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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.

The recently described red alga Tsunamia transpacifica (Stylonematophyceae) was previously isolated from plastic drift found at the pacific coast, but the natural habitat remains unknown. Here, we investigate ultrastructural details and the low molecular weight soluble carbohydrate composition to get further insight into the adaptation to this uncommon habitat. By means of high pressure freeze fixation, followed by freeze substitution, we could detect an up to 2-µm-thick cell wall surrounded by a distinct layer of extracellular polymeric substances (EPS), likely responsible for the adhering capacities of Tsunamia. The central position of the nucleus and multilobed parietal chloroplast, already observed by light microscopy, could be confirmed. The ultrastructure revealed large electron-dense bodies (EB) in the central cytoplasm, likely resembling degradation products of the chloroplast. Interestingly, these structures contained phosphorous and cobalt, and iron was found in smaller rounded electron-dense bodies by electron energy loss spectroscopy (EELS). Accumulation of these elements suggests a high biosorption activity of Tsunamia. Liquid chromatography-mass spectrometry (LC–MS) data showed the presence of two heterosides (floridoside and digeneaside) together with the polyol sorbitol, which are known as organic osmolytes and compatible solutes. Taken together, these are the first observations on ultrastructural details, element storage and accumulation of protective compounds are contributing to our understanding of the ultrastructural and osmotic solute basis for the ability of Tsunamia to thrive on plastic surfaces.

Purpose/objectives High dose rate (HDR) interstitial brachytherapy (ISBT) boost is integral for definitive radiation treatment of primary vaginal cancer. Technological advances with CT or MRI guidance provide improved precision and ability to treat more extensively invasive tumors over historical techniques, but reported experience is limited. We sought to provide updated outcome and toxicity data for women with primary vaginal cancer undergoing treatment with a modern ISBT technique. Material/methods Databases of primary vaginal carcinoma patients treated at two Canadian academic cancer institutions were combined including patient, tumor and treatment characteristics, and survival outcomes and toxicity data. Descriptive statistics, survival estimates based on the Kaplan-Meier method, and univariable/multivariable Cox proportional hazards regression analyses are reported. Results Between 2002 and 2017, 67 women with primary vaginal cancer were treated with 3D HDR ISBT. FIGO stage distribution was I (22.4%), II (50.8%), III (17.9%), IVa (9.0%). All patients received external beam radiotherapy and HDR ISBT of 500–750 cGy per fraction over 2–4 fractions. Median follow-up was 2.68 years (95% confidence interval: 2.04–6.04). Cumulative rate of grade 3–4 genitourinary/gastrointestinal toxicity was 10.4%. Four patients developed vaginal fistula. Progression-free survival at 2 and 3 years was 73.5% and 66.4% for all patients, 78.3% and 75.0% for stage I-II and 61.6% and 46.2% for stage III-IVa, respectively (log-rank p = 0.252). Conclusions Use of 3D image-guided HDR ISBT boost was safe and resulted in improved survival outcomes compared to historical rates in this series of primary vaginal cancer patients. Prospective study is warranted to better define clinical and dosimetric predictors of local control.

Interstitial lung disease (ILD) is a term used to describe a heterogeneous group of lung disorders with characteristic clinical and imaging features. Patients with ILD are at an increased risk of developing NSCLC, which is frequently medically comorbid, often precluding operative management. In this scenario, radiotherapy (RT) is generally recommended; however, ILD is known to increase the risk of RT-related toxicity. Recommendations for treatment with appropriately individualized risks and benefits are thus dependent on integration of patient-, ILD-, and cancer-specific factors. We aim to provide an overview of ILD for the thoracic oncologist, an assessment of risk of thoracic RT in patients with ILD, and evidence-based recommendations for treatment in a variety of clinical scenarios.

As the coronavirus disease 2019 (COVID-19) pandemic continues to disrupt nearly all facets of daily life, residency programs must ensure the safety and wellness of their residents while maintaining a commitment to their training and advancement. In addition to standard clinical training, radiation oncology residency programs integrate highly specialized elements specific to the delivery of radiation therapy. Few publications have addressed the significant effects of the pandemic on medical training and even fewer have addressed concerns specific to radiation oncology. We report our experience developing a resident-led adaptation of our training program in response to the COVID-19 pandemic with the aim of assisting other programs to meet this challenge.

