Biology subjects

Sutherland, C. J.

Publications and source records attributed to Sutherland, C. J..

7 recordsLinked to original sources

bioRxiv2026

The translatome of quiescent Plasmodium falciparum gametocytes reveals parasite pyridoxal 5'-phosphate (PLP) biosynthesis is essential for efficient mosquito stage development

The ability of Plasmodium falciparum gametocytes to remain quiescent within the vertebrate host but poised for rapid onward development in the mosquito is an adaptation essential to maximise the onward spread of malaria. In this dormant state, mature infectious stage V gametocytes are largely unaffected by most antimalarial drugs and our limited understanding of how gametocytes prepare for mosquito transmission has hindered the identification of new molecular targets for transmission-blocking therapeutics. In this study, we move beyond the total proteome of gametocytes and define the translatome of mature stage V gametocytes using L-azidohomoalanine incorporation into nascent proteins, click chemistry purification and proteomic analysis. We identify the proteins and pathways that gametocytes sustain in preparation for transmission during this dormant period and through genetic disruption, we validate this approach by demonstrating the importance of parasite pyridoxal 5-phosphate biosynthesis for mosquito transmission.

microbiology↗

bioRxiv2022

Drug resistance-associated mutations in Plasmodium UBP-1 disrupt ubiquitin hydrolysis

Deubiquitinating enzymes function to cleave ubiquitin moieties from modified proteins, serving to maintain the pool of free ubiquitin in the cell while simultaneously impacting the fate and function of a target protein. Like all eukaryotes, Plasmodium parasites rely on the dynamic addition and removal of ubiquitin for their own growth and survival. While humans possess around 100 DUBs, Plasmodium contains [~]20 putative ubiquitin hydrolases, many of which bear little to no resemblance to those of other organisms. In this study, we characterize PfUBP-1, a large ubiquitin hydrolase unique to Plasmodium spp that has been linked to endocytosis and drug resistance. We demonstrate its ubiquitin activity, linkage specificity and assess the repercussions of point mutations associated with drug resistance on catalytic activity and parasite fitness.

microbiology↗

bioRxiv2022

Mechanochemical tuning of a kinesin motor essential for malaria parasite transmission

Kinesins are a superfamily of molecular motors that undertake ATP-dependent microtubule-based movement or regulate microtubule dynamics. Plasmodium species, which cause malaria and kill hundreds of thousands annually, encode 8-9 kinesins in their genomes. Of these, two - kinesin-8B and kinesin-8X -are canonically classified as kinesin-8s, which in other eukaryotes typically modulate microtubule dynamics. Unexpectedly, Plasmodium kinesin-8B is required for development of the flagellated male gamete in the mosquito host, and its absence completely blocks parasite transmission. To understand the molecular basis of kinesin-8Bs essential role, we characterised the in vitro properties of the kinesin-8B motor domains from P. berghei and P. falciparum. Both motors drive plus-end directed ATP-dependent microtubule gliding, but also catalyse ATP-dependent microtubule depolymerisation. We determined the microtubule-bound structures of these motors using cryo-electron microscopy. P. berghei and P. falciparum kinesin-8B exhibit a very similar mode of microtubule interaction, in which Plasmodium-distinct sequences at the microtubule-kinesin interface influence motor function. Intriguingly, however, P. berghei kinesin-8B exhibits a non-canonical structural response to ATP analogue binding such that neck linker docking is not induced. Nevertheless, the neck linker region is absolutely required for motility and depolymerisation activities of these motors. Taken together, these data suggest that the mechanochemistry of Plasmodium kinesin-8Bs has been functionally tuned to efficiently contribute to flagella formation.

biochemistry↗

bioRxiv2020

A Novel Multiplex qPCR Assay for Detection of Plasmodium falciparum with Histidine-rich Protein 2 and 3 (pfhrp2 and pfhrp3) Deletions in Polyclonal Infections

BackgroundRapid diagnostic tests (RDTs) that detect the malaria antigen histidine-rich protein 2 (HRP2) are widely used in endemic areas globally to confirm Plasmodium falciparum infection in febrile patients. The emergence of parasites lacking the gene encoding HRP2 and escaping RDT detection threatens progress in malaria control and elimination. Many health facilities in malaria endemic countries are dependent on RDTs for diagnosis and some National Health Service hospitals without expert microscopists rely on them for diagnosis out of hours. It is vital to study the emergence and the extent of such parasites globally to guide diagnostic policy. Currently, verification of the presence of such parasites in a blood sample requires a series of PCR assays to confirm the presence of P. falciparum and in the absence of amplicons from pfhrp2 and/or pfhrp3, which encodes a cross-reactive protein isoform. These tests have different limits of detection and many laboratories have reported difficulty in confirming the absence of pfhrp2 and pfhrp3 with certainty. MethodsWe developed and validated a novel and rapid multiplex real time quantitative (qPCR) assay to detect pfhrp2, pfhrp3, confirmatory parasite and human reference genes simultaneously. We also applied the assay to detect pfhrp2 and pfhrp3 deletion in 462 field samples from different endemic countries and UK travellers. ResultsThe qPCR assay showed limit of detection and quantification of 0.76-1.5 parasites per l. The amplification efficiency, coefficient of determination (R2) and slope for the genes were 96-1.07%, 0.96-0.98 and -3.375 2 to -3.416 respectively. The assay demonstrated diagnostic sensitivity of 100% (n=19, 95% CI= (82.3%; 100%)) and diagnostic specificity of 100% (n=31; 95% CI= (88.8%; 100%)) in detecting pfhrp2 and pfhrp3 in. In addition, the qPCR assay estimates P. falciparum parasite density and can detect pfhrp2 and pfhrp3 deletions masked in polyclonal infections. We report pfhrp2 and pfhrp3 deletions in parasite isolates from Kenya, Tanzania and in UK travellers. ConclusionThe new qPCR assay is simple to use and offers significant advantages in speed and ease of interpretation. It is easily scalable to routine surveillance studies in countries where P. falciparum parasites lacking pfhrp2 and pfhrp3 are a threat to malaria control.

