Mutant Parasites Research Articles

Trypanin Disruption Affects the Motility and Infectivity of the Protozoan Trypanosoma cruzi.

The flagellum of Trypanosomatids is an organelle that contributes to multiple functions, including motility, cell division, and host–pathogen interaction. Trypanin was first described in Trypanosoma brucei and is part of the dynein regulatory complex. TbTrypanin knockdown parasites showed motility defects in procyclic forms; however, silencing in bloodstream forms was lethal. Since TbTrypanin mutants show drastic phenotypic changes in mammalian stages, we decided to evaluate if the Trypanosoma cruzi ortholog plays a similar role by using the CRISPR-Cas9 system to generate null mutants. A ribonucleoprotein complex of SaCas9 and sgRNA plus donor oligonucleotide were used to edit both alleles of TcTrypanin without any selectable marker. TcTrypanin −/− epimastigotes showed a lower growth rate, partially detached flagella, normal numbers of nuclei and kinetoplasts, and motility defects such as reduced displacement and speed and increased tumbling propensity. The epimastigote mutant also showed decreased efficiency of in-vitro metacyclogenesis. Mutant parasites were able to complete the entire life cycle in vitro; however, they showed a reduction in their infection capacity compared with WT and addback cultures. Our data show that T. cruzi life cycle stages have differing sensitivities to TcTrypanin deletion. In conclusion, additional work is needed to dissect the motility components of T. cruzi and to identify essential molecules for mammalian stages.

Reply to Prasad and Patankar, “Recognition of Two Distinct Pathways for Trafficking of Proteins to the Apicoplast”

In a recent study (1), we investigated the role of TgGS27 (a Qb SNARE) and TgTrs85 (a tethering factor, transport protein particle III [TRAPP III]-specific subunit) in trafficking at Golgi stacks of Toxoplasma gondii. We found that both TgGS27 and TgTrs85 are critical for intra-Golgi network trafficking. Furthermore, the proper localization of several apicoplast-residing proteins was examined in these parasite mutants, including two proteins containing both signal and transit peptides (TgCPN60 and TgACP) and others lacking a clear transit peptide (TgATrx1, TgFtsH1, and TgAPT1, the last of which lacks signal and transit peptides). We concluded that disruption of intra-Golgi network trafficking caused by the depletion of either TgGS27 or TgTrs85 impaired the proper delivery of the nucleus-encoded apicoplast-targeted (NEAT) proteins to the apicoplast. Indeed, the data based on these experiments could not provide direct evidence to tell whether these proteins were transported through the Golgi pathway because depletion of TgGS27 or TgTrs85 leads to profound effects on the function of Golgi stacks and obvious changes in the parasite morphology, as we described in the article.

The 3-phosphoinositide-dependent protein kinase 1 is an essential upstream activator of protein kinase A in malaria parasites.

Cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) signalling is essential for the proliferation of Plasmodium falciparum malaria blood stage parasites. The mechanisms regulating the activity of the catalytic subunit PfPKAc, however, are only partially understood, and PfPKAc function has not been investigated in gametocytes, the sexual blood stage forms that are essential for malaria transmission. By studying a conditional PfPKAc knockdown (cKD) mutant, we confirm the essential role for PfPKAc in erythrocyte invasion by merozoites and show that PfPKAc is involved in regulating gametocyte deformability. We furthermore demonstrate that overexpression of PfPKAc is lethal and kills parasites at the early phase of schizogony. Strikingly, whole genome sequencing (WGS) of parasite mutants selected to tolerate increased PfPKAc expression levels identified missense mutations exclusively in the gene encoding the parasite orthologue of 3-phosphoinositide-dependent protein kinase-1 (PfPDK1). Using targeted mutagenesis, we demonstrate that PfPDK1 is required to activate PfPKAc and that T189 in the PfPKAc activation loop is the crucial target residue in this process. In summary, our results corroborate the importance of tight regulation of PfPKA signalling for parasite survival and imply that PfPDK1 acts as a crucial upstream regulator in this pathway and potential new drug target.

In Leishmania major, the Homolog of the Oncogene PES1 May Play a Critical Role in Parasite Infectivity.

