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Ubiquitylation is a post-translational modification promoting protein degradation. Within the endomembrane system ubiquitylation marks proteins for lysosomal processing. Deubiquitinases (DUBs) cleave ubiquitin from modified proteins and, in the case of surface proteins, prevent lysosomal targeting and thus play a major role in controlling turnover. InTrypanosoma cruzi,the etiological agent of Chagas disease, acquisition of nutrients in parasites proliferating within the blood meal of insect vectors occurs via the cytostome, a unique structure connected to a long tubular cytopharynx. Contents are delivered to late endosomes and accumulate in reservosomes, equivalent to lysosomes. When starved,T. cruzidifferentiates into mammalian infective trypomastigotes, which are cell cycle arrested. Here we asked what roles ubiquitylation plays in this unique endocytic process by interrogation of theT. cruziortholog of VDU (von Hippel-Lindau-interacting deubiquitylating enzyme)/USP33 (ubiquitin-specific protease). We found that TcVDU expression level inversely correlated with transferrin endocytosis, and that overexpression led to a longer retention of the endocytic cargo near the cytostome. TcVDU itself was found enriched in the anterior region of the parasite, in proximity to endocytic cargo. Most importantly, TcVDU overexpression reduced parasite invasion capacity and led to increased release oftrans-sialidase. These alterations in the abundance of multiple surface proteins in TcVDU mutants indicate a key role of TcVDU in modulating theT. cruzisurface by affecting the endosomal traffic and consequently the host-parasite interface.Author summaryTrypanosoma cruziis the cause of Chagas disease that affects large populations of Central and South America. Disease is spreading to other continents due to poor control of blood transfusion from donors with chronic and undiagnosed infection, migration and drug treatment with limited performance and undesired side effects. Proliferating forms ofT. cruziacquire nutrients through the cytostome, a cell surface opening connected to a long tubular cytopharynx. Internalized material is endocytosed in the cytopharynx. This is distinct from other members of the Trypanosomatids, which endocytose material exclusively through the flagellar pocket. By using CRISPR gene editing and overexpression we demonstrated that a conserved deubiquitinase (VDU) is a key control element for traffic from the cytopharynx to endosomal compartments inT. cruzi. Changed levels of VDU modify endosomal traffic and largely impact the parasite surface, causing changes in infectivity.

Primary amoebic meningoencephalitis is a rare but fatal central nervous system (CNS) disease caused by the “brain-eating amoeba” Naegleria fowleri . A major obstacle is the requirement for drugs with the ability to cross the blood-brain barrier, which are used in extremely high doses, cause severe side effects, and are usually ineffective.

Euglena gracilis is a unicellular photosynthetic eukaryotic flagellate of the Discoba supergroup, which also encompasses Kinetoplastida and Diplonema. Plastids have green algal origin and are secondarily acquired. The nuclear genome is extremely large and many genes suggest multiple endosymbiotic/gene transfer events, i.e. derivation from prokaryotes of various lineages. E. gracilis is remarkably robust and can proliferate in environments contaminated with heavy metals and acids. Extraordinary metabolic plasticity and a mixotrophic lifestyle confers an ability to thrive in a broad range of environments, as well as facilitating production of many novel metabolites, making Euglena of considerable biotechnological importance.

The characterization of protein-protein interactions (PPIs) is of high value for understanding protein function. Two strategies are popular for identification of PPIs direct from the cellular environment: affinity capture (pulldown) isolates the protein of interest with an immobilized matrix that specifically captures the target and potential partners, whereas in BioID, genetic fusion of biotin ligase facilitates proximity biotinylation, and labeled proteins are isolated with streptavidin. Whilst both methods provide valuable insights, they can reveal distinct PPIs, but the basis for these differences is less obvious. Here, we compare both methods using four different trypanosome proteins as baits: poly(A)-binding proteins PABP1 and PABP2, mRNA export receptor MEX67, and the nucleoporin NUP158. With BioID, we found that the population of candidate interacting proteins decreases with more confined bait protein localization, but the candidate population is less variable with affinity capture. BioID returned more likely false positives, in particular for proteins with less confined localization, and identified low molecular weight proteins less efficiently. Surprisingly, BioID for MEX67 identified exclusively proteins lining the inner channel of the nuclear pore complex (NPC), consistent with the function of MEX67, whereas the entire NPC was isolated by pulldown. Similarly, for NUP158, BioID returned surprisingly few PPIs within NPC outer rings that were by contrast detected with pulldown but instead returned a larger cohort of nuclear proteins. These rather significant differences highlight a clear issue with reliance on a single method to identify PPIs and suggest that BioID and affinity capture are complementary rather than alternative approaches.

