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Repetitive elements in DNA sequences are a hallmark of Apicomplexan protozoa. A genome-wide screening for Tandem Repeats was conducted in Toxoplasma gondii and related Coccidian parasites with a novel strategy to assess compositional bias. A conserved pattern of GC skew and purine-pyrimidine bias was observed. Compositional bias was also present at the protein level. Glutamic acid was the most abundant amino acid in the purine (GA) rich cluster, while Serine prevailed in pyrimidine (CT) rich cluster. Purine rich repeats, and consequently glutamic acid abundance, correlated with high scores for intrinsically disordered protein regions/domains. Finally, variability was established for repetitive regions within a well-known rhoptry antigen (ROP1) and an uncharacterized hypothetical protein with similar features. The approach we present could be useful to identify potential antigens bearing repetitive elements., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Metacaspases are a distinct class of cysteine proteases predominantly found in plants, fungi, and protozoa, crucial for regulating programmed cell death (PCD). They possess unique structural features and differ markedly from caspases in their activation mechanisms and substrate specificities, with a notable preference for binding basic residues in substrates. In this study, we introduced vanillin-derived oximic compounds to explore their pharmaceutical potential. We evaluated these compounds for their inhibitory effects on TbMCA2, a metacaspase in Trypanosoma brucei, identifying AO-7, AO-12, and EO-20 as promising inhibitors. AO-12 showed significant potential as a non-competitive inhibitor with notable IC 50 values. Molecular docking studies were also conducted to evaluate the binding affinity of these compounds for TbMCA2. This research is particularly relevant given the urgent need for more effective and less toxic treatments for trypanosomiasis, a parasitic disease caused by trypanosomes. The absence of available vaccines and the limitations imposed by drug toxicity underscore the importance of these findings. Our study represents a significant advancement in developing therapeutic agents targeting metacaspases in trypanosomatids and highlights the necessity of understanding metacaspase regulation across various species. It provides valuable insights into inhibitor sensitivity and potential species-specific therapeutic strategies. In conclusion, this research opens promising avenues for novel therapeutic agents targeting metacaspases in trypanosomatids, addressing a critical gap in combating neglected diseases associated with these pathogens. Further research is essential to refine the efficacy and safety profiles of these compounds, aiming to deliver more accessible and effective therapeutic solutions to populations afflicted by these debilitating diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Entamoeba nuttalli is genetically the closest to Entamoeba histolytica, the causative agent of human amebiasis, and its natural host is Macaca species. A unique E. nuttalli specific surface protein (PTORS) containing 42 repeats of octapeptide was identified by comparative genomic analysis of Entamoeba species. We aimed to elucidate the function of this protein. When trophozoites from various E. nuttalli strains were examined by immunofluorescence microscopy and flow cytometry using a PTORS-specific monoclonal antibody, only a limited proportion of trophozoites were stained, indicating that the protein was not commonly expressed in all E. nuttalli trophozoite. The proportion of trophozoites expressing PTORS increased after passage in hamster livers, suggesting that the protein functions in the virulence of trophozoites in the liver tissue. Single-cell analysis revealed that in the cluster including trophozoites with PTORS gene expression, genes of virulence-related proteins were also upregulated. Trophozoites of E. histolytica transfected with PTORS showed enhanced adherence and subsequent phagocytic activity towards human Jurkat cells, independent of the lectin. E. histolytica trophozoites expressing PTORS formed larger liver abscesses in hamsters. These results demonstrate that PTORS is a novel virulence factor in Entamoeba species., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Ascorbate peroxidase (APX) has emerged as a promising target for chemotherapy because of its absence in humans and crucial role in the antioxidant defence of trypanosomatids. APXs, which are class I haeme-containing enzymes, reduces hydrogen peroxide using ascorbate to produce water and monodehydroascorbate, thereby preventing cell damage caused by H 2 O 2 ., Methods: We aimed to create an APX-knockout L. infantum line using CRISPR/Cas9. Despite unsuccessful attempts at full knockouts, we achieved deletion of chromosomal copies post-APX episomal insertion, yielding LiΔchrAPX::LbAPX parasites. We performed phenotypic characterization to assess the impact of these genetic modifications, which included the determination of APX transcript expression levels using quantitative PCR, drug sensitivity, infectivity, and parasite survival in macrophages., Results: Quantitative polymerase chain reaction (PCR) analysis revealed a 10- to 13-fold reduction in APX transcript expression in LiΔchrAPX::LbAPX compared with wild-type (LiWT) and APX-overexpressing (Li::Cas9::LbAPX) parasites, respectively. The episomes in those knockdown parasites remained stable even after 20 drug-free passages in vitro. Li::Cas9::LbAPX parasites showed increased resistance to trivalent antimony (Sb III ) and isoniazid, reduced tolerance to H 2 O 2 , and unchanged macrophage infectivity compared with LiWT. In contrast, LiΔchrAPX::LbAPX parasites were more sensitive to Sb III and isoniazid, exhibited greater susceptibility to H 2 O 2 -induced oxidative stress, and 72 h post-infection, showed fewer infected macrophages and intracellular amastigotes compared with LiWT parasites., Conclusions: Our findings hint at the indispensability of APX in L. infantum and raise the possibility of its potential as a therapeutic target for leishmaniasis., Competing Interests: Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Egress represents a crucial process employed by Neospora caninum in the establishment of infection. Dense granule proteins (GRAs), secreted by the dense granule, play significant roles in modifying the parasitophorous vacuole, maintenance of morphology, and regulating host-cell interactions. However, their precise involvement in tachyzoite egress remains inadequately characterized. In this study, we identified a homologous gene, Ncgra41, corresponding to the dense granule protein 41 (GRA41) of Toxoplasma gondii, which is associated with egress, utilizing NCBI and ToxoDB databases. NcGRA41 is localized extracellularly within dense granules and intracellularly within parasitic vacuoles. Deletion of NcGRA41 did not affect tachyzoites invasion or proliferation but significantly reduced egress capacity and pathogenicity in mice. The phenotypic characteristics were restored in a complementary strain. Further investigation revealed that the absence of NcGRA41 reduced gliding motility and the transcription level of the subtilisin-like protein (SUB1). A microneme secretion assay demonstrated a significant decrease in NcMIC1 secretion, along with reduced expression levels of NcMIC1, NcMIC4, and NcMIC8. These findings demonstrate that NcGRA41, a novel dense granule protein in N. caninum, modulates tachyzoites egress and influences pathogenicity by regulating microneme secretion., Competing Interests: Declarations Ethics approval The mice used in this study were handled in compliance with the recommendations outlined in the “Guidelines for the Care and Use of Laboratory Animals” of the Ministry of Science and Technology of China. Additionally, our research protocol received approval from the Laboratory Animal Welfare and Animal Experimental Ethical Committee of China Agricultural University (Certificate No.: CAU-AW31901202-2–1). Consent to participate Done and confirmed. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Faithful chromosome segregation in eukaryotes requires the assembly of a bipolar spindle and the faithful attachment of kinetochores to spindle microtubules, which are regulated by various spindle-associated proteins (SAPs) that play distinct functions in regulating spindle dynamics and microtubule-kinetochore attachment. The protozoan parasite Trypanosoma brucei employs evolutionarily conserved and kinetoplastid-specific proteins, including some kinetoplastid-specific nucleus- and spindle-associated proteins (NuSAPs), to regulate chromosome segregation. Here, we characterized NuSAP4 and its functional interplay with diverse SAPs in promoting chromosome segregation in T. brucei. NuSAP4 associates with the spindle during mitosis and concentrates at spindle poles where it interacts with SPB1 and MAP103. Knockdown of NuSAP4 impairs chromosome segregation by disrupting bipolar spindle assembly and spindle pole protein localization. These results uncover the mechanistic role of NuSAP4 in regulating chromosome segregation by promoting bipolar spindle assembly, and highlight the unusual features of mitotic regulation by spindle-associated proteins in this early divergent microbial eukaryote., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Chagas disease is caused by the eukaryote parasite Trypanosoma cruzi . Current treatment exhibits limited efficacy and selenium-based compounds emerged as promising candidates for new therapies which is surpassing its bioisoster, sulfur. We designed new thiosemicarbazones, thiazoles, selenosemicarbazones and selenazoles, using isosteric substitution. We synthesized 57 new chalcogen compounds which were evaluated against T. cruzi , C2C12 cells and cruzain, the main target of this parasite. Additionally, human cathepsin L, was tested for selectivity. Three compounds were selected, based on their activity against the intracellular amastigotes (EC 50 < 1 μM, SI > 10) and cruzain (IC 50 < 100 nM, SI > 5.55) which compared favorably with the approved drug, Benznidazole, and the well-established cruzain inhibitor K777. Seleno-compounds demonstrated enhanced activity and selenazoles showed a decrease in selenium-associated toxicity. Compound 4-methyl-2-(2-(1-(3-nitrophenyl)ethylidene)hydrazineyl)-1,3-selenazole ( Se 2h ) emerged as a promising candidate, and its binding to cruzain was investigated. Pharmacokinetic assessment was conducted, showing a favorable profile for subsequent in vivo assays.