Leishmaniasis is a neglected tropical disease caused by Leishmania spp. The improvement of existing treatments and the discovery of new drugs remain ones of the major goals in control and eradication of this disease. From the parasite genome, we have identified the homologue of the human oncogene PES1 in Leishmania major (LmjPES). It has been demonstrated that PES1 is involved in several processes such as ribosome biogenesis, cell proliferation and genetic transcription. Our phylogenetic studies showed that LmjPES encodes a highly conserved protein containing three main domains: PES N-terminus (shared with proteins involved in ribosomal biogenesis), BRCT (found in proteins related to DNA repair processes) and MAEBL-type domain (C-terminus, related to erythrocyte invasion in apicomplexan). This gene showed its highest expression level in metacyclic promastigotes, the infective forms; by fluorescence microscopy assay, we demonstrated the nuclear localization of LmjPES protein. After generating mutant parasites overexpressing LmjPES, we observed that these clones displayed a dramatic increase in the ratio of cell infection within macrophages. Furthermore, BALB/c mice infected with these transgenic parasites exhibited higher footpad inflammation compared to those inoculated with non-overexpressing parasites.

Epidemiology of mutant Plasmodium falciparum parasites lacking histidine-rich protein 2/3 genes in Eritrea 2 years after switching from HRP2-based RDTs

Eritrea was the first African country to complete a nationwide switch in 2016 away from HRP2-based RDTs due to high rates of false-negative RDT results caused by Plasmodium falciparum parasites lacking hrp2/hrp3 genes. A cross-sectional survey was conducted during 2019 enrolling symptomatic malaria patients from nine health facilities across three zones consecutively to investigate the epidemiology of P. falciparum lacking hrp2/3 after the RDT switch. Molecular analyses of 715 samples revealed the overall prevalence of hrp2-, hrp3-, and dual hrp2/3-deleted parasites as 9.4% (95%CI 7.4–11.7%), 41.7% (95% CI 38.1–45.3%) and 7.6% (95% CI 5.8–9.7%), respectively. The prevalence of hrp2- and hrp3-deletion is heterogeneous within and between zones: highest in Anseba (27.1% and 57.9%), followed by Gash Barka (6.4% and 37.9%) and Debub zone (5.2% and 43.8%). hrp2/3-deleted parasites have multiple diverse haplotypes, with many shared or connected among parasites of different hrp2/3 status, indicating mutant parasites have likely evolved from multiple and local parasite genetic backgrounds. The findings show although prevalence of hrp2/3-deleted parasites is lower 2 years after RDT switching, HRP2-based RDTs remain unsuitable for malaria diagnosis in Eritrea. Continued surveillance of hrp2/3-deleted parasites in Eritrea and neighbouring countries is required to monitor the trend.

Intermittent screening and treatment with artemisinin-combination therapy versus intermittent preventive treatment with sulphadoxine-pyrimethamine for malaria in pregnancy: a systematic review and individual participant data meta-analysis of randomised clinical trials.

BackgroundIn sub-Saharan Africa, the efficacy of intermittent preventive therapy in pregnancy with sulphadoxine-pyrimethamine (IPTp-SP) for malaria in pregnancy is threatened by parasite resistance. We conducted an individual-participant data (IPD) meta-analysis to assess the efficacy of intermittent screening with malaria rapid diagnostic tests (RDTs) and treatment of RDT-positive women with artemisinin-based combination therapy (ISTp-ACT) compared to IPTp-SP, and understand the importance of subpatent infections.MethodsWe searched MEDLINE and the Malaria-in-Pregnancy Library on May 6, 2021 for trials comparing ISTp-ACT and IPTp-SP. Generalised linear regression was used to compare adverse pregnancy outcomes (composite of small-for-gestational-age, low birthweight (LBW), or preterm delivery) and peripheral or placental Plasmodium falciparum at delivery. The effects of subpatent (PCR-positive, RDT/microscopy-negative) infections were assessed in both arms pooled using multi-variable fixed-effect models adjusting for the number of patent infections. PROSPERO registration: CRD42016043789.FindingsFive trials conducted between 2007 and 2014 contributed (10,821 pregnancies), two from high SP-resistance areas where dhfr/dhps quintuple mutant parasites are saturated, but sextuple mutants are still rare (Kenya and Malawi), and three from low-resistance areas (West-Africa). Four trials contributed IPD data (N=10,362). At delivery, the prevalence of any malaria infection (relative risk [RR]=1.08, 95% CI 1.00-1.16, I2=67.0 %) and patent infection (RR=1.02, 0.61-1.16, I2=0.0%) were similar. Subpatent infections were more common in ISTp recipients (RR=1.31, 1.05-1.62, I2=0.0%). There was no difference in adverse pregnancy outcome (RR=1.00, 0.96-1.05; studies=4, N=9,191, I2=54.5%). Subpatent infections were associated with LBW (adjusted RR=1.13, 1.07-1.19), lower mean birthweight (adjusted mean difference=32g, 15-49), and preterm delivery (aRR=1.35, 1.15-1.57).InterpretationISTp-ACT was not superior to IPTp-SP and may result in more subpatent infections than the existing IPTp-SP policy. Subpatent infections were associated with increased LBW and preterm delivery. More sensitive diagnostic tests are needed to detect and treat low-grade infections.FundingCenters for Disease Control and Prevention and Worldwide Antimalarial Resistance Network.