Many of the currently available anti-parasitic and anti-fungal frontline drugs have severe limitations, including adverse side effects, complex administration, and increasing occurrence of resistance. The discovery and development of new therapeutic agents is a costly and lengthy process. Therefore, repurposing drugs with already established clinical application offers an attractive, fast-track approach for novel treatment options. In this study, we show that the anti-cancer drug candidate MitoTam, a mitochondria-targeted analog of tamoxifen, efficiently eliminates a wide range of evolutionarily distinct pathogens

Leucyl aminopeptidases (LAPs) are involved in multiple cellular functions, which, in the case of infectious diseases, includes participation in the pathogen-host cell interface and pathogenesis. Th...

Many of the currently available anti-parasitic and anti-fungal frontline drugs have severe limitations, including adverse side effects, complex administration, and increasing occurrence of resistance. The discovery and development of new therapeutic agents is a costly and lengthy process. Therefore, repurposing drugs with already established clinical application offers an attractive, fast-track approach for novel treatment options. In this study, we show that the anti-cancer drug MitoTam, a mitochondria-targeted analog of tamoxifen, efficiently eliminates a wide range of evolutionarily distinct pathogens in vitro, including pathogenic fungi, Plasmodium falciparum, and several species of trypanosomatid parasites, causative agents of debilitating neglected tropical diseases. MitoTam treatment was also effective in vivo and significantly reduced parasitemia of two medically important parasites, Leishmania mexicana and Trypanosoma brucei, in their respective animal infection models. Functional analysis in the bloodstream form of T. brucei showed that MitoTam rapidly altered mitochondrial functions, particularly affecting cellular respiration, lowering ATP levels, and dissipating mitochondrial membrane potential. Our data suggest that the mode of action of MitoTam involves disruption of the inner mitochondrial membrane, leading to rapid organelle depolarization and cell death. Altogether, MitoTam is an excellent candidate drug against several important pathogens, for which there are no efficient therapies and for which drug development is not a priority.Author SummaryMitoTam, a mitochondrially targeted analog of tamoxifen, is a promising anti-cancer candidate drug acting by accumulating in and destabilizing cell mitochondria. In this study, we analyze its effect on a wide range of evolutionarily distinct and medically important pathogens. These include a) pathogenic fungi, Candida albicans, and Cryptococcus neoformans, ubiquitous opportunistic pathogens that cause life-threatening diseases in immunocompromised or immunologically deficient individuals; b) Plasmodium falciparum, the causative agent of human malaria; and c) several species of trypanosomatid parasites such as Trypanosoma cruzi, responsible for deadly Chagas disease in South America, Trypanosoma brucei, the causative agent of sleeping sickness in Africa, and Leishmania, the etiological agent of leishmaniasis, a spectrum of diseases ranging from usually self-healing but potentially disfiguring cutaneous and mucocutaneous leishmaniasis, to visceral leishmaniasis, which is invariably fatal if left untreated. We show that MitoTam efficiently kills these parasites in laboratory conditions and in the case of trypanosomes and leishmaniases suppress or at least slow down the infection in mouse model.

Euglena gracilis is a metabolically flexible, photosynthetic, and adaptable free-living protist of considerable environmental importance and biotechnological value. By label-free liquid chromatography tandem mass spectrometry, a total of 1,786 proteins were identified from the E. gracilis purified mitochondria, representing one of the largest mitochondrial proteomes so far described. Despite this apparent complexity, protein machinery responsible for the extensive RNA editing, splicing, and processing in the sister clades diplonemids and kinetoplastids is absent. This strongly suggests that the complex mechanisms of mitochondrial gene expression in diplonemids and kinetoplastids occurred late in euglenozoan evolution, arising independently. By contrast, the alternative oxidase pathway and numerous ribosomal subunits presumed to be specific for parasitic trypanosomes are present in E. gracilis. We investigated the evolution of unexplored protein families, including import complexes, cristae formation proteins, and translation termination factors, as well as canonical and unique metabolic pathways. We additionally compare this mitoproteome with the transcriptome of Eutreptiella gymnastica, illuminating conserved features of Euglenida mitochondria as well as those exclusive to E. gracilis. This is the first mitochondrial proteome of a free-living protist from the Excavata and one of few available for protists as a whole. This study alters our views of the evolution of the mitochondrion and indicates early emergence of complexity within euglenozoan mitochondria, independent of parasitism.