Malaria still causes over 600,000 deaths annually, with rising resistance to frontline drugs by Plasmodium falciparum increasing this number each year. New medicines with novel mechanisms of action are, therefore, urgently needed. In this work, we solved the cocrystal structure of the essential malarial kinase Pf CLK3 with the reversible inhibitor TCMDC-135051 ( 1 ), enabling the design of covalent inhibitors targeting a unique cysteine residue (Cys368) poorly conserved in the human kinome. Chloroacetamide 4 shows nanomolar potency and covalent inhibition in both recombinant protein and P. falciparum assays. Efficacy in parasites persisted after a 6 h washout, indicating an extended duration of action. Additionally, 4 showed improved kinase selectivity and a high selectivity index against HepG2 cells, with a low propensity for resistance (log MIR > 8.1). To our knowledge, compound 4 is the first covalent inhibitor of a malarial kinase, offering promising potential as a lead for a single-dose malaria cure.

The potency of frontline antimalarial drug artemisinin (ART) derivatives is triggered by heme-induced cleavage of the endoperoxide bond to form reactive heme-ART alkoxy radicals and covalent heme-ART adducts, which are highly toxic to the parasite. ART-resistant (ART-R) parasites with mutations in the Plasmodium falciparum Kelch-containing protein Kelch13 (PfKekch13) exhibit impaired hemoglobin uptake, reduced yield of hemoglobin-derived heme, and thus decreased ART activation. However, any direct involvement of PfKelch13 in heme-mediated ART activation has not been reported. Here, we show that the purified recombinant PfKelch13 wild-type (WT) protein displays measurable binding affinity for iron and heme, the main effectors for ART activation. The heme-binding property is also exhibited by the native PfKelch13 protein from parasite culture. The two ART-R recombinant PfKelch13 mutants (C580Y and R539T) display weaker heme binding affinities compared to the ART-sensitive WT and A578S mutant proteins, which further translates into reduced yield of heme-ART derivatives when ART is incubated with the heme molecules bound to the mutant PfKelch13 proteins. In conclusion, this study provides the first evidence for ART activation via the heme-binding propensity of PfKelch13. This mechanism may contribute to the modulation of ART-R levels in malaria parasites through a novel function of PfKelch13., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Macropinocytosis is an evolutionarily conserved endocytic process that plays a vital role in internalizing extracellular fluids and particles in cells. This non-selective endocytic pathway is crucial for various physiological functions such as nutrient uptake, sensing, signaling, antigen presentation, and cell migration. While macropinocytosis has been extensively studied in macrophages and cancer cells, the molecular mechanisms of macropinocytosis in pathogens are less understood. It has been known that Entamoeba histolytica, the causative agent of amebiasis, exploits macropinocytosis for survival and pathogenesis. Since macropinocytosis is initiated by actin polymerization, leading to the formation of membrane ruffles and the subsequent trapping of solutes in macropinosomes, actin cytoskeleton regulation is crucial. Thus, this study focuses on unraveling the role of well-conserved actin cytoskeleton regulators, Rho small GTPase family proteins, in macropinocytosis in E. histolytica. Through gene silencing of highly transcribed Ehrho/Ehrac genes and following flow cytometry analysis, we identified that silencing EhracM enhances dextran macropinocytosis and affects cellular migration persistence. Live imaging and interactome analysis unveiled the cytosolic and vesicular localization of EhRacM, along with its interaction with signaling and membrane traffic-related proteins, shedding light on EhRacM's multiple roles. Our findings provide insights into the specific regulatory mechanisms of macropinocytosis among endocytic pathways in E. histolytica, highlighting the significance of EhRacM in both macropinocytosis and cellular migration., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Shimoyama et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

As a model organism in the study of immunity to infection, Toxoplasma gondii has been instrumental in establishing key principles of host anti-microbial defense and its regulation. Here, we employed an attenuated uracil auxotroph strain of Type I Toxoplasma designated OMP to further untangle the early immune response to this parasitic pathogen. Experiments using αβ T cell-deficient Tcrb -/- mice unexpectedly revealed that an intact αβ T lymphocyte compartment was essential to survive infection with OMP. Subsequent antibody depletion and knockout mouse experiments demonstrated contributions from CD4 + T cells and most predominantly CD8 + T cells in resistance. Using transgenic knockout mice, we found only a partial requirement for IFN-γ and a lack of requirement for Toll-like receptor (TLR) adaptor MyD88 in resistance. In contrast to other studies on Toxoplasma , the ability to survive OMP infection did not require IL-12p40. Surprisingly, T cell-dependent IL-10 was found to be critical for survival, and deficiency of this cytokine triggered an abnormally high systemic inflammatory response. We also found that parasite molecule GRA24, a dense granule protein that triggers TLR-independent IL-12 production, acts as a virulence factor contributing to death of OMP-infected Tcrb -/- and IL-10 -/- mice. Furthermore, resistance against OMP was restored in Tcrb -/- mice via monoclonal depletion of IL-12p40, suggesting that GRA24-induced IL-12 underlies the fatal immunopathology observed. Collectively, our studies provide insight into a novel and rapidly arising T lymphocyte-dependent anti-inflammatory response to T. gondii which operates independently of MyD88 and IL-12 and that depends on the function of parasite-dense granule protein GRA24.IMPORTANCEAs a model infectious microbe and an important human pathogen, the apicomplexan Toxoplasma gondii has provided many important insights into innate and adaptive immunity to infection. We show here that a low virulence uracil auxotrophic Toxoplasma strain emerges as a virulent parasite in the absence of an intact T cell compartment. Both CD4 + and CD8 + T lymphocytes are required for optimal protection, in line with previous findings in other models of Toxoplasma infection. Nevertheless, several novel aspects of the response were identified in our study. Protection occurs independently of IL-12 and MyD88 and only partially requires IFN-γ. This is noteworthy particularly because the cytokines IL-12 and IFN-γ have previously been regarded as essential for protective immunity to T. gondii . Instead, we identified the anti-inflammatory effects of T cell-dependent IL-10 as the critical factor enabling host survival. The parasite dense granule protein GRA24, a host-directed mitogen-activated protein kinase activator, was identified as a major virulence factor in T cell-deficient hosts. Collectively, our results provide new and unexpected insights into host resistance to Toxoplasma ., Competing Interests: The authors declare no conflict of interest.