Zanthoxylum zanthoxyloides, syn. Fagara zanthoxyloides, is a tree growing in West Africa and is used in traditional medicine against a variety of diseases, including malaria. In the work reported here, root bark and stem bark extracts of this tree, as well as compounds isolated from the extracts, have been investigated for activity in vitro against chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum. In addition, toxicity against nauplii of the brine shrimp Artemia salina has been studied. Dichloromethane extracts of the root bark and stem bark, and a methanol extract of the stem bark, showed anti-parasitic activity towards chloroquine-sensitive as well as chloroquine-resistant P. falciparum, with IC50 values between 1 and 10 µg/mL. Among the isolated compounds, bis-dihydrochelerythrinyl ether, buesgenine, chelerythrine, γ-fagarine, skimmianine, and pellitorine were the most active, with IC50 values of less than 5 µg/mL. The dichloromethane extracts were toxic to brine shrimp nauplii, with LC50 values of less than 1 µg/mL. Methanol extracts were much less toxic (LC50 between 50 and 100 µg/mL). Among the isolated substances, bis-dihydrochelethrinyl ether was the most toxic (LC50 ca. 2 µg/mL).

The antibiotic actinonin kills malaria parasites (Plasmodium falciparum) by interfering with apicoplast function. Early evidence suggested that actinonin inhibited prokaryote-like post-translational modification in the apicoplast; mimicking its activity against bacteria. However, Amberg Johnson et al. (2017) identified the metalloprotease TgFtsH1 as the target of actinonin in the related parasite Toxoplasma gondii and implicated actinonin in the inhibition of P. falciparum growth. The authors were not, however, able to recover actinonin resistant malaria parasites, leaving the specific target of actinonin uncertain. We generated actinonin resistant P. falciparum by in vitro selection and identified a specific sequence change in PfftsH1 associated with resistance. Re-Introduction of this point mutation using CRISPr-Cas9 allelic replacement was sufficient to confer actinonin resistance in P. falciparum. Our data unequivocally identifies PfFtsH1 as the target of actinonin and suggests that actinonin should not be included in the highly valuable collection of “irresistible” drugs for combatting malaria.

The antibiotic actinonin kills malaria parasites (Plasmodium falciparum) by interfering with apicoplast function. Early evidence suggested that actinonin inhibited prokaryote-like post-translational modification in the apicoplast; mimicking its activity against bacteria. However, Amberg Johnson et al. (2017) identified the metalloprotease TgFtsH1 as the target of actinonin in the related parasite Toxoplasma gondii and implicated P. falciparum FtsH1 as a likely target in malaria parasites. The authors were not, however, able to recover actinonin resistant malaria parasites, leaving the specific target of actinonin uncertain. We generated actinonin resistant P. falciparum by in vitro selection and identified a specific sequence change in PfFtsH1 associated with resistance. Introduction of this point mutation using CRISPr-Cas9 allelic replacement was sufficient to confer actinonin resistance in P. falciparum. Our data unequivocally identify PfFtsH1 as the target of actinonin and suggests that actinonin should not be included in the highly valuable collection of ‘irresistible’ drugs for combatting malaria.

PURPOSE: As no completed randomized trials of surgery versus stereotactic ablative radiation therapy (SABR) in patients with early-stage non-small cell lung cancer are available, numerous propensity score studies have attempted to mimic the setting of clinical trials using nonrandomized data. We performed a meta-analysis of propensity score studies comparing SABR and surgery.METHODS AND MATERIALS: The MEDLINE and Embase databases were queried up to December 2016. Two authors independently reviewed the records for inclusion and extracted outcome measures. The study was conducted according to PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) and MOOSE (Meta-analysis of Observational Studies in Epidemiology) guidelines. The primary meta-analysis and secondary analyses were carried out using R (version 3.3.2) at a significance level of .05.RESULTS: Sixteen studies were included in the meta-analysis. Overall survival favored surgery (hazard ratio for SABR vs surgery, 1.48 [95% confidence interval, 1.26-1.72]; I2 = 80.5%). Lung cancer-specific survival was not significantly different between SABR and surgery (hazard ratio, 1.17 [95% confidence interval, 0.92-1.50]; I2 = 18.6%). On stratification, overall survival favored both lobectomy and sublobar resection over SABR, although lung cancer-specific survival was again not significantly different. On secondary analysis, the lymph node upstaging rate was 15.6% following surgery, with 11.4% of patients receiving chemotherapy. The propensity score-matching caliper distance and first-author specialty were found to be associated with survival endpoints on regression.CONCLUSIONS: For patients with early-stage non-small cell lung cancer who are eligible for either treatment, better overall survival was seen after surgery compared with SABR. However, lung cancer-specific survival was similar for both treatments. Prospective clinical trials are preferred to propensity analyses in evaluating the nature of non-cancer-related death after SABR.