Constant emergence of drug-resistant Plasmodium falciparum warrants urgent need for effective and inexpensive drugs. Herein, phthalimide (Pht) analogs possessing the bioactive scaffolds, benzimidazole and 1,2,3-triazole, were evaluated for in vitro and in vivo anti-plasmodial activity without any apparent hemolysis, or cytotoxicity. Analogs 4(a-e) inhibited the growth of 3D7 and RKL-9 strains at submicromolar concentrations. Defects were observed during parasite egress from or invasion of the red blood cells. Mitochondrial membrane depolarization was measured as one of the causes of cell death. Phts 4(a-e) in combination with artemisinin exhibited two-to three-fold increased efficacy. Biophysical and biochemical analysis suggest that Pht analogs mediate plasmodial growth inhibition by interacting with tubulin protein of the parasite. Lastly, Phts 4(a-e) significantly decreased parasitemia and extended host survival in murine model Plasmodium berghei ANKA infection. Combined, the data indicate that Pht analogs should be further explored, which could offer novel value to the antimalarial drug development pipeline.

For coronaviruses, RNA-dependent RNA polymerase (RdRp) is an essential enzyme that catalyses the replication from RNA template and therefore remains an attractive therapeutic target for anti-COVID drug discovery. In the present study, we performed a comprehensive

The nuclear lamina supports many functions, including maintaining nuclear structure and gene expression control, and correct spatio-temporal assembly is vital to meet these activities. Recently, multiple lamina systems have been described that, despite independent evolutionary origins, share analogous functions. In trypanosomatids the two known lamina proteins, NUP-1 and NUP-2, have molecular masses of 450 and 170 kDa, respectively, which demands a distinct architecture from the ∼60 kDa lamin-based system of metazoa and other lineages. To uncover organizational principles for the trypanosome lamina we generated NUP-1 deletion mutants to identify domains and their arrangements responsible for oligomerization. We found that both the N- and C-termini act as interaction hubs, and that perturbation of these interactions impacts additional components of the lamina and nuclear envelope. Furthermore, the assembly of NUP-1 terminal domains suggests intrinsic organizational capacity. Remarkably, there is little impact on silencing of telomeric variant surface glycoprotein genes. We suggest that both terminal domains of NUP-1 have roles in assembling the trypanosome lamina and propose a novel architecture based on a hub-and-spoke configuration., Summary: The arrangement of the lamina in trypanosomatids has architectural differences to the metazoan system.

The eukaryotic flagellum/cilium is a prominent organelle with conserved structure and diverse functions. Euglena gracilis, a photosynthetic and highly adaptable protist, employs its flagella for both locomotion and environmental sensing. Using proteomics of isolated E. gracilis flagella we identify nearly 1700 protein groups, which challenges previous estimates of the protein complexity of motile eukaryotic flagella. We not only identified several unexpected similarities shared with mammalian flagella, including an entire glycolytic pathway and proteasome, but also document a vast array of flagella-based signal transduction components that coordinate gravitaxis and phototactic motility. By contrast, the pellicle was found to consist of > 900 protein groups, containing additional structural and signalling components. Our data identify significant adaptations within the E. gracilis flagellum, many of which are clearly linked to the highly flexible lifestyle.

