-18)BACKGROUND: Malaria invasion of red blood cells involves multiple parasite-specific targets that are easily accessible to inhibitory compounds, making it an attractive target for antimalarial development. However, no current antimalarial agents act against host cell invasion. RESULTS: Here, we demonstrate that the clinically used macrolide antibiotic azithromycin, which is known to kill human malaria asexual blood-stage parasites by blocking protein synthesis in their apicoplast, is also a rapid inhibitor of red blood cell invasion in human (Plasmodium falciparum) and rodent (P. berghei) malarias. Multiple lines of evidence demonstrate that the action of azithromycin in inhibiting parasite invasion of red blood cells is independent of its inhibition of protein synthesis in the parasite apicoplast, opening up a new strategy to develop a single drug with multiple parasite targets. We identified derivatives of azithromycin and erythromycin that are better invasion inhibitors than parent compounds, offering promise for development of this novel antimalarial strategy. CONCLUSIONS: Safe and effective macrolide antibiotics with dual modalities could be developed to combat malaria and reduce the parasite's options for resistance.
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ItemNatural products from Zanthoxylum heitzii with potent activity against the malaria parasite(BMC, 2016-09-20)BACKGROUND: Zanthoxylum heitzii (Rutaceae) (olon) is used in traditional medicine in Central and West Africa to treat malaria. To identify novel compounds with anti-parasitic activity and validate medicinal usage, extracts and compounds isolated from this tree were tested against the erythrocytic stages of the human malaria parasite Plasmodium falciparum and for inhibition of transmission in rodent malaria parasite Plasmodium berghei. RESULTS: Hexane bark extract showed activity against P. falciparum (IC50 0.050 μg/ml), while leaf and seed extracts were inactive. Fractionation of the hexane bark extract led to the identification of three active constituents; dihydronitidine, pellitorine and heitziquinone. Dihydronitidine was the most active compound with an IC50 value of 0.0089 µg/ml (25 nM). This compound was slow acting, requiring 50 % longer exposure time than standard anti-malarials to reach full efficacy. Heitziquinone and pellitorine were less potent, with IC50 values of 3.55 μg/ml and 1.96 µg/ml, but were fast-acting. Plasmodium berghei ookinete conversion was also inhibited by the hexane extract (IC50 1.75 µg/ml), dihydronitidine (0.59 µg/ml) and heitziquinone (6.2 µg/ml). Water extracts of Z. heitzii bark contain only low levels of dihydronitidine and show modest anti-parasitic activity. CONCLUSIONS: Three compounds with anti-parasitic activity were identified in Z. heitzii bark extract. The alkaloid dihydronitidine is the most effective of these, accounting for the bulk of activity in both erythrocytic and transmission-blocking assays. These compounds may present good leads for development of novel anti-malarials and add to the understanding of the chemical basis of the anti-parasitic activity in these classes of natural product.
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ItemA novel genetic technique in Plasmodium berghei allows liver stage analysis of genes required for mosquito stage development and demonstrates that de novo heme synthesis is essential for liver stage development in the malaria parasite(PUBLIC LIBRARY SCIENCE, 2017-06)The combination of drug resistance, lack of an effective vaccine, and ongoing conflict and poverty means that malaria remains a major global health crisis. Understanding metabolic pathways at all parasite life stages is important in prioritising and targeting novel anti-parasitic compounds. The unusual heme synthesis pathway of the rodent malaria parasite, Plasmodium berghei, requires eight enzymes distributed across the mitochondrion, apicoplast and cytoplasm. Deletion of the ferrochelatase (FC) gene, the final enzyme in the pathway, confirms that heme synthesis is not essential in the red blood cell stages of the life cycle but is required to complete oocyst development in mosquitoes. The lethality of FC deletions in the mosquito stage makes it difficult to study the impact of these mutations in the subsequent liver stage. To overcome this, we combined locus-specific fluorophore expression with a genetic complementation approach to generate viable, heterozygous oocysts able to produce a mix of FC expressing and FC deficient sporozoites. These sporozoites show normal motility and can invade liver cells, where FC deficient parasites can be distinguished by fluorescence and phenotyped. Parasites lacking FC exhibit a severe growth defect within liver cells, with development failure detectable in the early to mid stages of liver development in vitro. FC deficient parasites could not complete liver stage development in vitro nor infect naïve mice, confirming liver stage arrest. These results validate the heme pathway as a potential target for prophylactic drugs targeting liver stage parasites. In addition, we demonstrate that our simple genetic approach can extend the phenotyping window beyond the insect stages, opening considerable scope for straightforward reverse genetic analysis of genes that are dispensable in blood stages but essential for completing mosquito development.
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ItemTargeting of a Transporter to the Outer Apicoplast Membrane in the Human Malaria Parasite Plasmodium falciparum(PUBLIC LIBRARY SCIENCE, 2016-07-21)Apicoplasts are vestigial plastids in apicomplexan parasites like Plasmodium, the causative agent of malaria. Apicomplexan parasites are dependant on their apicoplasts for synthesis of various molecules that they are unable to scavenge in sufficient quantity from their host, which makes apicoplasts attractive drug targets. Proteins known as plastid phosphate translocators (pPTs) are embedded in the outer apicoplast membrane and are responsible for the import of carbon, energy and reducing power to drive anabolic synthesis in the organelle. We investigated how a pPT is targeted into the outer apicoplast membrane of the human malaria parasite P. falciparum. We showed that a transmembrane domain is likely to act as a recessed signal anchor to direct the protein into the endomembrane system, and that a tyrosine in the cytosolic N-terminus of the protein is essential for targeting, but one or more, as yet unidentified, factors are also essential to direct the protein into the outer apicoplast membrane.