Decreased Susceptibility to Dihydrofolate Reductase Inhibitors Associated With Genetic Polymorphisms in Ugandan Plasmodium falciparum Isolates.

The Plasmodium falciparum dihydrofolate reductase (PfDHFR) inhibitors pyrimethamine and cycloguanil (the active metabolite of proguanil) have important roles in malaria chemoprevention, but drug resistance challenges their efficacies. A new compound, P218, was designed to overcome resistance, but drug-susceptibility data for P falciparum field isolates are limited. We studied ex vivo PfDHFR inhibitor susceptibilities of 559 isolates from Tororo and Busia districts, Uganda, from 2016 to 2020, sequenced 383 isolates, and assessed associations between genotypes and drug-susceptibility phenotypes. Median half-maximal inhibitory concentrations (IC50s) were 42 100 nM for pyrimethamine, 1200 nM for cycloguanil, 13000 nM for proguanil, and 0.6 nM for P218. Among sequenced isolates, 3 PfDHFR mutations, 51I (100%), 59R (93.7%), and 108N (100%), were very common, as previously seen in Uganda, and another mutation, 164L (12.8%), had moderate prevalence. Increasing numbers of mutations were associated with decreasing susceptibility to pyrimethamine, cycloguanil, and P218, but not proguanil, which does not act directly against PfDHFR. Differences in P218 susceptibilities were modest, with median IC50s of 1.4 nM for parasites with mixed genotype at position 164 and 5.7 nM for pure quadruple mutant (51I/59R/108N/164L) parasites. Resistance-mediating PfDHFR mutations were common in Ugandan isolates, but P218 retained excellent activity against mutant parasites.

Associations between Varied Susceptibilities to PfATP4 Inhibitors and Genotypes in Ugandan Plasmodium falciparum Isolates.

Among novel compounds under recent investigation as potential new antimalarial drugs are three independently developed inhibitors of the Plasmodium falciparum P-type ATPase (PfATP4): KAE609 (cipargamin), PA92, and SJ733. We assessed ex vivo susceptibilities to these compounds of 374 fresh P. falciparum isolates collected in Tororo and Busia districts, Uganda, from 2016 to 2019. Median IC50s were 65 nM for SJ733, 9.1 nM for PA92, and 0.5 nM for KAE609. Sequencing of pfatp4 for 218 of these isolates demonstrated many nonsynonymous single nucleotide polymorphisms; the most frequent mutations were G1128R (69% of isolates mixed or mutant), Q1081K/R (68%), G223S (25%), N1045K (16%), and D1116G/N/Y (16%). The G223S mutation was associated with decreased susceptibility to SJ733, PA92, and KAE609. The D1116G/N/Y mutations were associated with decreased susceptibility to SJ733, and the presence of mutations at both codons 223 and 1116 was associated with decreased susceptibility to PA92 and SJ733. In all of these cases, absolute differences in susceptibilities of wild-type (WT) and mutant parasites were modest. Analysis of clones separated from mixed field isolates consistently identified mutant clones as less susceptible than WT. Analysis of isolates from other sites demonstrated the presence of the G223S and D1116G/N/Y mutations across Uganda. Our results indicate that malaria parasites circulating in Uganda have a number of polymorphisms in PfATP4 and that modestly decreased susceptibility to PfATP4 inhibitors is associated with some mutations now present in Ugandan parasites.