For coronaviruses, RNA-dependent RNA polymerase (RdRp) is an essential enzyme that catalyses the replication from RNA template and therefore remains an attractive therapeutic target for anti-COVID drug discovery. In the present study, we performed a comprehensive in silico screening for 16,776 potential molecules from recently established drug libraries based on two important pharmacophores (3-amino-4-phenylbutan-2-ol and piperazine). Based on initial assessment, 4042 molecules were obtained suitable as drug candidates, which were following Lipinski’s rule. Molecular docking implemented for the analysis of molecular interactions narrowed this number of compounds down to 19. Subsequent to screening filtering criteria and considering the critical parameters viz. docking score and MM-GBSA binding free energy, 1-(4-((2S,3S)-3-amino-2-hydroxy-4-phenylbutyl)piperazin-1-yl)-3-phenylurea (compound 1) was accomplished to score highest in comparison to the remaining 18 shortlisted drug candidates. Notably, compound 1 displayed higher docking score (−8.069 kcal/mol) and MM-GBSA binding free energy (−49.56 kcal/mol) than the control drug, remdesivir triphosphate, the active form of remdesivir as well as adenosine triphosphate. Furthermore, a molecular dynamics simulation was carried out (100 ns), which substantiated the candidacy of compound 1 as better inhibitor. Overall, our systematic in silico study predicts the potential of compound 1 to exhibit a more favourable specific activity than remdesivir triphosphate. Hence, we suggest compound 1 as a novel potential drug candidate, which should be considered for further exploration and validation of its potential against SARS-CoV-2 in wet lab experimental studies. Communicated by Ramasawamy H. Sarma

In trypanosomes the orthologs of human USP7 and VDU1 control abundance of a cohort of surface proteins, including invariant surface glycoproteins (ISGs) by functioning as deubiquitinases (DUBs) Silencing TbUsp7 partially inhibits endocytosis and invariant surface glycoprotein turnover. As a component of cullin E3 ubiquitin ligases, S-phase kinase-associated protein 1 (Skp1) has crucial roles in cell cycle progression, transcriptional regulation, signal transduction and other processes in animals and fungi. Unexpectedly, trypanosomes possess multiple Skp1 paralogs, including a divergent paralog designated SkpZ. SkpZ is implicated in suramin-sensitivity and endocytosis and decreases in abundance following TbUsp7 knockdown and physically interacts with TbUsp7 and TbTpr86. The latter is a tetratricopeptide-repeat protein also implicated in suramin sensitivity and located close to the flagellar pocket/endosomes, consistent with a role in endocytosis. Further, silencing SkpZ reduced abundance of TbUsp7 and TbTpr86 and many trans-membrane domain surface proteins. Our data indicate that TbTpr86, TbUsp7 and SkpZ form the ‘TUS’ complex that regulates abundance of a significant cohort of trypanosome surface proteins.

Differentiation is a central aspect of the parasite life cycle and encompasses adaptation to both host and environment. If we accept that evolution cannot anticipate an organism's needs as it enters a new environment, how do parasite differentiation pathways arise? The transition between vertebrate and insect stage African trypanosomes is probably one of the better studied and involves a cell-cycle arrested or 'stumpy' form that activates metabolic pathways advantageous to the parasite in the insect host. However, a range of stimuli and stress conditions can trigger similar changes, leading to formation of stumpy-like cellular states. We propose that the origin and optimisation of this differentiation program represents repurposing of a generic stress response to gain considerable gain-of-fitness associated with parasite transmission.

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.

Leucyl aminopeptidases (LAPs) are involved in multiple cellular functions, which, in the case of infectious diseases, includes participation in the pathogen-host cell interface and pathogenesis. Thus, LAPs are considered good candidate drug targets, and the major M17-LAP from

Reliable determination of protein complex composition or changes to protein levels in whole cells is challenging. Despite the multitude of methods now available for labeling, analysis, and the statistical processing of data, this large variety is of itself an issue: Which approach is most appropriate, where do you set cutoffs, and what is the most cost-effective strategy? One size does not fit all for such work, but some guidelines can help in terms of reducing cost, improving data quality, and ultimately advancing investigations. Here we describe two protocols and algorithms for facile sample preparation for mass spectrometric analysis, robust data processing, and considerations of how to interpret large proteomic datasets in a productive and robust manner.