We previously reported that the Plasmodium falciparum putative serine/threonine protein phosphatase 7 (PP7) is a high-confidence substrate of the cAMP-dependent protein kinase (PKA). Here we explore the function of PP7 in asexual P. falciparum blood stage parasites. We show that conditional disruption of PP7 leads to a severe growth arrest. We show that PP7 is a calcium-dependent phosphatase that interacts with calmodulin and calcium-dependent protein kinase 1 (CDPK1), consistent with a role in calcium signaling. Notably, PP7 was found to be dispensable for erythrocyte invasion, but was crucial for ring-stage development, with PP7-null parasites arresting shortly following invasion and showing no transition to ameboid forms. Phosphoproteomic analysis revealed that PP7 may regulate certain PKAc substrates. Its interaction with calmodulin and CDPK1 further emphasizes a role in calcium signaling, while its impact on early ring development and PKAc substrate phosphorylation underscores its importance in parasite development., Importance: Plasmodium falciparum causes malaria and is responsible for more than 600,000 deaths each year. Although effective drugs are available to treat disease, the spread of drug-resistant parasites endangers their future efficacy. It is hoped that a better understanding of the biology of malaria parasites will help us to discover new drugs to tackle the resistance problem. Our work is focused on the cell signaling mechanisms that control the development of the parasite throughout its complex life cycle. All signal transduction pathways are ultimately regulated by reversible protein phosphorylation by protein kinase and protein phosphatase enzymes. In this study, we investigate the function of calcium-dependent protein phosphatase PP7 and show that it is essential for the development of ring-stage parasites following the invasion of human erythrocytes. Our results contribute to the understanding of the erythrocytic stages of the parasite life cycle that cause malaria pathology., Competing Interests: The authors declare no conflict of interest.

Toxoplasma gondii possesses a highly polarized secretory pathway that contains both broadly conserved eukaryotic organelles and unique apicomplexan organelles, which play essential roles in the parasite's lytic cycle. As in other eukaryotes, the T. gondii Golgi apparatus sorts and modifies proteins prior to their distribution to downstream organelles. Many of the typical trafficking factors found involved in these processes are missing from apicomplexan genomes, suggesting that these parasites have evolved unique proteins to fill these roles. Here, we identify a Golgi-localizing protein (ULP1), which is structurally similar to the eukaryotic trafficking factor p115/Uso1. We demonstrate that depletion of ULP1 leads to a dramatic reduction in parasite fitness that is the result of defects in microneme secretion, invasion, replication, and egress. Using ULP1 as bait for TurboID proximity labeling and immunoprecipitation, we identify 11 more Golgi-associated proteins and demonstrate that ULP1 interacts with the T. gondii -conserved oligomeric Golgi (COG) complex. These proteins include both conserved trafficking factors and parasite-specific proteins. Using a conditional knockdown approach, we assess the effect of each of these 11 proteins on parasite fitness. Together, this work reveals a diverse set of T. gondii Golgi-associated proteins that play distinct roles in the secretory pathway. As several of these proteins are absent outside of the Apicomplexa, they represent potential targets for the development of novel therapeutics against these parasites., Importance: Apicomplexan parasites such as Toxoplasma gondii infect a large percentage of the world's population and cause substantial human disease. These widespread pathogens use specialized secretory organelles to infect their host cells, modulate host cell functions, and cause disease. While the functions of the secretory organelles are now better understood, the Golgi apparatus of the parasite remains largely unexplored, particularly regarding parasite-specific innovations that may help direct traffic intracellularly. In this work, we characterize ULP1, a protein that is unique to parasites but shares structural similarity to the eukaryotic trafficking factor p115/Uso1. We show that ULP1 plays an important role in parasite fitness and demonstrate that it interacts with the conserved oligomeric Golgi (COG) complex. We then use ULP1 proximity labeling to identify 11 additional Golgi-associated proteins, which we functionally analyze via conditional knockdown. This work expands our knowledge of the Toxoplasma Golgi apparatus and identifies potential targets for therapeutic intervention., Competing Interests: The authors declare no conflict of interest.

Dinoflagellate chromosomes are extraordinary, as their organization is independent of architectural nucleosomes unlike typical eukaryotes and shows a cholesteric liquid crystal state. 5-hydroxymethyluridine (5hmU) is present at unusually high levels and its function remains an enigma in dinoflagellates chromosomal DNA for several decades. Here, we demonstrate that 5hmU contents vary among different dinoflagellates and are generated through thymidine hydroxylation. Importantly, we identified the enzyme, which is a putative dinoflagellate TET/JBP homolog, catalyzing 5hmU production using both in vivo and in vitro biochemical assays. Based on the near-chromosomal level genome assembly of dinoflagellate Amphidinium carterae , we depicted a comprehensive 5hmU landscape and found that 5hmU loci are significantly enriched in repeat elements. Moreover, inhibition of 5hmU via dioxygenase inhibitor leads to transcriptional activation of 5hmU-marked transposable elements, implying that 5hmU appears to serve as an epigenetic mark for silencing transposon. Together, our results revealed the biogenesis, genome-wide landscape, and molecular function of dinoflagellate 5hmU, providing mechanistic insight into the function of this enigmatic DNA mark., Competing Interests: Competing interests statement:The authors declare no competing interest.

In Toxoplasma gondii , the conoid comprises a cone with spiraling tubulin fibers, preconoidal rings, and intraconoidal microtubules. This dynamic organelle undergoes extension and retraction through the apical polar ring (APR) during egress, gliding, and invasion. The forces involved in conoid extrusion are beginning to be understood, and its role in directing F-actin flux to the pellicular space, thereby controlling parasite motility, has been proposed. However, the contribution of the APR and its interactions with the conoid remain unclear. To gain insight into the APR architecture, ultrastructure expansion microscopy was applied to pinpoint known and newly identified APR proteins (APR2 to APR7). Our results revealed that the APR is constructed as a fixed multilayered structure. Notably, conditional depletion of APR2 resulted in significant impairments in motility and invasion. Electron microscopy and cryoelectron tomography revealed that depletion of APR2 alters APR integrity, affecting conoid extrusion and causing cytosolic leakage of F-actin. These findings implicate the APR structure in directing the apico-basal flux of F-actin to regulate parasite motility and invasion., Competing Interests: Competing interests statement:The authors declare no competing interest.

Plasmodium falciparum malaria parasites invade and multiply inside red blood cells (RBCs), the most iron-rich compartment in humans. Like all cells, P. falciparum requires nutritional iron to support essential metabolic pathways, but the critical mechanisms of iron acquisition and trafficking during RBC infection have remained obscure. Parasites internalize and liberate massive amounts of heme during large-scale digestion of RBC hemoglobin within an acidic food vacuole (FV) but lack a heme oxygenase to release porphyrin-bound iron. Although most FV heme is sequestered into inert hemozoin crystals, prior studies indicate that trace heme escapes biomineralization and is susceptible to nonenzymatic degradation within the oxidizing FV environment to release labile iron. Parasites retain a homolog of divalent metal transporter 1 (DMT1), a known mammalian iron transporter, but its role in P. falciparum iron acquisition has not been tested. Our phylogenetic studies indicate that P. falciparum DMT1 (PfDMT1) retains conserved molecular features critical for metal transport. We localized this protein to the FV membrane and defined its orientation in an export-competent topology. Conditional knockdown of PfDMT1 expression is lethal to parasites, which display broad cellular defects in iron-dependent functions, including impaired apicoplast biogenesis and mitochondrial polarization. Parasites are selectively rescued from partial PfDMT1 knockdown by supplementation with exogenous iron, but not other metals. These results support a cellular paradigm whereby PfDMT1 is the molecular gatekeeper to essential iron acquisition by blood-stage malaria parasites and suggest that therapeutic targeting of PfDMT1 may be a potent antimalarial strategy., Competing Interests: Competing interests statement:The authors declare no competing interest.