Untargeted Metabolomics Uncovers the Essential Lysine Transporter in Toxoplasma gondii.

Apicomplexan parasites are responsible for devastating diseases, including malaria, toxoplasmosis, and cryptosporidiosis. Current treatments are limited by emerging resistance to, as well as the high cost and toxicity of existing drugs. As obligate intracellular parasites, apicomplexans rely on the uptake of many essential metabolites from their host. Toxoplasma gondii, the causative agent of toxoplasmosis, is auxotrophic for several metabolites, including sugars (e.g., myo-inositol), amino acids (e.g., tyrosine), lipidic compounds and lipid precursors (cholesterol, choline), vitamins, cofactors (thiamine) and others. To date, only few apicomplexan metabolite transporters have been characterized and assigned a substrate. Here, we set out to investigate whether untargeted metabolomics can be used to identify the substrate of an uncharacterized transporter. Based on existing genome- and proteome-wide datasets, we have identified an essential plasma membrane transporter of the major facilitator superfamily in T. gondii—previously termed TgApiAT6-1. Using an inducible system based on RNA degradation, TgApiAT6-1 was depleted, and the mutant parasite’s metabolome was compared to that of non-depleted parasites. The most significantly reduced metabolite in parasites depleted in TgApiAT6-1 was identified as the amino acid lysine, for which T. gondii is predicted to be auxotrophic. Using stable isotope-labeled amino acids, we confirmed that TgApiAT6-1 is required for efficient lysine uptake. Our findings highlight untargeted metabolomics as a powerful tool to identify the substrate of orphan transporters.

Downregulation of FeSOD-A expression in Leishmania infantum alters trivalent antimony and miltefosine susceptibility

BackgroundSuperoxide dismutase (SOD), a central component of the antioxidant defence system of most organisms, removes excess superoxide anions by converting them to oxygen and hydrogen peroxide. As iron (Fe) SOD is absent in the human host, this enzyme is a promising molecular target for drug development against trypanosomatids.ResultsWe obtained Leishmania infantum mutant clones with lower FeSOD-A expression and investigated their phenotypes. Our attempts to delete this enzyme-coding gene using three different methodologies (conventional allelic replacement or two different CRISPR/methods) failed, as FeSOD-A gene copies were probably retained by aneuploidy or gene amplification. Promastigote forms of WT and mutant parasites were used in quantitative reverse-transcription polymerase chain reaction (RT-qPCR) and western blot analyses, and these parasite forms were also used to assess drug susceptibility. RT-qPCR and western blot analyses revealed that FeSOD-A transcript and protein levels were lower in FeSOD-A−/−/+L. infantum mutant clones than in the wild-type (WT) parasite. The decrease in FeSOD-A expression in L. infantum did not interfere with the parasite growth or susceptibility to amphotericin B. Surprisingly, FeSOD-A−/−/+L. infantum mutant clones were 1.5- to 2.0-fold more resistant to trivalent antimony and 2.4- to 2.7-fold more resistant to miltefosine. To investigate whether the decrease in FeSOD-A expression was compensated by other enzymes, the transcript levels of five FeSODs and six enzymes from the antioxidant defence system were assessed by RT-qPCR. The transcript level of the enzyme ascorbate peroxidase increased in both the FeSOD-A−/−/+ mutants tested. The FeSOD-A−/−/+ mutant parasites were 1.4- to 1.75-fold less tolerant to oxidative stress generated by menadione. Infection analysis using THP-1 macrophages showed that 72 h post-infection, the number of infected macrophages and their intracellular multiplication rate were lower in the FeSOD-A−/−/+ mutant clones than in the WT parasite.ConclusionsThe unsuccessful attempts to delete FeSOD-A suggest that this gene is essential in L. infantum. This enzyme plays an important role in the defence against oxidative stress and infectivity in THP-1 macrophages. FeSOD-A-deficient L. infantum parasites deregulate their metabolic pathways related to antimony and miltefosine resistance.Graphic

Surveillance of molecular markers of Plasmodium falciparum artemisinin resistance (kelch13 mutations) in Papua New Guinea between 2016 and 2018