Insecticide-impregnated bed nets have saved millions from fatal malaria, but their effectiveness is waning due to mosquito insecticide resistance. A new strategy (Paton et al., Nature, 2019) to deliver parasiticidal compounds into mosquitoes to kill transmission-stage parasites could enhance the effectiveness of bed nets and get around the perennial problems of resistance.

Malaria is a devastating parasitic disease caused by parasites from the genus Plasmodium. Therapeutic resistance has been reported against all clinically available antimalarials, threatening our ability to control the disease and therefore there is an ongoing need for the development of novel antimalarials. Towards this goal, we identified the 2-(N-phenyl carboxamide) triazolopyrimidine class from a high throughput screen of the Janssen Jumpstarter library against the asexual stages of the P. falciparum parasite. Here we describe the structure activity relationship of the identified class and the optimisation of asexual stage activity while maintaining selectivity against the human HepG2 cell line. The most potent analogues from this study were shown to exhibit equipotent activity against P. falciparum multidrug resistant strains and P. knowlesi asexual parasites. Asexual stage phenotyping studies determined the triazolopyrimidine class arrests parasites at the trophozoite stage, but it is likely these parasites are still metabolically active until the second asexual cycle, and thus have a moderate to slow onset of action. Non-NADPH dependent degradation of the central carboxamide and low aqueous solubility was observed in in vitro ADME profiling. A significant challenge remains to correct these liabilities for further advancement of the 2-(N-phenyl carboxamide) triazolopyrimidine scaffold as a potential moderate to slow acting partner in a curative or prophylactic antimalarial treatment.

Rapid emergence of resistance to atovaquone, which targets electron transport in the malaria parasite mitochondrion, relegated its use to prophylaxis and even cast a shadow over the development of drugs targeting other parasite mitochondrial pathways. Here we argue for a renewed focus on the mitochondrion as a drug target, focusing particularly on the issues of resistance. We posit a hypothesis for why atovaquone resistance emerges so quickly, and we explain how facile acquisition of resistance is apparently offset by an inability of parasites to spread this resistance. We also explore the utility and resistance issues for emerging new drugs targeting parasite mitochondria, concluding that the mitochondrion is indeed an excellent target.

© 2020 Elsevier Inc. Purpose: Simulation-based medical education is an effective tool for medical teaching, but simulation-based medical education deployment in radiation oncology (RO) is limited. Flexible nasopharyngoscopy (FNP), an essential skill for RO residents, requires practice that typically occurs on volunteer patients, introducing the potential for stress and discomfort. We sought to develop a high-fidelity simulator and intervention that provides RO residents the opportunity to develop FNP skills in a low-pressure environment. Methods and Materials: Computed tomography images were used to create an anatomically accurate 3-dimensional–printed model of the head and neck region. An intervention incorporating didactic instruction, multimedia content, and FNP practice on the model was designed and administered to RO residents attending the Anatomy and Radiology Contouring Bootcamp. Participants completed pre- and postintervention evaluations of the training session and model fidelity, and self-assessments of FNP skill and confidence performing FNP. Participants were video recorded performing FNP pre- and postintervention. Videos were scored by a blinded observer on a predefined rubric. Changes in scores were evaluated using the Wilcoxon signed-rank test. Results: Twenty-four participants from 17 institutions and 4 countries completed the intervention, 50% were women, and most were senior residents. Postintervention, FNP confidence and FNP performance improved significantly (mean ± standard deviation on a 10-point scale: 1.8 ± 1.8, P < .001; 2.2 ± 2.0, P < .001, respectively). Participants felt the model was helpful (mean ± standard deviation on a 5-point scale: 4.2 ± 0.6), anatomically correct (4.1 ± 0.9), and aided in spatial comprehension (4.3 ± 0.8). Overall satisfaction for the intervention was high (4.3 ± 0.8). Participants strongly agreed the intervention should be integrated into RO training programs (4.3 ± 0.8). Conclusions: A 3-dimensional–printed model and associated intervention were effective at improving FNP performance and the teaching method was rated highly by participants. RO residents may benefit from broader dissemination of this technique to improve trainee performance.