To combat the permanent exposure to potential pathogens every organism relies on an immune system. Important factors in innate immunity are antimicrobial peptides (AMPs) that are structurally highly diverse. Some AMPs are known to belong to the saposin-like proteins (SAPLIPs), a group of polypeptides with a broad functional spectrum. The model organism Dictyostelium discoideum possesses a remarkably large arsenal of potential SAPLIPs, which are termed amoebapore-like peptides (Apls), but the knowledge about these proteins is very limited. Here, we report about the biochemical characterization of AplE1, AplE2, AplK1, and AplK2, which are derived from the two precursor proteins AplE and AplK, thereby resembling prosaposins of vertebrates. We produced these Apls as recombinant polypeptides in Escherichia coli using a self-splicing intein to remove an affinity tag used for purification. All recombinant Apls exhibited pore-forming activity in a pH-dependent manner, as evidenced by liposome depolarization, showing higher activities the more acidic the setting was. Lipid preference was detected for negatively charged phospholipids and in particular for cardiolipin. Antimicrobial activity against various bacteria was found to be inferior in classical microdilution assays. However, all of the Apls studied permeabilized the cytoplasmic membrane of live Bacillus subtilis . Collectively, we assume that the selected Apls interact by their cationic charge with negatively charged bacterial membranes in acidic environments such as phagolysosomes and eventually lyse the target cells by pore formation.

Acidocalcisomes of Trypanosoma brucei are membrane-bounded organelles characterized by their acidity and high content of polyphosphate and cations, like calcium and magnesium. They have important roles in cation and phosphorus storage, osmoregulation, autophagy initiation, calcium signaling, and virulence. Acidocalcisomes of T. brucei possess several membrane transporters, pumps, and channels, some of which were identified by proteomic and immunofluorescence analyses and validated as acidocalcisome proteins by their colocalization with the acidocalcisome marker vacuolar proton pyrophosphatase (VP1). Here, we report that a set of membrane transporters and enzymes, which were proposed to be present in acidocalcisomes by the morphological appearance of tagged proteins, colocalize with VP1, validating their character as acidocalcisome proteins., Importance: Acidocalcisomes are acidic organelles rich in polyphosphate and calcium present in a variety of eukaryotes and important for osmoregulation and calcium signaling. Several proteins were postulated to localize to acidocalcisomes based on their morphological characteristics. We provide validation of the localization of ten10 acidocalcisome proteins by their co-localization with enzymatic markers. These findings reveal the roles of acidocalcisomes in the storage of toxic metals, and the presence of enzymes involved in palmitoylation and polyphosphate metabolism., Competing Interests: The authors declare no conflict of interest.

Entamoeba histolytica is an enteric protozoan parasite that causes human amebic colitis and extraintestinal abscesses. As a prerequisite for parasite colonization and invasion, adherence of E. histolytica is predominantly mediated by galactose (Gal)- and N-acetyl-d-galactosamine (GalNAc)-inhibitable lectins. The intermediate subunit (Igl) of Gal-/GalNAc-inhibitable lectin is a cysteine-rich protein containing multiple CXXC motifs and is considered a key factor affecting trophozoite's pathogenicity. However, details of the function of Igl during parasite adherence remain unclear. Here, using segmentally expressed Igl proteins and a CHO cell model transfected with Igl fragments, we identified a carbohydrate-recognition domain (CRD)-like region between amino acids 989 and 1,088. Through single- and double-point mutations in the Igl segments, two core CXXC motifs responsible for carbohydrate recognition in the CRD-like region, which are highly conserved among several lectins, were confirmed. In addition to adhesion, the roles of CRD-like region and its core CXXC motifs in various pathogenic effects were further explored. To our knowledge, this is the first report showing an adhesion-related region in E. histolytica Igl. The identification and characterization of this CRD-like region provides further insights into molecular mechanisms underlying E. histolytica pathogenicity and also aids in the determination of a potential drug target in this parasite., Importance: Entamoeba histolytica adhesion mainly depends on galactose (Gal)-/N-acetyl-d-galactosamine (GalNAc)-inhibitable lectins, subsequently triggering a series of amebic reactions. Among the three subunits of Gal-/GalNAc-inhibitable lectin, heavy subunit and intermediate subunit (Igl) have exhibited lectin activity, but that of Igl remains poorly understood. In this study, we confirmed a carbohydrate-recognition domain (CRD)-like limiting region in E. histolytica Igl and further identified its two core CXXC motifs responsible for carbohydrate recognition. Moreover, the role of Igl's CRD-like region and its CXXC motifs in hemolysis and pathogenic effects was explored. This is the first study to determine an adhesion-related region in E. histolytica Igl protein, providing a new reference direction for subsequent research studies. Since the potential homogeneity of galectin-2 in several mammals and Igl CRD-like region, it could be meaningful to relate the corresponding pathogeneses and phenotypes of these two proteins. Except for adhesion, studies on the involvement of Igl CRD-like region in different parasite-host interactions are also promising., Competing Interests: The authors declare no conflict of interest.

Among sequenced organisms, the genome of Dictyostelium discoideum is unique in that it encodes for a massive amount of repeat-rich sequences in the coding region of genes. This results in the Dictyostelium proteome encoding for thousands of repeat-rich proteins, with nearly 24% of the Dictyostelium proteome encoding Q/N-rich regions that are predicted to be prion like in nature. To begin investigating the role of prion-like proteins in Dictyostelium , we decided to investigate ERF3, the Dictyostelium ortholog of the well-characterized yeast prion protein Sup35. ERF3 lacks the Q/N-rich region required for prion formation in yeast, raising the question of whether this protein aggregates and has prion-like properties in Dictyostelium . Here, we found that ERF3 formed aggregates in response to acute cellular stress. However, unlike bona fide prions, we were unable to detect transmission of aggregates to progeny. We further found that aggregation of this protein is driven by the ordered C-terminal domain independently of the disordered N-terminal domain. Finally, we also observed aggregation of ERF3 under conditions that induce multicellular development, suggesting that this phenomenon may play a role in Dictyostelium development. Together, these findings suggest a role for regulated protein aggregation in Dictyostelium cells under stress and during development.IMPORTANCEPrion-like proteins have both beneficial and deleterious effects on cellular health, and many organisms have evolved distinct mechanisms to regulate the behaviors of these proteins. The social amoeba Dictyostelium discoideum contains the highest proportion of proteins predicted to be prion like and has mechanisms to suppress their aggregation. However, the potential roles and regulation of these proteins remain largely unknown. Here, we demonstrate that aggregation of the Dictyostelium translation termination factor ERF3 is induced by both acute cellular stress and by multicellular development. These findings imply that protein aggregation may have a regulated and functional role in the Dictyostelium stress response and during multicellular development., Competing Interests: The authors declare no conflict of interest.

