Characterisation of the Plasmodium falciparum export complex

Plasmodium parasites extensively remodel the mammalian host cells they infect, namely the erythrocytes and hepatocytes. This is achieved through exporting hundreds of parasite proteins into the host where they play many virulence related roles including altering membrane permeability to acquire nutrients for rapid growth and for increasing immune evasion. Proteins are secreted from the parasite into the vacuole that surrounds them, where they must then pass across the parasitophorous vacuole membrane (PVM) to gain access to the host cytoplasm. It has previously been shown that a protein complex, the Plasmodium Translocon of EXported proteins (PTEX), is found on the PVM and is most likely the protein translocon responsible for this export process. To validate and further dissect PTEX function, a conditional expression system that utilizes the glmS riboswitch was employed to conditionally knockdown the levels of PTEX150 within parasites. Using this approach, even a relatively low level of PTEX150 knockdown lead to a significant decrease in the ability of the parasites to export proteins across the PVM. This failure of protein export across the PVM arrested growth and eventually caused parasite death. Interestingly, the export of all classes of protein cargoes tested, including PfEMP1, were blocked upon PTEX150 knockdown suggesting PTEX may serve as a single portal for export. This demonstrates the importance of PTEX to the malaria parasite and validates it as a potent drug target, since blocking it would prevent hundreds of exported proteins from reaching their functional destinations. To further investigate the specific function of PTEX150 we performed C-terminal truncations in order to generate a parasite line with a partially defective protein. These lines showed destabilisation of the PTEX complex, suggesting that the C-terminus of PTEX150, while not essential for PTEX function or the absolute binding of the other components, is required for stabilising the PTEX complex. Interestingly, these parasites did not show a defect in either their ability to grow or export proteins. To further investigate the effect of PTEX150 knockdown on parasite health the new permeability pathways (NPPs) were investigated. To do this a novel method was developed to investigate NPP function using luciferase. This method is highly sensitive, reducing the amount of parasites required as well as removing the need to purify parasites from culture. The effect of PTEX150 knockdown on NPPs was unclear using this method. Due to the high sensitivity and ease of this method it was developed as a HTS to discover NPP inhibitors. Using this method the malaria box of 400 compounds was screened. Importantly, two compounds, MMV020439and MMV007571, showed inhibitory activity significantly higher than the control compound NPPB

The molecular basis of antimalarial drug resistance in Plasmodium vivax

Plasmodium vivax is the most geographically widespread cause of human malaria and is responsible for the majority of cases outside of the African continent. While great progress has been made towards eliminating human malaria, drug resistant parasite strains pose a threat towards continued progress. Resistance has arisen to multiple antimalarials in P. vivax, including to chloroquine, which is currently the first line therapy for P. vivax in most regions. Despite its importance, an understanding of the molecular mechanisms of drug resistance in this species remains elusive, in large part due to the complex biology of P. vivax and the lack of in vitro culture. In this review, we will cover the extent and challenges of measuring clinical and in vitro drug resistance in P. vivax. We will consider the roles of candidate drug resistance genes. We will highlight the development of molecular approaches for studying P. vivax biology that provide the opportunity to validate the role of putative drug resistance mutations as well as identify novel mechanisms of drug resistance in this understudied parasite. Validated molecular determinants and markers of drug resistance are essential for the rapid and cost-effective monitoring of drug resistance in P. vivax, and will be useful for optimizing drug regimens and for informing drug policy in control and elimination settings

Additional toxins for feral pig (Sus scrofa) control: Identifying and testing Achilles' heels

A literature review was conducted in order to identify unique weaknesses in the physiology or metabolism of pigs that could be targeted with specific chemicals (i.e. an ‘Achilles’ heel’ search). A promising weakness identified was the species’ susceptibility to methaemoglobin-forming compounds, most likely related to their uniquely low levels of methaemoglobin reductase. Further examination revealed that sodium nitrite is a cost-effective, readily available methaemoglobin-forming compound that is highly toxic to domestic pigs, which has caused numerous accidental poisonings. Pen trials on pigs showed that sodium nitrite delivered by gavage (>90 mg kg−1) and freely consumed in bait (>400 mg kg−1) caused rapid and lethal rises in methaemoglobin. Sodium nitrite appeared to be more humane than currently used toxins, with deaths following bait consumption being considerably quicker and with fewer symptoms (within 80 min of clinical signs beginning; clinical signs including infrequent vomiting, lethargy, ataxia and dyspnoea). The review also identified a second deficiency in the metabolism of pigs, namely high sensitivity to selective inhibition of cytochrome P450 liver enzymes. This leads to potentially lethal interactions between various drugs, such as two antibiotics, monensin and tiamulin. A pen trial confirmed that the antibiotic combination in a single gavage dose was reliably and rapidly lethal to pigs. However, its utility as a pig toxin is low, because it was unpalatable to pigs when delivered in bait and appeared to cause pain and suffering (leading to the early termination of pen trials). The findings presented here demonstrate the potential of sodium nitrite as an additional feral pig toxin

Identification of inhibitors that dually target the new permeability pathway and dihydroorotate dehydrogenase in the blood stage of Plasmodium falciparum