Significance Malaria parasites require certain host nutrients for growth and survival. In this project, we examined the role of the human vitamin biotin in all stages of the malaria life cycle. We cultured blood- and liver-stage malaria parasites in the absence of biotin and found that, whereas blood-stage replication was unaffected, liver-stage parasites deprived of biotin were no longer capable of establishing a blood-stage infection. Interestingly, biotin depletion resulted in more severe developmental defects than the genetic disruption of parasite biotin metabolism. This finding suggests that host biotin metabolism also contributes to parasite development. Because neither the parasite nor the human host can synthesize biotin, parasite infectivity may be affected by the nutritional status of the host., Acetyl-CoA carboxylase (ACC) is a biotin-dependent enzyme that is the target of several classes of herbicides. Malaria parasites contain a plant-like ACC, and this is the only protein predicted to be biotinylated in the parasite. We found that ACC is expressed in the apicoplast organelle in liver- and blood-stage malaria parasites; however, it is activated through biotinylation only in the liver stages. Consistent with this observation, deletion of the biotin ligase responsible for ACC biotinylation does not impede blood-stage growth, but results in late liver-stage developmental defects. Biotin depletion increases the severity of the developmental defects, demonstrating that parasite and host biotin metabolism are required for normal liver-stage progression. This finding may link the development of liver-stage malaria parasites to the nutritional status of the host, as neither the parasite nor the human host can synthesize biotin.

Mitochondrial ATP synthase is driven by chemiosmotic oxidation of pyruvate derived from glycolysis. Blood-stage malaria parasites eschew chemiosmosis, instead relying almost solely on glycolysis for their ATP generation, which begs the question of whether mitochondrial ATP synthase is necessary during the blood stage of the parasite life cycle. We knocked out the mitochondrial ATP synthase β subunit gene in the rodent malaria parasite, Plasmodium berghei , ablating the protein that converts ADP to ATP. Disruption of the β subunit gene of the ATP synthase only marginally reduced asexual blood-stage parasite growth but completely blocked mouse-to-mouse transmission via Anopheles stephensi mosquitoes. Parasites lacking the β subunit gene of the ATP synthase generated viable gametes that fuse and form ookinetes but cannot progress beyond this stage. Ookinetes lacking the β subunit gene of the ATP synthase had normal motility but were not viable in the mosquito midgut and never made oocysts or sporozoites, thereby abrogating transmission to naive mice via mosquito bite. We crossed the self-infertile ATP synthase β subunit knockout parasites with a male-deficient, self-infertile strain of P. berghei , which restored fertility and production of oocysts and sporozoites, which demonstrates that mitochondrial ATP synthase is essential for ongoing viability through the female, mitochondrion-carrying line of sexual reproduction in P. berghei malaria. Perturbation of ATP synthase completely blocks transmission to the mosquito vector and could potentially be targeted for disease control.

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.

Malaria parasites contain a relict plastid, the apicoplast, which is considered an excellent drug target due to its bacterial-like ancestry. Numerous parasiticidals have been proposed to target the apicoplast, but few have had their actual targets substantiated. Isopentenyl pyrophosphate (IPP) production is the sole required function of the apicoplast in the blood stage of the parasite life cycle, and IPP supplementation rescues parasites from apicoplast-perturbing drugs. Hence, any drug that kills parasites when IPP is supplied in culture must have a nonapicoplast target. Here, we use IPP supplementation to discriminate whether 23 purported apicoplast-targeting drugs are on- or off-target. We demonstrate that a prokaryotic DNA replication inhibitor (ciprofloxacin), several prokaryotic translation inhibitors (chloramphenicol, doxycycline, tetracycline, clindamycin, azithromycin, erythromycin, and clarithromycin), a tRNA synthase inhibitor (mupirocin), and two IPP synthesis pathway inhibitors (fosmidomycin and FR900098) have apicoplast targets. Intriguingly, fosmidomycin and FR900098 leave the apicoplast intact, whereas the others eventually result in apicoplast loss. Actinonin, an inhibitor of bacterial posttranslational modification, does not produce a typical delayed-death response but is rescued with IPP, thereby confirming its apicoplast target. Parasites treated with putative apicoplast fatty acid pathway inhibitors could not be rescued, demonstrating that these drugs have their primary targets outside the apicoplast, which agrees with the dispensability of the apicoplast fatty acid synthesis pathways in the blood stage of malaria parasites. IPP supplementation provides a simple test of whether a compound has a target in the apicoplast and can be used to screen novel compounds for mode of action.