Eukaryotic cells depend on dynamic changes in shape to fulfill a wide range of cellular functions, maintain essential biological processes, and regulate cellular behavior. The single-celled, predatory ciliate Lacrymaria exhibits extraordinary dynamic shape-shifting using a flexible "neck" that can stretch 7-8 times the length of its body to capture prey. The molecular mechanism behind this morphological change remains a mystery. We have observed that when in an active state, Lacrymaria repeatedly extends and contracts its neck to enable 360-degree space search and prey capture. This remarkable morphological change involves a unique actin-myosin system rather than the Ca 2+ -dependent system found in other contractile ciliates. Two cytoskeletons are identified in the cortex of the Lacrymaria cell, namely the myoneme cytoskeleton and the microtubule cytoskeleton. The myoneme cytoskeleton is composed of centrin-myosin proteins, exhibiting distinct patterns between the neck and body, with their boundary seemingly associated with the position of the macronucleus. A novel giant protein forming a ladder-like structure was discovered as a component of the microtubule cytoskeleton. Thick centrin-myosin fibers are situated very close to the right side of the ladders in the neck but are far away from such structures in the body. This arrangement enables the decoupling of the neck and body. Plasmodium-like unconventional actin has been discovered in Lacrymaria, and this may form highly dynamic short filaments that could attach to the giant protein and myosin, facilitating coordination between the two cytoskeletons in the neck. In summary, this fascinating organism employs unconventional cytoskeletal components to accomplish its extraordinary dynamic shape-shifting., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Kinetochores form the interface between chromosomes and spindle microtubules and are thus under tight control by a complex regulatory circuitry. The Aurora B kinase plays a central role within this circuitry by destabilizing improper kinetochore-microtubule attachments and relaying the attachment status to the spindle assembly checkpoint. Intriguingly, Aurora B is conserved even in kinetoplastids, a group of early-branching eukaryotes which possess a unique set of kinetochore proteins. It remains unclear how their kinetochores are regulated to ensure faithful chromosome segregation. Here, we show in Trypanosoma brucei that Aurora B activity controls the metaphase-to-anaphase transition through phosphorylation of the divergent Bub1-like protein KKT14. Depletion of KKT14 overrides the metaphase arrest resulting from Aurora B inhibition, while expression of non-phosphorylatable KKT14 delays anaphase onset. Finally, we demonstrate that re-targeting Aurora B to the outer kinetochore suffices to promote mitotic exit but causes extensive chromosome missegregation in anaphase. Our results indicate that Aurora B and KKT14 are involved in an unconventional circuitry controlling cell cycle progression in trypanosomes., (© 2024 Ballmer et al.)

Toxoplasma gondii is an obligate intracellular parasite that can infect humans and diverse animals. Fatty acids are critical for the growth and proliferation of T. gondii , which has at least two pathways to synthesize fatty acids, including the type II de novo synthesis pathway in the apicoplast and the elongation pathway in the endoplasmic reticulum (ER). Acetyl-CoA is the key substrate for both fatty acid synthesis pathways. In the apicoplast, acetyl-CoA is mainly provided by the pyruvate dehydrogenase complex. However, how the ER acquires acetyl-CoA is not fully understood. Here, we identified a putative acetyl-CoA transporter (TgAT1) that localized to the ER of T. gondii . Deletion of TgAT1 impaired parasite growth and invasion in vitro and attenuated tachyzoite virulence in vivo . Metabolic tracing using 13 C-acetate found that loss of TgAT1 reduced the incorporation of 13 C into certain fatty acids, suggesting reduced activities of elongation. Truncation of AT1 was previously reported to confer resistance to the antimalarial compound GNF179 in Plasmodium falciparum . Interestingly, GNF179 had much weaker inhibitory effect on Toxoplasma than on Plasmodium . In addition, deletion of AT1 did not affect the susceptibility of Toxoplasma to GNF179, suggesting that this compound might be taken up differently or has different inhibitory mechanisms in these parasites. Together, our data show that TgAT1 has important roles for parasite growth and fatty acid synthesis, but its disruption does not confer GNF179 resistance in T. gondii .

The regulation of gene expression in Plasmodium spp., the causative agents of malaria, relies on precise transcriptional control. Malaria parasites encode a limited repertoire of sequence-specific transcriptional regulators dominated by the apicomplexan APETALA 2 (ApiAP2) protein family. ApiAP2 DNA-binding proteins play critical roles at all stages of the parasite life cycle. Recent studies have provided mechanistic insight into the functional roles of many ApiAP2 proteins. Two major areas that have advanced significantly are the identification of ApiAP2-containing protein complexes and the role of ApiAP2 proteins in malaria parasite sexual development. In this review, we present recent advances on the functional biology of ApiAP2 proteins and their role in regulating gene expression across the blood stages of the parasite life cycle., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Trypanosoma brucei  is a human and animal pathogen that depends on flagellar motility for transmission and infection. The trypanosome flagellum is built around a canonical "9+2" axoneme, containing nine doublet microtubules (DMTs) surrounding two singlet microtubules. Each DMT contains a 13-protofilament A-tubule and a 10-protofilament B-tubule, connected to the A-tubule by a conserved, non-tubulin inner junction (IJ) filament made up of alternating PACRG and FAP20 subunits. Here we investigate FAP20 in procyclic form  T. brucei . A FAP20-NeonGreen fusion protein localized to the axoneme as expected. Surprisingly, FAP20 knockdown led to a catastrophic failure in flagellum assembly and concomitant lethality. This differs from other organisms, where FAP20 is required for normal flagellum motility, but generally dispensable for flagellum assembly and viability. Transmission electron microscopy demonstrates failed flagellum assembly in FAP20 mutants is associated with a range of DMT defects and defective assembly of the paraflagellar rod, a lineage-specific flagellum filament that attaches to DMT 4-7 in trypanosomes. Our studies reveal a lineage-specific requirement for FAP20 in trypanosomes, offering insight into adaptations for flagellum stability and motility in these parasites and highlighting pathogen versus host differences that might be considered for therapeutic intervention in trypanosome diseases., Competing Interests: Conflicts of interest: The authors declare no financial conflict of interest.

The enigmatic haptomonad forms of Leishmania parasites adhere to the sandfly stomodeal valve, damaging this feeding valve and promoting parasite transmission. Yanase et al. recently identified the first parasite proteins involved in adhesion and showed their essentiality in binding to and damaging the valve., Competing Interests: Declaration of interests The author declares no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Peroxisomal protein import has been identified as a valid target in trypanosomiases, an important health threat in Central and South America. The importomer is built of multiple peroxins (Pex) and structural characterization of these proteins facilitates rational inhibitor development. We report crystal structures of the Trypanosoma brucei and T. cruzi tetratricopeptide repeat domain (TPR) of the cytoplasmic peroxisomal targeting signal 1 (PTS1) receptor Pex5. The structure of the TPR domain of TbPex5 represents an apo-form of the receptor which, together with the previously determined structure of the complex of TbPex5 TPR and PTS1 demonstrate significant receptor dynamics associated with signal peptide recognition. The structure of the complex of TPR domain of TcPex5 with PTS1 provided in this study details the molecular interactions that guide signal peptide recognition at the atomic level in the pathogenic species currently perceived as the most relevant among Trypanosoma. Small - angle X - ray scattering (SAXS) data obtained in solution supports the crystallographic findings on the compaction of the TPR domains of TbPex5 and TcPex5 upon interaction with the cargo., Competing Interests: Declaration of competing interest None. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The functional mitochondrion is vital for the propagation of the malaria parasite in the human host. Members of the SPFH protein family, Prohibitins (PHBs), are known to play crucial roles in maintaining mitochondrial homeostasis and cellular functions. Here, we have functionally characterized the homologue of the Plasmodium falciparumProhibitin-2 (PfPhb2) protein. A transgenic parasite line, generated using the selection-linked integration (SLI) strategy for C-terminal tagging, was utilized for cellular localization as well as for inducible knock-down of PfPhb2. We show that PfPhb2 localizes in the parasite mitochondrion during the asexual life cycle. Inducible knock-down of PfPhb2 by GlmS ribozyme caused no significant effect on the growth and multiplication of parasites. However, depletion of PfPhb2 under mitochondrial-specific stress conditions, induced by inhibiting the essential mitochondrial AAA-protease, ClpQ protease, results in enhanced inhibition of parasite growth, mitochondrial ROS production, mitochondrial membrane potential loss and led to mitochondrial fission/fragmentation, ultimately culminating in apoptosis-like cell-death. Further, PfPhb2 depletion renders the parasites more susceptible to mitochondrial targeting drug proguanil. These data suggest the functional involvement of PfPhb2 along with ClpQ protease in stabilization of various mitochondrial proteins to maintain mitochondrial homeostasis and functioning. Overall, we show that PfPhb2 has an anti-apoptotic role in maintaining mitochondrial homeostasis in the parasite., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