Plasmodium falciparum resistance to artemisinin-based combination therapy (ACT) is a global threat to malaria control and elimination efforts. Mutations in the P. falciparum kelch13 gene (Pfk13) that are associated with delayed parasite clearance have emerged on the Thai-Cambodian border since 2008. There is growing evidence of widespread Pfk13 mutations throughout South-East Asia and they have independently emerged in other endemic regions. In Papua New Guinea (PNG), Pfk13 “C580Y” mutant parasites with reduced in vitro sensitivity to artemisinin have been isolated in Wewak, a port town in East Sepik Province. However, the extent of any local spread of these mutant parasites in other parts of PNG is unknown. We investigated the prevalence of Pfk13 mutations in multiple malaria-endemic regions of PNG. P. falciparum isolates (n = 1152) collected between 2016 and 2018 and assessed for Pfk13 variation by sequencing. Of 663 high quality Pfk13 sequences a total of five variants were identified. They included C580Y, a mutation at a previously documented polymorphic locus: N499K, and three previously undescribed mutations: R471C, K586E and Y635C. All variants were found in single isolates, indicating that these Pfk13 mutations were rare in the areas surveyed. Notably, C580Y was absent from Maprik district, which neighbours Wewak where C580Y mutant parasites were previously identified. The single C580Y isolate was found in the port town of Lae, Morobe Province, a potential entry site for the importation of drug resistant parasites into PNG. Although sample size in this location was small (n = 5), our identification of a C580Y mutant in this second location is concerning, highlighting the urgent need for further surveillance in Lae. Other Pfk13 mutants were rare in PNG between 2016 and 2018. Continued surveillance for molecular markers of drug resistance is critically important to inform malaria control in PNG.

Balanced impacts of fitness and drug pressure on the evolution of PfMDR1 polymorphisms in Plasmodium falciparum

BackgroundAnti-malarial drug resistance may be limited by decreased fitness in resistant parasites. Important contributors to resistance are mutations in the Plasmodium falciparum putative drug transporter PfMDR1.MethodsImpacts on in vitro fitness of two common PfMDR1 polymorphisms, N86Y, which is associated with sensitivity to multiple drugs, and Y184F, which has no clear impact on drug sensitivity, were evaluated to study associations between resistance mediators and parasite fitness, measured as relative growth in competitive culture experiments. NF10 P. falciparum lines engineered to represent all PfMDR1 N86Y and Y184F haplotypes were co-cultured for 40 days, and the genetic make-up of the cultures was characterized every 4 days by pyrosequencing. The impacts of culture with anti-malarials on the growth of different haplotypes were also assessed. Lastly, the engineering of P. falciparum containing another common polymorphism, PfMDR1 D1246Y, was attempted.ResultsCo-culture results were as follows. With wild type (WT) Y184 fixed (N86/Y184 vs. 86Y/Y184), parasites WT and mutant at 86 were at equilibrium. With mutant 184 F fixed (N86/184F vs. 86Y/184F), mutants at 86 overgrew WT. With WT N86 fixed (N86/Y184 vs. N86/184F), WT at 184 overgrew mutants. With mutant 86Y fixed (86Y/Y184 vs. 86Y/184F), WT and mutant at 86 were at equilibrium. Parasites with the double WT were in equilibrium with the double mutant, but 86Y/Y184 overgrew N86/184F. Overall, WT N86/mutant 184F parasites were less fit than parasites with all other haplotypes. Parasites engineered for another mutation, PfMDR1 1246Y, were unstable in culture, with reversion to WT over time. Thus, the N86 WT is stable when accompanied by the Y184 WT, but incurs a fitness cost when accompanied by mutant 184F. Culturing in the presence of chloroquine favored 86Y mutant parasites and in the presence of lumefantrine favored N86 WT parasites; piperaquine had minimal impact.ConclusionsThese results are consistent with those for Ugandan field isolates, suggest reasons for varied haplotypes, and highlight the interplay between drug pressure and fitness that is guiding the evolution of resistance-mediating haplotypes in P. falciparum.

Cytochrome b Drug Resistance Mutation Decreases Babesia Fitness in the Tick Stages But Not the Mammalian Erythrocytic Cycle.