Background The clinical symptoms of malaria are caused by the asexual replication of Plasmodium parasites in the blood of the vertebrate host. To spread to new hosts, however, the malaria parasite must differentiate into sexual forms, termed gametocytes, which are ingested by a mosquito vector. Sexual differentiation produces either female or male gametocytes, and involves significant morphological and biochemical changes. These transformations prepare gametocytes for the rapid progression to gamete formation and fertilisation, which occur within 20 min of ingestion. Here we present the transcriptomes of asexual, female, and male gametocytes in P. berghei, and a comprehensive statistically-based differential-expression analysis of the transcriptional changes that underpin this sexual differentiation. Results RNA-seq analysis revealed numerous differences in the transcriptomes of female and male gametocytes compared to asexual stages. Overall, there is net downregulation of transcripts in gametocytes compared to asexual stages, with this trend more marked in female gametocytes. Our analysis identified transcriptional changes in previously-characterised gametocyte-specific pathways, which validated our approach. We also detected many previously-unreported female- and male-specific pathways and genes. Transcriptional biases in stage and gender were then used to investigate sex-specificity and sexual dimorphism of Plasmodium in an evolutionary context. Sex-related gene expression is well conserved between Plasmodium species, but relatively poorly conserved in related organisms outside this genus. This pattern of conservation is most evident in genes necessary for both male and female gametocyte formation. However, this trend is less pronounced for male-specific genes, which are more highly conserved outside the genus than genes specific to female development. Conclusions We characterised the transcriptional changes that are integral to the development of the female and male sexual forms of Plasmodium. These differential-expression patterns provide a vital insight into understanding the gender-specific characteristics of this essential stage that is the primary target for treatments that block parasite transmission. Our results also offer insight into the evolution of sex genes through Alveolata, and suggest that many Plasmodium sex genes evolved within the genus. We further hypothesise that male gametocytes co-opted pre-existing cellular machinery in their evolutionary history, whereas female gametocytes evolved more through the development of novel, parasite-specific pathways. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-4100-0) contains supplementary material, which is available to authorized users.

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., Author summary Combating, and ultimately eliminating, malaria requires drugs that hit the parasite at all stages of its life cycle. Deletions of parasite metabolism genes can expose where and when a certain metabolic pathway is essential, and hence vulnerable to drugs. However, once a parasite gene has been deleted, it is difficult to examine that parasite beyond the point in the life cycle where that gene is essential because the parasites die. We developed a simple genetic complementation strategy to bridge malaria parasites through the mosquito life stage when they carry a genetic defect that otherwise specifically stops them completing this stage. Our strategy allows the phenotypic analysis of any such defect in the subsequent life stages, in any genetically tractable parasite species. It does not require specific background lines, or the chemical or physical treatment necessary for conditional knockout approaches and can be applied to any of the ~50 genes already known to be specifically required for development in the mosquito. This includes genes of the heme synthesis pathway. Malaria parasites are obliged to synthesize the iron-managing molecule heme to complete their development in mosquitoes. We used our genetic complementation strategy to bridge malaria parasites lacking the final enzyme in the heme synthesis pathway through the mosquito phase and back into a vertebrate. We discovered that parasite-generated heme is crucial for the parasite to complete the liver phase of vertebrate development; absence of this enzyme prevents parasites from progressing to the symptomatic blood phase. Our results indicate that blocking parasite heme production should work as a prophylactic intervention to prevent the progression of incipient malaria infections into the deadly blood phase.