The integrity of genomes of the two crucial organelles of the malaria parasite - an apicoplast and mitochondrion in each cell - must be maintained by DNA repair mediated by proteins targeted to these compartments. We explored the localisation and function of Plasmodium falciparum base excision repair (BER) DNA N-glycosylase homologs PfEndoIII and PfOgg1. These N-glycosylases would putatively recognise DNA lesions prior to the action of apurinic/apyrimidinic (AP)-endonucleases. Both Ape1 and Apn1 endonucleases have earlier been shown to function solely in the parasite mitochondrion. Immunofluorescence localisation showed that PfEndoIII was exclusively mitochondrial. PfOgg1 was not seen clearly in mitochondria when expressed as a PfOgg1 leader -GFP fusion, although chromatin immunoprecipitation assays showed that it could interact with both mitochondrial and apicoplast DNA. Recombinant PfEndoIII functioned as a DNA N-glycosylase as well as an AP-lyase on thymine glycol (Tg) lesions. We further studied the importance of Ogg1 in the malaria life cycle using reverse genetic approaches in Plasmodium berghei. Targeted disruption of PbOgg1 resulted in loss of 8-oxo-G specific DNA glycosylase/lyase activity. PbOgg1 knockout did not affect blood, mosquito or liver stage development but caused reduced blood stage infection after inoculation of sporozoites in mice. A significant reduction in erythrocyte infectivity by PbOgg1 knockout hepatic merozoites was also observed, thus showing that PbOgg1 ensures smooth transition from liver to blood stage infection. Our results strengthen the view that the Plasmodium mitochondrial genome is an important site for DNA repair by the BER pathway., (Copyright © 2024 Australian Society for Parasitology. All rights reserved.)

Autophagy regulates the formation of primary cilia, which in turn affects autophagy. The relationship between autophagy and cilia is known to be bidirectional although the specific mechanisms involved have yet to be elucidated. In this study, we found for the first time that ATG8 protein localizes in the basal body of the dorsal kineties and the base of the ventral cirri in Euplotes amieti. ATG8 protein maintains the structural integrity of cilia and plays a role in the construction of the cortical ciliature and microtubule cytoskeleton associated with cilia. ATG8 gene interference leads to the degradation of IFT88, the transport protein in cilia, thus inhibiting the generation of cilia, and affecting the swing of cilia. This influences the swimming speed and cilia pattern, leading to death in Euplotes amieti., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Purpose: Merozoites are the only extracellular form of blood stage parasites, making it a worthwhile target. Multiple invasins that are stored in the merozoite apical organelles, are secreted just prior to invasion, and mediates its interaction with RBC. A comprehensive identification of all these secreted invasins is lacking and this study addresses that gap., Experimental Design: Pf3D7 merozoites were enriched and triggered to discharge apical organelle contents by exposure to ionic conditions mimicking that of blood plasma. The secreted proteins were separated from cellular contents and both the fractions were subjected to proteomic analysis. Also, the identified secreted proteins were subjected to GO, PPI network analysis, and AI-based in silico approach to understand their vaccine candidacy., Results: A total of 63 proteins were identified in the secretory fraction with membrane and apical organellar localization. This includes various MSPs, micronemal EBAs and rhoptry bulb proteins, which play a crucial role in initial and late merozoite attachment, and majority of them qualified as vaccine candidates., Conclusion and Clinical Relevance: We, for the first time, report the secretory repertoire of merozoite and its status for vaccine candidacy. This information can be utilized to develop better invasion blocking multisubunit vaccines, comprising of immunological epitopes from several secreted invasins., (© 2024 Wiley‐VCH GmbH.)

Stress granules (SGs) are stress-induced RNA condensates consisting of stalled initiation complexes resulting from translational inhibition. The biochemical composition and function of SGs are highly diverse, and this diversity has been attributed to different stress conditions, signalling pathways involved and specific cell types. Interestingly, mRNA decay components, which are found in ubiquitous cytoplasmic foci known as processing bodies (PB), have also been identified in SG proteomes. A major challenge in current SG studies is to understand the cause of SG diversity, as well as the function of SG under different stress conditions. Trypanosoma brucei is a single-cellular parasite that causes Human African Trypanosomiasis (sleeping sickness). In this study, we showed that by varying the supply of extracellular carbon sources during starvation, cellular ATP levels changed rapidly, resulting in SGs of different compositions and dynamics. We identified a subset of SG components, which dissociated from the SGs in response to cellular ATP depletion. Using expansion microscopy, we observed sub-granular compartmentalization of PB- and SG-components within the stress granules. Our results highlight the importance of cellular ATP in SG composition and dynamics, providing functional insight to SGs formed under different stress conditions., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Aye et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Visceral leishmaniasis is a life-threatening parasitic disease, but current antileishmanial drugs have severe drawbacks. Antifungal azoles inhibit the activity of cytochrome P450 (CYP) 51 enzymes which are responsible for removing the C14α-methyl group of lanosterol, a key step in ergosterol biosynthesis in Leishmania. However, they exhibit varying degrees of antileishmanial activities in culture, suggesting the existence of unrecognized molecular targets. Our previous study reveals that, in Leishmania, lanosterol undergoes parallel C4- and C14-demethylation to form 4α,14α-dimethylzymosterol and T-MAS, respectively. In the current study, CYP5122A1 is identified as a sterol C4-methyl oxidase that catalyzes the sequential oxidation of lanosterol to form C4-oxidation metabolites. CYP5122A1 is essential for both L. donovani promastigotes in culture and intracellular amastigotes in infected mice. CYP5122A1 overexpression results in growth delay, increased tolerance to stress, and altered expression of lipophosphoglycan and proteophosphoglycan. CYP5122A1 also helps to determine the antileishmanial effect of antifungal azoles in vitro. Dual inhibitors of CYP51 and CYP5122A1 possess superior antileishmanial activity against L. donovani promastigotes whereas CYP51-selective inhibitors have little effect on promastigote growth. Our findings uncover the critical biochemical and biological role of CYP5122A1 in L. donovani and provide an important foundation for developing new antileishmanial drugs by targeting both CYP enzymes., (© 2024. The Author(s).)

Background: Babesia duncani is a pathogen within the phylum Apicomplexa that causes human babesiosis. It poses a significant threat to public health, as it can be transmitted not only through tick bites but also via blood transfusion. Consequently, an understanding of the gene functions of this pathogen is necessary for the development of drugs and vaccines. However, the absence of conditional gene knockdown tools has hindered the research on this pathogen. The auxin-inducible degron (AID) system is a rapid, reversible conditional knockdown system widely used in gene function studies. Thus, there is an urgent need to establish the AID system in B. duncani to study essential gene functions., Methods: The endogenous genes of the Skp1-Cullin-F-box (SCF) complex in B. duncani were identified and confirmed through multiple sequence alignment and conserved domain analysis. The expression of the F-box protein TIR1 from Oryza sativa (OsTIR1) was achieved by constructing a transgenic parasite strain using a homologous recombination strategy. Polymerase chain reaction (PCR), western blot, and indirect immunofluorescence assay (IFA) were used to confirm the correct monoclonal parasite strain. The degradation of enhanced green fluorescent protein (eGFP) tagged with an AID degron was detected through western blot and live-cell fluorescence microscopy after treatment of indole-3-acetic acid (IAA)., Results: In this study, Skp1, Cul1, and Rbx1 of the SCF complex in B. duncani were identified through sequence alignment and domain analysis. A pure BdTIR1 strain with expression of the OsTIR1 gene was constructed through homologous recombination and confirmed. This strain showed no significant differences from the wild type (WT) in terms of growth rate and proportions of different parasite forms. The eGFP tagged with an AID degron was successfully induced for degradation using 500 μM IAA. Grayscale analysis of western blot indicated a 61.3% reduction in eGFP expression levels, while fluorescence intensity analysis showed a 77.5% decrease in fluorescence intensity. Increasing the IAA concentration to 2 mM accelerated eGFP degradation and enhanced the extent of degradation., Conclusions: This study demonstrated the functionality of the AID system in regulating protein levels by inducing rapid degradation of eGFP using IAA, providing an important research tool for studying essential gene functions related to invasion, egress, and virulence of B. duncani. Moreover, it also offers a construction strategy for apicomplexan parasites that have not developed an AID system., (© 2024. The Author(s).)