We examine in detail the AP-2 adaptin complex from the malaria parasite Plasmodium falciparum. In most studied organisms, AP-2 is involved in bringing material into the cell from outside, a process called endocytosis. Previous work shows that changes to the μ subunit of AP-2 can contribute to drug resistance. Our experiments show that AP-2 is essential for parasite development in blood but does not have any role in clathrin-mediated endocytosis. This suggests that a specialized function for AP-2 has developed in malaria parasites, and this may be important for understanding its impact on drug resistance., The efficacy of current antimalarial drugs is threatened by reduced susceptibility of Plasmodium falciparum to artemisinin, associated with mutations in pfkelch13. Another gene with variants known to modulate the response to artemisinin encodes the μ subunit of the AP-2 adaptin trafficking complex. To elucidate the cellular role of AP-2μ in P. falciparum, we performed a conditional gene knockout, which severely disrupted schizont organization and maturation, leading to mislocalization of key merozoite proteins. AP-2μ is thus essential for blood-stage replication. We generated transgenic P. falciparum parasites expressing hemagglutinin-tagged AP-2μ and examined cellular localization by fluorescence and electron microscopy. Together with mass spectrometry analysis of coimmunoprecipitating proteins, these studies identified AP-2μ-interacting partners, including other AP-2 subunits, the K10 kelch-domain protein, and PfEHD, an effector of endocytosis and lipid mobilization, but no evidence was found of interaction with clathrin, the expected coat protein for AP-2 vesicles. In reverse immunoprecipitation experiments with a clathrin nanobody, other heterotetrameric AP-complexes were shown to interact with clathrin, but AP-2 complex subunits were absent.

In endolysosomal networks, two hetero-hexameric tethers called HOPS and CORVET are found widely throughout eukaryotes. The unicellular ciliate Tetrahymena thermophila possesses elaborate endolysosomal structures, but curiously both it and related protozoa lack the HOPS tether and several other trafficking proteins, while retaining the related CORVET complex. Here, we show that Tetrahymena encodes multiple paralogs of most CORVET subunits, which assemble into six distinct complexes. Each complex has a unique subunit composition and, significantly, shows unique localization, indicating participation in distinct pathways. One pair of complexes differ by a single subunit (Vps8), but have late endosomal versus recycling endosome locations. While Vps8 subunits are thus prime determinants for targeting and functional specificity, determinants exist on all subunits except Vps11. This unprecedented expansion and diversification of CORVET provides a potent example of tether flexibility, and illustrates how ‘backfilling’ following secondary losses of trafficking genes can provide a mechanism for evolution of new pathways. This article has an associated First Person interview with the first author of the paper.

Introduced about a century ago, suramin remains a frontline drug for the management of early-stage East African trypanosomiasis (sleeping sickness). Cellular entry into the causative agent, the protozoan parasite Trypanosoma brucei, occurs through receptor-mediated endocytosis involving the parasite's invariant surface glycoprotein 75 (ISG75), followed by transport into the cytosol via a lysosomal transporter. The molecular basis of the trypanocidal activity of suramin remains unclear, but some evidence suggests broad, but specific, impacts on trypanosome metabolism (i.e. polypharmacology). Here we observed that suramin is rapidly accumulated in trypanosome cells proportionally to ISG75 abundance. Although we found little evidence that suramin disrupts glycolytic or glycosomal pathways, we noted increased mitochondrial ATP production, but a net decrease in cellular ATP levels. Metabolomics highlighted additional impacts on mitochondrial metabolism, including partial Krebs' cycle activation and significant accumulation of pyruvate, corroborated by increased expression of mitochondrial enzymes and transporters. Significantly, the vast majority of suramin-induced proteins were normally more abundant in the insect forms compared with the blood stage of the parasite, including several proteins associated with differentiation. We conclude that suramin has multiple and complex effects on trypanosomes, but unexpectedly partially activates mitochondrial ATP-generating activity. We propose that despite apparent compensatory mechanisms in drug-challenged cells, the suramin-induced collapse of cellular ATP ultimately leads to trypanosome cell death.

Reliable determination of protein complex composition or changes to protein levels in whole cells is challenging. Despite the multitude of methods now available for labeling, analysis, and the statistical processing of data, this large variety is of itself an issue: Which approach is most appropriate, where do you set cutoffs, and what is the most cost-effective strategy? One size does not fit all for such work, but some guidelines can help in terms of reducing cost, improving data quality, and ultimately advancing investigations. Here we describe two protocols and algorithms for facile sample preparation for mass spectrometric analysis, robust data processing, and considerations of how to interpret large proteomic datasets in a productive and robust manner.