Malaria parasites retain a relict plastid (apicoplast) from a photosynthetic ancestor. The apicoplast is a useful drug target but the specificity of compounds believed to target apicoplast fatty acid biosynthesis has become uncertain, as this pathway is not essential in blood stages of the parasite. Herbicides that inhibit the plastid acetyl Coenzyme A (Co-A) carboxylase of plants also kill Plasmodium falciparum in vitro, but their mode of action remains undefined. We characterised the gene for acetyl Co-A carboxylase in P. falciparum. The P. falciparum acetyl-CoA carboxylase gene product is expressed in blood stage parasites and accumulates in the apicoplast. Ablation of the gene did not render parasites insensitive to herbicides, suggesting that these compounds are acting off-target in blood stages of P. falciparum.

Summary Plasmodium parasites remodel their vertebrate host cells by translocating hundreds of proteins across an encasing membrane into the host cell cytosol via a putative export machinery termed PTEX. Previously PTEX150, HSP101 and EXP2 have been shown to be bona fide members of PTEX. Here we validate that PTEX88 and TRX2 are also genuine members of PTEX and provide evidence that expression of PTEX components are also expressed in early gametocytes, mosquito and liver stages, consistent with observations that protein export is not restricted to asexual stages. Although amenable to genetic tagging, HSP101, PTEX150, EXP2 and PTEX88 could not be genetically deleted in Plasmodium berghei, in keeping with the obligatory role this complex is postulated to have in maintaining normal blood-stage growth. In contrast, the putative thioredoxin-like protein TRX2 could be deleted, with knockout parasites displaying reduced grow-rates, both in vivo and in vitro, and reduced capacity to cause severe disease in a cerebral malaria model. Thus, while not essential for parasite survival, TRX2 may help to optimize PTEX activity. Importantly, the generation of TRX2 knockout parasites that display altered phenotypes provides a much-needed tool to dissect PTEX function.

Most current antimalarials for treatment of clinical Plasmodium falciparum malaria fall into two broad drug families and target the food vacuole of the trophozoite stage. No antimalarials have been shown to target the brief extracellular merozoite form of blood-stage malaria. We studied a panel of 12 drugs, 10 of which have been used extensively clinically, for their invasion, schizont rupture, and growth-inhibitory activity using high-throughput flow cytometry and new approaches for the study of merozoite invasion and early intraerythrocytic development. Not surprisingly, given reported mechanisms of action, none of the drugs inhibited merozoite invasion in vitro . Pretreatment of erythrocytes with drugs suggested that halofantrine, lumefantrine, piperaquine, amodiaquine, and mefloquine diffuse into and remain within the erythrocyte and inhibit downstream growth of parasites. Studying the inhibitory activity of the drugs on intraerythrocytic development, schizont rupture, and reinvasion enabled several different inhibitory phenotypes to be defined. All drugs inhibited parasite replication when added at ring stages, but only artesunate, artemisinin, cycloheximide, and trichostatin A appeared to have substantial activity against ring stages, whereas the other drugs acted later during intraerythrocytic development. When drugs were added to late schizonts, only artemisinin, cycloheximide, and trichostatin A were able to inhibit rupture and subsequent replication. Flow cytometry proved valuable for in vitro assays of antimalarial activity, with the free merozoite population acting as a clear marker for parasite growth inhibition. These studies have important implications for further understanding the mechanisms of action of antimalarials, studying and evaluating drug resistance, and developing new antimalarials.

We have synthesized new derivatives of the macrolide antibiotics erythromycin and azithromycin. Novel deoxysugar moieties were attached to these standard antibiotics by biotransformation using a heterologous host. The resulting compounds were tested against several standard laboratory and clinically isolated bacterial strains. In addition, they were also tested in vitro against standard and drug-resistant strains of human malaria parasites (Plasmodium falciparum) and the liver stages of the rodent malaria parasite (Plasmodium berghei). Antibacterial activity of modified erythromycin and azithromycin showed no improvement over the unmodified macrolides, but the modified compounds showed a 10-fold increase in effectiveness after a short-term exposure against blood stages of malaria. The new compounds also remained active against azithromycin-resistant strains of P. falciparum and inhibited growth of liver-stage parasites at concentrations similar to those used for primaquine. Our findings show that malaria parasites have two distinct responses to macrolide antibiotics, one reflecting the prokaryotic origin of the apicoplast and a second, as-yet uncharacterized response that we attribute to the eukaryotic nature of the parasite. This is the first report for macrolides that target two different functions in the Plasmodium parasites. Fil: Goodman, Christopher D.. The University of Melbourne; Australia Fil: Useglio, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Peirú, Salvador. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Labadie, Guillermo Roberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Química Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Química Rosario; Argentina Fil: McFadden, Geoff. The University of Melbourne; Australia Fil: Rodriguez, Eduardo Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Gramajo, Hugo Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina

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, pellitories 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. Electronic supplementary material The online version of this article (doi:10.1186/s12936-016-1533-x) contains supplementary material, which is available to authorized users.