Babesia spp. are obligate intracellular parasites that invade host cells to complete their asexual development and transmission. Here, we identified a transcription factor AP2-M (BXIN&#95;0799) in Babesia sp. Xinjiang (Bxj), a member of the Apicomplexan AP2 family, which regulates gene expression related to red blood cell (RBC) invasion and cell cycle progression. Our genome-wide analysis of (Cut-Tag) data shows that AP2-M specifically recognized DNA motifs in the promoters of target genes. AP2-M target genes included other AP2 gene family members and epigenetic markers, which could modulate gene expression involved in RBC invasion, merozoite morphology, and cell cycle phases, as indicated by RNA sequencing, proteomics, and single-cell RNA sequencing (scRNA-seq) data from an ap2-m gene disrupted strain (AP2-M (-)). We conclude that AP2-M appeared to contribute to the process of red blood cell invasion, maintain merozoite morphology, and cell cycle progression through GS and MS phases., (© 2024 Federation of American Societies for Experimental Biology.)

Leishmania infantum belongs to the L. donovani complex, which includes species associated with visceral leishmaniasis. Traditionally, antimonial compounds have served as the primary antiparasitic treatment for all clinical forms of leishmaniasis. However, the global spread of resistance to these compounds has posed a significant challenge in the treatment in some regions. In this study, we aimed to investigate resistance to trivalent sodium stibogluconate in vitro using promastigotes from a wild strain of L. infantum. We compared the growth rates and proteomic profiles of wild-type and resistant line conducting label-free quantitative mass spectrometry-based proteomic analyses. Statistical and bioinformatics analyses were employed to evaluate the significance of protein concentration changes, protein identity annotation, GO term analysis, biosynthetic pathways, and protein-protein interactions. Our findings revealed that the resistant line displayed a notable reduction in growth rate. Proteomic data unveiled similar protein concentrations per cell in both groups but with differing molecule copy numbers. We identified 165 proteins with increased concentration, these were associated with transcription and translation activities, lipid metabolism, energy metabolism, and peroxisome biogenesis. In the decreased protein groups were 56 proteins linked to metal acquisition and metabolism, particularly iron. These results suggest a novel perspective on antimonial resistance, highlighting the importance of post-transcriptional and post-translational regulation, alongside energy expenditure compensation and alterations in organelle membrane lipid composition in antimonial-resistant parasites. Overall, our study provides insights into the proteomic profile of stibogluconate-resistant strain, contributing to our general understanding of the complex landscape of antiparasitic resistance in L. infantum. SIGNIFICANCE: Species within the Leishmania donovani complex are implicated in cases of visceral leishmaniasis in the world. Leishmania infantum is a species that predominates in regions spanning the Mediterranean Basin, the Middle East, Central Asia, South and Central America. Antimonials were the first treatment for leishmaniasis, however in the last decades, the resistance has emerged in subregions like India, where it is not a therapeutic option. In contrast, sodium stibogluconate (SbIII) remains the first-line treatment in the Americas. Unfortunately, the emergence of resistance has outpaced the development of new therapeutic options, thereby becoming a critical point in the struggle against the disease. In this study we performed an in-depth proteomic analysis with liquid chromatography mass-mass spectrometry (LC-MS/MS) on L. infantum with Sb-induced resistance in vitro. Results showed a complex proteomic adaptation in the resistant line, involving transcriptional and translational proteins, energy compensation, and homeostasis maintenance. These insights contribute to understanding the molecular adaptation in the parasite and provide information to new investigations related to therapeutics development., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024. Published by Elsevier B.V.)

The cytosolic T-complex protein-1 ring complex (TRiC), also referred as chaperonin containing TCP-1(CCT), comprising eight different subunits stacked in double toroidal rings, binds to around 10 % of newly synthesized polypeptides and facilitates their folding in ATP dependent manner. In Leishmania, among five subunits of TCP1 complex, identified either by transcriptome or by proteome analysis, only LdTCP1γ has been well characterized. It forms biologically active homo-oligomeric complex and plays role in protein folding and parasite survival. Lack of information regarding rest of the TCP1 subunits and its structural configuration laid down the necessity to study individual subunits and their role in parasite pathogenicity. The present study involves the cloning, expression and biochemical characterization of TCP1ε subunit (LdTCP1ε) of Leishmania donovani, the causative agent of visceral leishmaniasis. LdTCP1ε exhibited significant difference in primary structure as compared to LdTCP1γ and was evolutionary close to LdTCP1 zeta subunit. Recombinant protein (rLdTCP1ε) exhibited two major bands of 132 kDa and 240 kDa on native-PAGE that corresponds to the dimeric and tetrameric assembly of the epsilon subunit, which showed the chaperonin activity (ATPase and luciferase refolding activity). LdTCP1ε also displayed an increased expression upto 2.7- and 1.8-fold in the late log phase and stationary phase promastigotes and exhibited majorly vesicular localization. The study, thus for the first time, provides an insight for the presence of highly diverge but functionally active dimeric/tetrameric TCP1 epsilon subunit in Leishmania parasite., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Artemisinin-based combination therapy (ACT) is the mainstay of effective treatment of Plasmodium falciparum malaria. However, the long-term utility of ACTs is imperiled by widespread partial artemisinin resistance in Southeast Asia and its recent emergence in parts of East Africa. This underscores the need to identify chemotypes with new modes of action (MoAs) to circumvent resistance to ACTs. In this study, we characterized the asexual blood stage antiplasmodial activity and resistance mechanisms of LDT-623, a 4-aminoquinoline (4-AQ). We also detected LDT-623 activity against multiple stages (liver schizonts, stage IV-V gametocytes, and ookinetes) of Plasmodium's life cycle, a feature unlike other 4-AQs such as chloroquine (CQ) and piperaquine (PPQ). Using heme fractionation profiling and drug uptake studies in PfCRT-containing proteoliposomes, we observed inhibition of hemozoin formation and PfCRT-mediated transport, which constitute characteristic features of 4-AQs' MoA. We also found minimal cross-resistance to LDT-623 in a panel of mutant pfcrt or pfmdr1 lines, but not the PfCRT F145I mutant that is highly resistant to PPQ resistance yet is very unfit. No P. falciparum parasites were recovered in an in vitro resistance selection study, suggesting a high barrier for resistance to emerge. Finally, a competitive growth assay comprising >50 barcoded parasite lines with mutated resistance mediators or major drug targets found no evidence of cross-resistance. Our findings support further exploration of this promising 4-AQ., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ferreira et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Trypanosomatids are single-celled parasites responsible for human and animal disease. Typically, colonization of an insect host is required for transmission. Stable attachment of parasites to insect tissues via their single flagellum coincides with differentiation and morphological changes. Although attachment is a conserved stage in trypanosomatid life cycles, the molecular mechanisms are not well understood. To study this process, we elaborate upon an in vitro model in which the swimming form of the trypanosomatid Crithidia fasciculata rapidly differentiates following adhesion to artificial substrates. Live imaging of cells transitioning from swimming to attached shows parasites undergoing a defined sequence of events, including an initial adhesion near the base of the flagellum immediately followed by flagellar shortening, cell rounding, and the formation of a hemidesmosome-like attachment plaque between the tip of the shortened flagellum and the substrate. Quantitative proteomics of swimming versus attached parasites suggests differential regulation of cyclic adenosine monophosphate (cAMP)-based signaling proteins. We have localized two of these proteins to the flagellum of swimming C. fasciculata ; however, both are absent from the shortened flagellum of attached cells. Pharmacological inhibition of cAMP phosphodiesterases increased cAMP levels in the cell and prevented attachment. Further, treatment with inhibitor did not affect the growth rate of either swimming or established attached cells, indicating that its effect is limited to a critical window during the early stages of adhesion. These data suggest that cAMP signaling is required for attachment of C. fasciculata and that flagellar signaling domains may be reorganized during differentiation and attachment.IMPORTANCETrypanosomatid parasites cause significant disease burden worldwide and require insect vectors for transmission. In the insect, parasites attach to tissues, sometimes dividing as attached cells or producing motile, infectious forms. The significance and cellular mechanisms of attachment are relatively unexplored. Here, we exploit a model trypanosomatid that attaches robustly to artificial surfaces to better understand this process. This attachment recapitulates that observed in vivo and can be used to define the stages and morphological features of attachment as well as conditions that impact attachment efficiency. We have identified proteins that are enriched in either swimming or attached parasites, supporting a role for the cyclic AMP signaling pathway in the transition from swimming to attached. As this pathway has already been implicated in environmental sensing and developmental transitions in trypanosomatids, our data provide new insights into activities required for parasite survival in their insect hosts., Competing Interests: The authors declare no conflict of interest.