The kinetoplastida and their close relatives are unicellular organisms prevalent within the biosphere and important for significant impacts on global health, economy and ecosystems. They are, under most models, an early branching lineage. Individual species adapted to highly diverse environments by adopting complex life styles; parasitic species can infect a wide range of eukaryotic hosts, while many relatives are free-living and some autotrophic from acquiring a plastid for photosynthesis. Adaptation is especially evident in the evolution of kinetoplastid cell surface architecture and is supported by endomembrane trafficking and serves as a platform for interaction with its environment. Here we summarize and discuss recent genomic and experimental studies of the protein trafficking system in kinetoplastids, with focus on the composition and function of the surface as well as mechanisms for constructing, maintaining and regulating the cell surface proteome. We hope this provides a broad view of how protein trafficking contributes to the intricate and dynamic host-parasite interfaces that are critical for successful environmental adaptation of this highly important lineage.

In endolysosomal networks, two hetero-hexameric tethers called HOPS and CORVET are found widely throughout eukaryotes. The unicellular ciliate Tetrahymena thermophila possesses elaborate endolysosomal structures, but curiously both it and related protozoa lack the HOPS tether and several other trafficking proteins, while retaining the related CORVET complex. Here, we show that Tetrahymena encodes multiple paralogs of most CORVET subunits, which assemble into six distinct complexes. Each complex has a unique subunit composition and, significantly, shows unique localization, indicating participation in distinct pathways. One pair of complexes differ by a single subunit (Vps8), but have late endosomal versus recycling endosome locations. While Vps8 subunits are thus prime determinants for targeting and functional specificity, determinants exist on all subunits except Vps11. This unprecedented expansion and diversification of CORVET provides a potent example of tether flexibility, and illustrates how ‘backfilling’ following secondary losses of trafficking genes can provide a mechanism for evolution of new pathways. This article has an associated First Person interview with the first author of the paper.

SummaryThe efficacy of current antimalarial drugs is threatened by reduced susceptibility of Plasmodium falciparum to artemisinin. In the Mekong region this is associated with mutations in the kelch propeller-encoding domain of pfkelch13, but variants of other parasite proteins are also thought to modulate the response to drug. Evidence from human and rodent studies suggests that the μ-subunit of the AP-2 adaptin trafficking complex is one such protein of interest. We generated transgenic Plasmodium falciparum parasites encoding the I592T variant of pfap2μ, orthologous to the I568T mutation associated with in vivo artemisinin resistance in P. chabaudi. When exposed to a four-hour pulse of dihydroartemisin in the ring-stage survival assay, two P. falciparum clones expressing AP-2μ I592T displayed significant and reproducible survival of 8.0% and 10.3%, respectively, compared to P. falciparum, K10. Conditional knockout indicates that the AP-2 trafficking complex in P. falciparum is essential for the fidelity of merozoite biogenesis and membrane organisation in the mature schizont. We also show that while other heterotetrameric AP-complexes and secretory factors interact with clathrin, AP-2 complex subunits do not. Thus, the AP-2 complex may be diverted from a clathrin-dependent endocytic role seen in most eukaryotes into a Plasmodium-specific function. These findings represent striking divergences from eukaryotic dogma and support a role for intracellular traffic in determining artemisinin sensitivity in vitro, confirming the existence of multiple functional routes to reduced ring-stage artemisinin susceptibility. Therefore, the utility of pfkelch13 variants as resistance markers is unlikely to be universal, and phenotypic surveillance of parasite susceptibility in vivo may be needed to identify threats to our current combination therapies.