Antibiotics inhibiting protein translation have long been used to treat and prevent infections by apicomplexan parasites. These compounds kill parasites by inhibiting organellar translation, and most act specifically against the apicoplast, a relict plastid in apicomplexans. Drug resistance in Plasmodium and other apicomplexans dictates a need for development of novel targets. Some apicoplast inhibitors have a delayed onset of action, so they cannot replace fast-acting drugs, although they still fulfil important roles in treating and preventing infections. The plethora of bacterial-like actors in the translation machinery of parasite mitochondria and plastids presents validated targets with strong potential for selectivity. Here we discuss existing drugs that inhibit organellar translation, and explore targets that may be further exploited in antiparasitic drug design.

Summary Malaria parasites can synthesize fatty acids via a type II fatty acid synthesis (FASII) pathway located in their apicoplast. The FASII pathway has been pursued as an anti‐malarial drug target, but surprisingly little is known about its role in lipid metabolism. Here we characterize the apicoplast glycerol 3‐phosphate acyltransferase that acts immediately downstream of FASII in human (Plasmodium falciparum) and rodent (Plasmodium berghei) malaria parasites and investigate how this enzyme contributes to incorporating FASII fatty acids into precursors for membrane lipid synthesis. Apicoplast targeting of the P. falciparum and P. berghei enzymes are confirmed by fusion of the N‐terminal targeting sequence to GFP and 3′ tagging of the full length protein. Activity of the P. falciparum enzyme is demonstrated by complementation in mutant bacteria, and critical residues in the putative active site identified by site‐directed mutagenesis. Genetic disruption of the P. falciparum enzyme demonstrates it is dispensable in blood stage parasites, even in conditions known to induce FASII activity. Disruption of the P. berghei enzyme demonstrates it is dispensable in blood and mosquito stage parasites, and only essential for development in the late liver stage, consistent with the requirement for FASII in rodent malaria models. However, the P. berghei mutant liver stage phenotype is found to only partially phenocopy loss of FASII, suggesting newly made fatty acids can take multiple pathways out of the apicoplast and so giving new insight into the role of FASII and apicoplast glycerol 3‐phosphate acyltransferase in malaria parasites.

The causative agent of malaria, Plasmodium, possesses three translationally active compartments: the cytosol, the mitochondrion and a relic plastid called the apicoplast. Aminoacyl-tRNA synthetases to charge tRNA are thus required for all three compartments. However, the Plasmodiumfalciparum genome encodes too few tRNA synthetases to supply a unique enzyme for each amino acid in all three compartments. We have investigated the subcellular localisation of three tRNA synthetases (AlaRS, GlyRS and ThrRS), which occur only once in the nuclear genome, and we show that each of these enzymes is dually localised to the P. falciparum cytosol and the apicoplast. No mitochondrial fraction is apparent for these three enzymes, which suggests that the Plasmodium mitochondrion lacks at least these three tRNA synthetases. The unique Plasmodium ThrRS is the presumed target of the antimalarial compound borrelidin. Borrelidin kills P. falciparum parasites quickly without the delayed death effect typical of apicoplast translation inhibitors and without an observable effect on apicoplast morphology. By contrast, mupirocin, an inhibitor of the apicoplast IleRS, kills with a delayed death effect that inhibits apicoplast growth and division. Because inhibition of dual targeted tRNA synthetases should arrest translation in all compartments of the parasite, these enzymes deserve further investigation as potential targets for antimalarial drug development.

Expression of a bacterial transporter protein in Toxoplasma gondii results in parasite susceptibility to Formidomycin, a drug targeting isoprenoid precursor synthesis., 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.