Plasmodium parasites are responsible for the devastating disease malaria that affects hundreds of millions of people each year. Blood stage parasites establish new permeability pathways (NPPs) in infected red blood cell membranes to facilitate the uptake of nutrients and removal of parasite waste products. Pharmacological inhibition of the NPPs is expected to lead to nutrient starvation and accumulation of toxic metabolites resulting in parasite death. Here, we have screened a curated library of antimalarial compounds, the MMV Malaria Box, identifying two compounds that inhibit NPP function. Unexpectedly, metabolic profiling suggested that both compounds also inhibit dihydroorotate dehydrogense (DHODH), which is required for pyrimidine synthesis and is a validated drug target in its own right. Expression of yeast DHODH, which bypasses the need for the parasite DHODH, increased parasite resistance to these compounds. These studies identify two potential candidates for therapeutic development that simultaneously target two essential pathways in Plasmodium, NPP and DHODH

<i>Plasmodium falciparum</i> Transfected with Ultra Bright NanoLuc Luciferase Offers High Sensitivity Detection for the Screening of Growth and Cellular Trafficking Inhibitors

<div><p>Drug discovery is a key part of malaria control and eradication strategies, and could benefit from sensitive and affordable assays to quantify parasite growth and to help identify the targets of potential anti-malarial compounds. Bioluminescence, achieved through expression of exogenous luciferases, is a powerful tool that has been applied in studies of several aspects of parasite biology and high throughput growth assays. We have expressed the new reporter NanoLuc (Nluc) luciferase in <i>Plasmodium falciparum</i> and showed it is at least 100 times brighter than the commonly used firefly luciferase. Nluc brightness was explored as a means to achieve a growth assay with higher sensitivity and lower cost. In addition we attempted to develop other screening assays that may help interrogate libraries of inhibitory compounds for their mechanism of action. To this end parasites were engineered to express Nluc in the cytoplasm, the parasitophorous vacuole that surrounds the intraerythrocytic parasite or exported to the red blood cell cytosol. As proof-of-concept, these parasites were used to develop functional screening assays for quantifying the effects of Brefeldin A, an inhibitor of protein secretion, and Furosemide, an inhibitor of new permeation pathways used by parasites to acquire plasma nutrients.</p></div

<i>Plasmodium falciparum</i> stably expressing Nluc.

<p>(A) The Nluc gene was cloned in the pEF vector for stable expression in <i>P. falciparum</i>. (B) Aliquots (100 µL) of cultures at 1% hematocrit and 5% parasitemia corresponding to 500,000 <i>P. falciparum</i> infected RBCs transfected with pEF-Nluc were mixed with 1 volume of Nano-Glo Luciferase Assay Reagent and reporter activity measured (1∶1). Nano-Glo Luciferase Assay Reagent was further diluted in 10-fold increments in Luciferase Cell Culture Lysis Reagent and used to determine reporter activity of the same culture. As a negative control, wild type parasites (wt) were mixed 1∶1 with Nano-Glo Luciferase Assay Reagent. (C) Parasites stably transfected with pEF-Nluc were diluted in 2-fold increments in RPMI + RBC maintaining 0.5% hematocrit. For each sample, 10 µL of the culture dilutions were mixed with 40 µL of Nano-Glo diluted 1∶400 in water and measured in the luminometer for 2 s with the gain adjusted 10% below saturation for the brightest sample. The solid line represents the mean RLU after linear regression. The dashed line represents the background +3 standard deviations. (D) Nluc expressing parasites were cultured in varying concentrations of chloroquine (CQ) and their growth determined by the LDH standard method or by measuring reporter activity using Nano-Glo Luciferase Assay Reagent at its standard dilution (1∶1) or diluted 1∶1000 as described in (C). Similarly, wild type parasites were transiently transfected with pPfNluc and their growth determined using Nano-Glo Luciferase Assay Reagent (Transient). IC₅₀ was calculated by non-linear regression and represents the mean of 3 experiments. (E) The IC₅₀s determined in D were plotted with 95% confidence intervals (CI).</p

NanoLuc (Nluc) luminescence in <i>Plasmodium falciparum.</i>

<p>(A) Diagrams of firefly and Nluc reporter vectors. (B) pPf86 and pPfNluc were transfected in trophozoite stage parasites and luciferase activity in relative light units (RLU) determined 4 days later. Note that the RLU of each luciferase was measured in its own optimal substrate ie, Nluc with Nano-Glo and Firefly with D-luciferin. Mock-transfected parasites were used as negative control and to determine background luminescence, which was then subtracted from firefly and Nluc activities. The result represents the mean of 3 independent transfections ± standard deviation.</p

Quantification of Nluc in cellular compartments of infected RBCs.

<p>(A) Schematic of the iRBC’s compartments where RBC represents the exported fraction that is released after Equinatoxin treatment. The PV compartment was then released following treatment with 0.01% saponin and finally, the Parasite fraction was lysed by Nano-Glo Luciferase Assay Reagent. (B) Trophozoite stage parasites transfected with either the original Nluc, secreted SP-Nluc or the exported PEXEL-Nluc fusions were fractionated as shown in (A) and luciferase activity measured. Nluc activities as a percentage of the total for each parasite line are shown and represent the mean of 3 experiments +/−SEM. Similar to (B), (C) Schizonts and (D) Ring stage parasites were also fractionated. Statistical significance (* p&lt;0.05) was determined by 2 way ANOVA test comparing the percentage of reporter activity of each sub-cellular fraction among the 3 cell lines.</p

Summary of the assay parameters from the NPP assay.

<p>Summary of the assay parameters from the NPP assay.</p

Summary of the assay parameters from the growth assays.

1<p>refers to luminescence measured from transiently transfected parasites using the standard Nano-Glo dilution.</p><p>Summary of the assay parameters from the growth assays.</p