© The Author(s) 2019 ., [Background]: Photosynthetic euglenids are major contributors to fresh water ecosystems. Euglena gracilis in particular has noted metabolic flexibility, reflected by an ability to thrive in a range of harsh environments. E. gracilis has been a popular model organism and of considerable biotechnological interest, but the absence of a gene catalogue has hampered both basic research and translational efforts., [Results]: We report a detailed transcriptome and partial genome for E. gracilis Z1. The nuclear genome is estimated to be around 500 Mb in size, and the transcriptome encodes over 36,000 proteins and the genome possesses less than 1% coding sequence. Annotation of coding sequences indicates a highly sophisticated endomembrane system, RNA processing mechanisms and nuclear genome contributions from several photosynthetic lineages. Multiple gene families, including likely signal transduction components, have been massively expanded. Alterations in protein abundance are controlled post-transcriptionally between light and dark conditions, surprisingly similar to trypanosomatids., [Conclusions]: Our data provide evidence that a range of photosynthetic eukaryotes contributed to the Euglena nuclear genome, evidence in support of the ‘shopping bag’ hypothesis for plastid acquisition. We also suggest that euglenids possess unique regulatory mechanisms for achieving extreme adaptability, through mechanisms of paralog expansion and gene acquisition., This work was supported by the Yousef Jameel Academic Program (through the Yousef Jameel PhD Scholarship), the Cambridge Commonwealth, European and International Trust, the Cambridge University Student Registry, the Cambridge Philosophical Society (all to TEE), the Medical Research Council (Grant #: P009018/1 to MCF), and German Aerospace Center - DLR, Cologne, on the behalf of Federal Ministry of Education and Research (BMBF), Germany (Grant no: 50WB1128 and 50WB1528 to ML), the European Research Council CZ LL1601 BFU2013-40866-P (to DPD) and the Czech Ministry of Education, Youth and Sports - National Sustainability Program II (Project BIOCEV-FAR) LQ 1604, by the project BIOCEV (CZ.1.05/1.1.00/02.0109), by the Centre for research of pathogenicity and virulence of parasites CZ.02.1.01/0.0/0.0/16_019/0000759 and by the Czech Science Foundation project nr. 16-25280S (to VH, AV and PS).

The M17 leucyl-aminopeptidase of Trypanosoma cruzi (LAPTc) is a novel drug target for Chagas disease. The objective of this work was to obtain recombinant LAPTc (rLAPTc) in Escherichia coli. A LAPTc gene was designed, optimized for its expression in E. coli, synthesized and cloned into the vector pET-19b. Production of rLAPTc in E. coli BL21(DE3)pLysS, induced for 20 h at 25 °C with 1 mM IPTG, yielded soluble rLAPTC that was catalytically active. The rLAPTc enzyme was purified in a single step by IMAC. The recombinant protein was obtained with a purity of 90% and a volumetric yield of 90 mg per liter of culture. The enzymatic activity has an optimal pH of 9.0, and preference for Leu-p-nitroanilide (appKM = 74 µM, appkcat = 4.4 s−1). The optimal temperature is 50 °C, and the cations Mg2+, Cd2+, Ba2+, Ca2+ and Zn2+ at 4 mM inhibited the activity by 60% or more, while Mn2+ inhibited by only 15% and addition of Co2+ activated by 40%. The recombinant enzyme is insensitive toward the protease inhibitors PMSF, TLCK, E-64 and pepstatin A, but is inhibited by EDTA and bestatin. Bestatin is a non-competitive inhibitor of the enzyme with a Ki value of 881 nM. The enzyme is a good target for inhibitor identification.

Euglena gracilis is a well-studied biotechnologically exploitable phototrophic flagellate harbouring secondary green plastids. Here we describe its plastid proteome obtained by high-resolution proteomics. We identified 1,345 candidate plastid proteins and assigned functional annotations to 774 of them. More than 120 proteins are affiliated neither to the host lineage nor the plastid ancestor and may represent horizontal acquisitions from various algal and prokaryotic groups. Reconstruction of plastid metabolism confirms both the presence of previously studied/predicted enzymes/pathways and also provides direct evidence for unusual features of its metabolism including uncoupling of carotenoid and phytol metabolism, a limited role in amino acid metabolism and the presence of two sets of the SUF pathway for FeS cluster assembly. Most significantly, one of these was acquired by lateral gene transfer (LGT) from the chlamydiae. Plastidial paralogs of membrane trafficking-associated proteins likely mediating a poorly understood fusion of transport vesicles with the outermost plastid membrane were identified, as well as derlin-related proteins that potentially act as protein translocases of the middle membrane, supporting an extremely simplified TIC complex. The proposed innovations may be also linked to specific features of the transit peptide-like regions described here. Hence the Euglena plastid is demonstrated to be a product of several genomes and to combine novel and conserved metabolism and transport processes.