Human babesiosis is an emerging tick-borne malaria-like illness caused by Babesia parasites following their development in erythrocytes. Here, we show that a mutation in the Babesia microti mitochondrial cytochrome b (Cytb) that confers resistance to the antibabesial drug ELQ-502 decreases parasite fitness in the arthropod vector. Interestingly, whereas the mutant allele does not affect B. microti fitness during the mammalian blood phase of the parasite life cycle and is genetically stable as parasite burden increases, ELQ-502-resistant mutant parasites developing in the tick vector are genetically unstable with a high rate of the wild-type allele emerging during the nymphal stage. Furthermore, we show that B. microti parasites with this mutation are transmitted from the tick to the host, raising the possibility that the frequency of Cytb resistance mutations may be decreased by passage through the tick vector, but could persist in the environment if present when ticks feed.

17-AAG-Induced Activation of the Autophagic Pathway in Leishmania Is Associated with Parasite Death.

The heat shock protein 90 (Hsp90) is thought to be an excellent drug target against parasitic diseases. The leishmanicidal effect of an Hsp90 inhibitor, 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), was previously demonstrated in both in vitro and in vivo models of cutaneous leishmaniasis. Parasite death was shown to occur in association with severe ultrastructural alterations in Leishmania, suggestive of autophagic activation. We hypothesized that 17-AAG treatment results in the abnormal activation of the autophagic pathway, leading to parasite death. To elucidate this process, experiments were performed using transgenic parasites with GFP-ATG8-labelled autophagosomes. Mutant parasites treated with 17-AAG exhibited autophagosomes that did not entrap cargo, such as glycosomes, or fuse with lysosomes. ATG5-knockout (Δatg5) parasites, which are incapable of forming autophagosomes, demonstrated lower sensitivity to 17-AAG-induced cell death when compared to wild-type (WT) Leishmania, further supporting the role of autophagy in 17-AAG-induced cell death. In addition, Hsp90 inhibition resulted in greater accumulation of ubiquitylated proteins in both WT- and Δatg5-treated parasites compared to controls, in the absence of proteasome overload. In conjunction with previously described ultrastructural alterations, herein we present evidence that treatment with 17-AAG causes abnormal activation of the autophagic pathway, resulting in the formation of immature autophagosomes and, consequently, incidental parasite death.

Plasmodium transcription repressor AP2-O3 regulates sex-specific identity of gene expression in female gametocytes.

Male and female gametocytes are sexual precursor cells essential for mosquito transmission of malaria parasite. Differentiation of gametocytes into fertile gametes (known as gametogenesis) relies on the gender‐specific transcription program. How the parasites establish distinct repertoires of transcription in the male and female gametocytes remains largely unknown. Here, we report that an Apetala2 family transcription factor AP2‐O3 operates as a transcription repressor in the female gametocytes. AP2‐O3 is specifically expressed in the female gametocytes. AP2‐O3‐deficient parasites produce apparently normal female gametocytes. Nevertheless, these gametocytes fail to differentiate into fully fertile female gametes, leading to developmental arrest in fertilization and early development post‐fertilization. AP2‐O3 disruption causes massive upregulation of transcriptionally dormant male genes and simultaneously downregulation of highly transcribed female genes in the female gametocytes. AP2‐O3 targets a substantial proportion of the male genes by recognizing an 8‐base DNA motif. In addition, the maternal AP2‐O3 is removed after fertilization, which is required for the zygote to ookinete development. Therefore, the global transcriptional repression of the male genes in the female gametocytes is required for safeguarding female‐specific transcriptome and essential for the mosquito transmission of Plasmodium.

Human Aurora kinase inhibitor Hesperadin reveals epistatic interaction between Plasmodium falciparum PfArk1 and PfNek1 kinases

Mitosis has been validated by numerous anti-cancer drugs as being a druggable process, and selective inhibition of parasite proliferation provides an obvious opportunity for therapeutic intervention against malaria. Mitosis is controlled through the interplay between several protein kinases and phosphatases. We show here that inhibitors of human mitotic kinases belonging to the Aurora family inhibit P. falciparum proliferation in vitro with various potencies, and that a genetic selection for mutant parasites resistant to one of the drugs, Hesperadin, identifies a resistance mechanism mediated by a member of a different kinase family, PfNek1 (PF3D7_1228300). Intriguingly, loss of PfNek1 catalytic activity provides protection against drug action. This points to an undescribed functional interaction between Ark and Nek kinases and shows that existing inhibitors can be used to validate additional essential and druggable kinase functions in the parasite.