Live cell imaging of human malaria parasites Plasmodium falciparum during gametocytogenesis revealed that the apicoplast does not grow, whereas the mitochondrion undergoes remarkable morphological development. A close connection of the two organelles is consistently maintained. The apicoplast and mitochondrion are not components of the male gametes, suggesting maternal inheritance.

Fatty acid biosynthesis pathways in protozoan parasites are reviewed with a view to targeting this metabolism for drug therapy. The type II fatty acid biosynthesis pathways derived from bacteria in protozoan relict plastids and mitochondria are examined in different groups with emphasis on apicomplexa. The suitability of different enzymes from the type II fatty acid biosynthesis pathway for drug intervention, and the state-of-play with known and potential inhibitors is explored. The type I acid biosynthesis pathways that occur in select protozoan parasites and their potential for inhibition using anti-tumour and obesity management compounds currently in development are also examined. Pathways used by parasites to scavenge and modify host lipids are also described briefly and their potential for therapeutics discussed.

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.

Many anti-bacterial drugs inhibit growth of malaria parasites by targeting their bacterium-derived endosymbiotic organelles, the mitochondrion and plastid. Several of these drugs are either in use or being developed as therapeutics or prophylactics, so it is paramount to understand more about their target of action and modality. To this end, we measured in vitro growth and visualized nuclear division and the development of the mitochondrion and apicoplast in Plasmodium falciparum treated with five drugs targeting bacterial housekeeping pathways. This revealed two distinct classes of drug effect. Ciprofloxacin, rifampicin, and thiostrepton had an immediate effect: slowing parasite growth, retarding organellar development and preventing nuclear division. Classic delayed-death, in which the drug has no apparent effect until division and reinvasion of a new host by the daughter merozoites, was only observed for two drugs: clindamycin and tetracycline. These cells had apparently normal division and segregation of organelles in the first cycle but severe defects in apicoplast growth, subtle changes in the mitochondrion and a failure to complete cytokinesis during the second cycle. In two cases, the drug response in P. falciparum directly conflicted with reported responses for the related parasite Toxoplasma gondii, suggesting significant differences in apicoplast biology between the two parasites.

Apicomplexan parasitic diseases impose devastating impacts on much of the world's population. The increasing prevalence of drug resistant parasites and the growing number of immuno-compromised individuals are exacerbating the problem to the point that the need for novel, inexpensive drugs is greater now than ever. Discovery of a prokaryotic, Type II fatty acid synthesis (FAS) pathway associated with the plastid-like organelle (apicoplast) of Plasmodium and Toxoplasma has provided a wealth of novel drug targets. Since this pathway is both essential and fundamentally different from the cytosolic Type I pathway of the human host, apicoplast FAS has tremendous potential for the development of parasite-specific inhibitors. Many components of this pathway are already the target for existing antibiotics and herbicides, which should significantly reduce the time and cost of drug development. Continuing interest--both in the pharmaceutical and herbicide industries--in fatty acid synthesis inhibitors proffers an ongoing stream of potential new anti-parasitic compounds. It has now emerged that not all apicomplexan parasites have retained the Type II fatty acid biosynthesis pathway. No fatty acid biosynthesis enzymes are encoded in the genome of Theileria annulata or T. parva, suggesting that fatty acid synthesis is lacking in these parasites. The human intestinal parasite Cryptosporidium parvum appears to have lost the apicoplast entirely; instead relying on an unusual cytosolic Type I FAS. Nevertheless, newly developed anti-cancer and anti-obesity drugs targeting human Type I FAS may yet prove efficacious against Cryptosporidium and other apicomplexans that rely on this Type I FAS pathway.

Transmission blocked by drug resistance Resistance to the antimalarial drug atovaquone might prove to be this parasite's weak spot. Resistance develops rapidly via mutations in the drug's target: the parasite's mitochondrial cytochrome b complex. Goodman et al. have discovered that although resistant Plasmodium berghei parasites persist in mice, in blood-sucking malarial mosquitoes, the mutations disable female parasites too much for them to reproduce. The human-specific Plasmodium falciparum can only be investigated experimentally in mosquitoes, but a similar effect was seen. Thus, atovaquone-resistant parasites cannot be transmitted to another mammal or person. Science , this issue p. 349

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. Electronic supplementary material The online version of this article (doi:10.1186/s12915-015-0162-0) contains supplementary material, which is available to authorized users.