Structural dissection of EssC, a membrane-bound component of the bacterial Type VII secretion system, reveals two β-sandwich domains at the N-terminus and two ATPase domains at the C-terminus. A structure for the potential pore of the secretion system is proposed., The membrane-bound protein EssC is an integral component of the bacterial Type VII secretion system (T7SS), which is a determinant of virulence in important Gram-positive pathogens. The protein is predicted to consist of an intracellular repeat of forkhead-associated (FHA) domains at the N-terminus, two transmembrane helices and three P-loop-containing ATPase-type domains, D1–D3, forming the C-terminal intracellular segment. We present crystal structures of the N-terminal FHA domains (EssC-N) and a C-terminal fragment EssC-C from Geobacillus thermodenitrificans, encompassing two of the ATPase-type modules, D2 and D3. Module D2 binds ATP with high affinity whereas D3 does not. The EssC-N and EssC-C constructs are monomeric in solution, but the full-length recombinant protein, with a molecular mass of approximately 169 kDa, forms a multimer of approximately 1 MDa. The observation of protomer contacts in the crystal structure of EssC-C together with similarity to the DNA translocase FtsK, suggests a model for a hexameric EssC assembly. Such an observation potentially identifies the key, and to date elusive, component of pore formation required for secretion by this recently discovered secretion system. The juxtaposition of the FHA domains suggests potential for interacting with other components of the secretion system. The structural data were used to guide an analysis of which domains are required for the T7SS machine to function in pathogenic Staphylococcus aureus. The extreme C-terminal ATPase domain appears to be essential for EssC activity as a key part of the T7SS, whereas D2 and FHA domains are required for the production of a stable and functional protein.

Photosynthetic euglenids are major components of aquatic ecosystems and relatives of trypanosomes.Euglena gracilishas considerable biotechnological potential and great adaptability, but exploitation remains hampered by the absence of a comprehensive gene catalogue. We address this by genome, RNA and protein sequencing: theE. gracilisgenome is >2Gb, with 36,526 predicted proteins. Large lineage-specific paralog families are present, with evidence for flexibility in environmental monitoring, divergent mechanisms for metabolic control, and novel solutions for adaptation to extreme environments. Contributions from photosynthetic eukaryotes to the nuclear genome, consistent with the shopping bag model are found, together with transitions between kinetoplastid and canonical systems. Control of protein expression is almost exclusively post-transcriptional. These data are a major advance in understanding the nuclear genomes of euglenids and provide a platform for investigating the contributions ofE. gracilisand its relatives to the biosphere.

Type VII secretion systems (T7SS) are found in many bacteria and secrete proteins involved in virulence and bacterial competition. In Staphylococcus aureus the small ubiquitin-like EsaB protein has been previously implicated as having a regulatory role in the production of the EsxC substrate. Here we show that in the S. aureus RN6390 strain, EsaB does not genetically regulate production of any T7 substrates or components, but is indispensable for secretion activity. Consistent with EsaB being an essential component of the T7SS, loss of either EsaB or EssC are associated with upregulation of a common set of iron acquisition genes. However, a further subset of genes were dysregulated only in the absence of EsaB. Quantitative western blotting indicates that EsaB is present at very low levels in cells. Substitution of a highly conserved threonine for alanine or arginine resulted in a loss of EsaB activity and destabilisation of the protein. Taken together our findings show that EsaB is essential for T7SS activity in RN6390.

Type VII secretion systems (T7SS) are found in many bacteria and secrete proteins involved in virulence and bacterial competition. In Staphylococcus aureus the small ubiquitin-like EsaB protein has been previously implicated as having a regulatory role in the production of the EsxC substrate. Here we show that in the S. aureus RN6390 strain, EsaB does not genetically regulate production of any T7 substrates or components, but is indispensable for secretion activity. Consistent with EsaB being a core component of the T7SS, loss of either EsaB or EssC are associated with upregulation of a common set of iron acquisition genes. However, a further subset of genes were dysregulated only in the absence of EsaB. In addition, fractionation revealed that although an EsaB fusion to yellow fluorescent protein partially localised to the membrane, it was still membrane-localised when the T7SS was absent. Taken together our findings suggest that EsaB has T7SS-dependent and T7SS-independent roles in S. aureus.