Plasmodium berghei K13 Mutations Mediate In Vivo Artemisinin Resistance That Is Reversed by Proteasome Inhibition.

The recent emergence of Plasmodium falciparum parasite resistance to the first line antimalarial drug artemisinin is of particular concern. Artemisinin resistance is primarily driven by mutations in the P. falciparum K13 protein, which enhance survival of early ring-stage parasites treated with the artemisinin active metabolite dihydroartemisinin in vitro and associate with delayed parasite clearance in vivo However, association of K13 mutations with in vivo artemisinin resistance has been problematic due to the absence of a tractable model. Herein, we have employed CRISPR/Cas9 genome editing to engineer selected orthologous P. falciparum K13 mutations into the K13 gene of an artemisinin-sensitive Plasmodium berghei rodent model of malaria. Introduction of the orthologous P. falciparum K13 F446I, M476I, Y493H, and R539T mutations into P. berghei K13 yielded gene-edited parasites with reduced susceptibility to dihydroartemisinin in the standard 24-h in vitro assay and increased survival in an adapted in vitro ring-stage survival assay. Mutant P. berghei K13 parasites also displayed delayed clearance in vivo upon treatment with artesunate and achieved faster recrudescence upon treatment with artemisinin. Orthologous C580Y and I543T mutations could not be introduced into P. berghei, while the equivalents of the M476I and R539T mutations resulted in significant growth defects. Furthermore, a Plasmodium-selective proteasome inhibitor strongly synergized dihydroartemisinin action in these P. berghei K13 mutant lines, providing further evidence that the proteasome can be targeted to overcome artemisinin resistance. Taken together, our findings provide clear experimental evidence for the involvement of K13 polymorphisms in mediating susceptibility to artemisinins in vitro and, most importantly, under in vivo conditions.IMPORTANCE Recent successes in malaria control have been seriously threatened by the emergence of Plasmodium falciparum parasite resistance to the frontline artemisinin drugs in Southeast Asia. P. falciparum artemisinin resistance is associated with mutations in the parasite K13 protein, which associates with a delay in the time required to clear the parasites upon drug treatment. Gene editing technologies have been used to validate the role of several candidate K13 mutations in mediating P. falciparum artemisinin resistance in vitro under laboratory conditions. Nonetheless, the causal role of these mutations under in vivo conditions has been a matter of debate. Here, we have used CRISPR/Cas9 gene editing to introduce K13 mutations associated with artemisinin resistance into the related rodent-infecting parasite, Plasmodium berghei Phenotyping of these P. berghei K13 mutant parasites provides evidence of their role in mediating artemisinin resistance in vivo, which supports in vitro artemisinin resistance observations. However, we were unable to introduce some of the P. falciparum K13 mutations (C580Y and I543T) into the corresponding amino acid residues, while other introduced mutations (M476I and R539T equivalents) carried pronounced fitness costs. Our study provides evidence of a clear causal role of K13 mutations in modulating susceptibility to artemisinins in vitro and in vivo using the well-characterized P. berghei model. We also show that inhibition of the P. berghei proteasome offsets parasite resistance to artemisinins in these mutant lines.

Attenuated P. falciparum Parasite Shows Cytokine Variations in Humanized Mice.

A recently developed humanized mouse has been used to assess the immune response evoked against the isolated attenuated C9 parasite clone (C9-M; carrying a single insertion disrupting the open reading frame (ORF) of PF3D7_1305500) of Plasmodium falciparum. Significant human RBC engraftment was achieved by ameliorating the residual non-adaptive immune response using clodronate-loaded liposome treatment. Controlled reactive professional phagocytic leukocytes in immunodeficient mice allowed for sizeable human blood chimerism and injected huRBCs acted as bona fide host cells for P. falciparum. huRBC-reconstituted immunodeficient mice received infectious challenge with attenuated P. falciparum C9 parasite mutants (C9-M), complemented (C9-C), and wild type (NF54) progenitors to study the role of immune effectors in the clearance of the parasite from mouse circulation. C9-M and NF54 parasites grew and developed in the huRBC-reconstituted humanized NSG mice. Further, the presence of mutant parasites in deep-seated tissues suggests the escape of parasites from the host's immune responses and thus extended the survival of the parasite. Our results suggest an evasion mechanism that may have been employed by the parasite to survive the mouse's residual non-adaptive immune responses. Collectively, our data suggest that huRBCs reconstituted NSG mice infected with attenuated P. falciparum is a valuable tool to explore the role of C9 mutation in the growth and survival of parasite mutants and their response to the host's immune responses. This mouse might help in identifying novel chemotherapeutic targets to develop new anti-malarial drugs.