genetics↗

bioRxiv2019

Modification of an atypical clathrin-independent AP-2 adaptin complex of Plasmodium falciparum reduces susceptibility to artemisinin

The efficacy of current antimalarial drugs is threatened by reduced susceptibility of Plasmodium falciparum to artemisinin. In the Mekong region this is associated with mutations in the kelch propeller-encoding domain of pfkelch13, but variants of other parasite proteins are also thought to modulate the response to drug. Evidence from human and rodent studies suggests that the -subunit of the AP-2 adaptin trafficking complex is one such protein of interest. We generated transgenic Plasmodium falciparum parasites encoding the I592T variant of pfap2, orthologous to the I568T mutation associated with in vivo artemisinin resistance in P. chabaudi. When exposed to a four-hour pulse of dihydroartemisin in the ring-stage survival assay, two P. falciparum clones expressing AP-2 I592T displayed significant and reproducible survival of 8.0% and 10.3%, respectively, compared to <2% for the 3D7 parental line (P = 0.0011 for each clone). In immunoprecipitation and localisation studies of HA-tagged AP-2, we identified interacting partners including AP-2, AP-1/2{beta}, AP-2{sigma} and a kelch-domain protein encoded on chromosome 10 of P. falciparum, K10. Conditional knockout indicates that the AP-2 trafficking complex in P. falciparum is essential for the fidelity of merozoite biogenesis and membrane organisation in the mature schizont. We also show that while other heterotetrameric AP-complexes and secretory factors interact with clathrin, AP-2 complex subunits do not. Thus, the AP-2 complex may be diverted from a clathrin-dependent endocytic role seen in most eukaryotes into a Plasmodium-specific function. These findings represent striking divergences from eukaryotic dogma and support a role for intracellular traffic in determining artemisinin sensitivity in vitro, confirming the existence of multiple functional routes to reduced ring-stage artemisinin susceptibility. Therefore, the utility of pfkelch13 variants as resistance markers is unlikely to be universal, and phenotypic surveillance of parasite susceptibility in vivo may be needed to identify threats to our current combination therapies.

microbiology↗

bioRxiv2019

Rapid and iterative genome editing in the zoonotic malaria parasite Plasmodium knowlesi: New tools for P. vivax research

Tackling relapsing Plasmodium vivax and zoonotic Plasmodium knowlesi infections is critical to reducing malaria incidence and mortality worldwide. Understanding the biology of these important and related parasites was previously constrained by the lack of robust molecular and genetic approaches. Here, we establish CRISPR-Cas9 genome editing in a culture-adapted P. knowlesi strain and define parameters for optimal homology-driven repair. We establish a scalable protocol for the production of repair templates by PCR and demonstrate the flexibility of the system by tagging proteins with distinct cellular localisations. Using iterative rounds of genome-editing we generate a transgenic line expressing P. vivax Duffy binding protein (PvDBP), a lead vaccine candidate. We demonstrate that PvDBP plays no role in reticulocyte restriction but can alter the macaque/human host cell tropism of P. knowlesi. Critically, antibodies raised against the P. vivax antigen potently inhibit proliferation of this strain, providing an invaluable tool to support vaccine development.

microbiology↗

bioRxiv2018

MOSQUITO TRANSMISSION AND HUMAN HEPATOCYTE INFECTIONS WITH Plasmodium ovale curtisi AND P. ovale wallikeri

Human ovale malaria is caused by the two closely related species Plasmodium ovale curtisi and P. ovale wallikeri. Both species are known to relapse from quiescent hepatic forms months or years after the primary infection occurred. Although some studies have succeeded in establishing mosquito transmission for ovale malaria, none have specifically described transmission and human hepatocyte infection of both sibling species. Here we describe a simplified protocol for successful transmission of both P. ovale curtisi and P. ovale wallikeri to Anopheles coluzzii mosquitoes, and streamlined monitoring of infection using sensitive parasite DNA detection, by loop-activated amplification, in blood-fed mosquitoes. In one experimental infection with P. ovale curtisi and one with P. ovale wallikeri, viable sporozoites were isolated from mosquito salivary glands, and used to successfully infect cultured human hepatocytes. This protocol provides a method for the utilisation of pre-treatment clinical blood samples from ovale malaria patients, collected in EDTA, for mosquito infection studies and generation of the hepatic life cycle stages of P. ovale curtisi and P. ovale wallikeri. We also demonstrate the utility of LAMP as a rapid and sensitive alternative to dissection for estimating the prevalence of infection in Anopheles mosquitoes fed with Plasmodium-infected blood.

microbiology↗