EIF2α phosphorylation is regulated in intracellular amastigotes for the generation of infective Trypanosoma cruzi trypomastigote forms.

Trypanosomatids regulate gene expression mainly at the post-transcriptional level through processing, exporting and stabilising mRNA and control of translation. In most eukaryotes, protein synthesis is regulated by phosphorylation of eukaryotic initiation factor 2 (eIF2) at serine 51. Phosphorylation halts overall translation by decreasing availability of initiator tRNAmet to form translating ribosomes. In trypanosomatids, the N-terminus of eIF2α is extended with threonine 169 the homologous phosphorylated residue. Here, we evaluated whether eIF2α phosphorylation varies during the Trypanosoma cruzi life cycle, the etiological agent of Chagas' disease. Total levels of eIF2α are diminished in infective and non-replicative trypomastigotes compared with proliferative forms from the intestine of the insect vector or amastigotes from mammalian cells, consistent with decreased protein synthesis reported in infective forms. eIF2α phosphorylation increases in proliferative intracellular forms prior to differentiation into trypomastigotes. Parasites overexpressing eIF2αT169A or with an endogenous CRISPR/Cas9-generated eIF2αT169A mutation were created and analysis revealed alterations to the proteome, largely unrelated to the presence of μORF in epimastigotes. eIF2αT169A mutant parasites produced fewer trypomastigotes with lower infectivity than wild type, with increased levels of sialylated mucins and oligomannose glycoproteins, and decreased galactofuranose epitopes and the surface protease GP63 on the cell surface. We conclude that eIF2α expression and phosphorylation levels affect proteins relevant for intracellular progression of T. cruzi.

Lathosterol Oxidase (Sterol C-5 Desaturase) Deletion Confers Resistance to Amphotericin B and Sensitivity to Acidic Stress in Leishmania major.

Lathosterol oxidase (LSO) catalyzes the formation of the C-5-C-6 double bond in the synthesis of various types of sterols in mammals, fungi, plants, and protozoa. In Leishmania parasites, mutations in LSO or other sterol biosynthetic genes are associated with amphotericin B resistance. To investigate the biological roles of sterol C-5-C-6 desaturation, we generated an LSO-null mutant line (lso- ) in Leishmania major, the causative agent for cutaneous leishmaniasis. lso- parasites lacked the ergostane-based sterols commonly found in wild-type L. major and instead accumulated equivalent sterol species without the C-5-C-6 double bond. These mutant parasites were replicative in culture and displayed heightened resistance to amphotericin B. However, they survived poorly after reaching the maximal density and were highly vulnerable to the membrane-disrupting detergent Triton X-100. In addition, lso- mutants showed defects in regulating intracellular pH and were hypersensitive to acidic conditions. They also had potential alterations in the carbohydrate composition of lipophosphoglycan, a membrane-bound virulence factor in Leishmania All these defects in lso- were corrected upon the restoration of LSO expression. Together, these findings suggest that the C-5-C-6 double bond is vital for the structure of the sterol core, and while the loss of LSO can lead to amphotericin B resistance, it also makes Leishmania parasites vulnerable to biologically relevant stress.IMPORTANCE Sterols are essential membrane components in eukaryotes, and sterol synthesis inhibitors can have potent effects against pathogenic fungi and trypanosomatids. Understanding the roles of sterols will facilitate the development of new drugs and counter drug resistance. LSO is required for the formation of the C-5-C-6 double bond in the sterol core structure in mammals, fungi, protozoans, plants, and algae. Functions of this C-5-C-6 double bond are not well understood. In this study, we generated and characterized a lathosterol oxidase-null mutant in Leishmania major Our data suggest that LSO is vital for the structure and membrane-stabilizing functions of leishmanial sterols. In addition, our results imply that while mutations in lathosterol oxidase can confer resistance to amphotericin B, an important antifungal and antiprotozoal agent, the alteration in sterol structure leads to significant defects in stress response that could be exploited for drug development.