The natural product 9-methoxystrobilurin G (9MG) from Favolaschia spp basidiomycetes is a potent and selective antimalarial. The mechanism of action of 9MG is unknown. We induced 9MG resistance in Plasmodium falciparum 3D7 and Dd2 strains and identified mutations associated with resistance by genome sequencing. All 9MG-resistant clones possessed missense mutations in the cytochrome b (CYTB) gene, a key component of mitochondrial complex III. The mutations map to the quinol oxidation site of CYTB, which is also the target of antimalarials such as atovaquone. In a complementary approach to identify protein targets of 9MG, a photoactivatable derivative of 9MG was synthesized and applied in chemoproteomic-based target profiling. Three components of mitochondrial complex III (QCR7, QCR9, and COX15) were specifically enriched consistent with 9MG targeting CYTB and complex III function in P. falciparum . Inhibition of complex III activity by 9MG was confirmed by ubiquinone cytochrome c reductase assay using P. falciparum extract. The findings from this study may be useful for developing novel antimalarials targeting CYTB.

Unknown factors regulate mitochondrial U-insertion/deletion (U-indel) RNA editing in procyclic-form (PCF) and bloodstream-form (BSF) T. brucei. This editing, directed by anti-sense gRNAs, creates canonical protein-encoding mRNAs and may developmentally control respiration. Canonical editing by gRNAs that specify protein-encoding mRNA sequences occurs amid massive non-canonical editing of unclear sources and biological significance. We found PCF-specific repression at a major early checkpoint in mRNA ND7, involving helicase KREH2-dependent opposite modulation of canonical and non-canonical 'terminator' gRNA utilization. Terminator-programmed editing derails canonical editing and installs proposed repressive structure in 30% of the ND7 transcriptome. BSF-to-PCF differentiation in vitro recreated this negative control. Remarkably, KREH2-RNAi knockdown relieved repression and increased editing progression by reverting canonical/terminator gRNA utilization. ND7 transcripts lacking early terminator-directed editing in PCF exhibited similar negative editing control along the mRNA sequence, suggesting global modulation of gRNA utilization fidelity. The terminator is a 'moonlighting' gRNA also associated with mRNA COX3 canonical editing, so the gRNA transcriptome seems multifunctional. Thus, KREH2 is the first identified repressor in developmental editing control. This and our prior work support a model whereby KREH2 activates or represses editing in a stage and substrate-specific manner. KREH2's novel dual role tunes mitochondrial gene expression in either direction during development., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Cryptosporidium spp. are important zoonotic parasites that can cause moderate to severe diarrhea in humans and animals. Among the three Cryptosporidium species infecting the intestines of calves, Cryptosporidium parvum has a broad host range and causes severe diarrhea in calves, while Cryptosporidium bovis and Cryptosporidium ryanae mainly infect calves without obvious clinical symptoms. Comparative genomic analysis revealed differences in the copy number of genes encoding the nonfinancial disclosure quality (NFDQ) secretory protein family among the three species, suggesting that this protein family may be associated with the host range or pathogenicity of Cryptosporidium spp. To understand the function of cgd8&#95;10 encoded NFDQ1, tagged and knockout strains were constructed and characterized in this study., Methods: To determine the localization of NFDQ1, we used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology to tag the C-terminus of NFDQ1 with three hemagglutinin epitopes (3 × HA). The tagged strain was constructed, and the genomic insertion was confirmed by polymerase chain reaction (PCR). Immunofluorescence assays were performed to observe the localization of NFDQ1 both in extracellular sporozoites and at various intracellular developmental stages. Immunoelectron microscopy was used to study the ultrastructural localization of NFDQ1. Then, the ΔNFDQ1 strain was generated by CRISPR/Cas9 and the in vitro growth assay on HCT-8 cells was used to analyze of phenotypic changes after knockout NFDQ1 in parasites., Results: The NFDQ1 tagging and knockout stains were successfully constructed by CRISPR/Cas9 technology and the insertions of transgenic strains were validated by PCR. The expression of NFDQ1 was validated in parasite by western blot. Immunofluorescence and immune-electron microscopy assay showed that NFDQ1 expressed in both asexual and sexual stages of C. parvum, where it was localized to the cytoplasm of the parasite. Upon ablation of NFDQ1, the ΔNFDQ1 strain showed an apparent growth retardation during sexual replication in vitro., Conclusions: NFDQ1 is a cytoplasmic protein without specific localization to secretory organelles, and it may participate in C. parvum growth during sexual reproduction. Future study should determine the role of NFDQ1 following C. parvum infection in vivo., (© 2024. The Author(s).)

Malaria, caused by Plasmodium parasites, imposes a significant health burden and live-attenuated parasites are being pursued as vaccines. Here, we report on the creation of a genetically attenuated parasite by the deletion of Plasmodium LINUP, encoding a liver stage nuclear protein. In the rodent parasite Plasmodium yoelii, LINUP expression was restricted to liver stage nuclei after the onset of liver stage schizogony. Compared to wildtype P. yoelii, P. yoelii LINUP gene deletion parasites (linup - ) exhibited no phenotype in blood stages and mosquito stages but suffered developmental arrest late in liver stage schizogony with a pronounced defect in exo-erythrocytic merozoite formation. This defect caused severe attenuation of the liver stage-to-blood stage transition and immunization of mice with linup - parasites conferred robust protection against infectious sporozoite challenge. LINUP gene deletion in the human parasite Plasmodium falciparum also caused a severe defect in late liver stage differentiation. Importantly, P. falciparum linup - liver stages completely failed to transition from the liver stage to a viable blood stage infection in a humanized mouse model. These results suggest that P. falciparum LINUP is an ideal target for late liver stage attenuation that can be incorporated into a late liver stage-arresting replication competent whole parasite vaccine., (© 2024. The Author(s).)

Mycobacterium tuberculosis (MTB) is a pathogenic bacterium, belonging to the family Mycobacteriaceae , that causes tuberculosis (TB). Toxoplasma gondii macrophage migration inhibitory factor (TgMIF), a protein homolog of macrophage migration inhibitory factor, has been explored for its potential to modulate immune responses during MTB infections. We observed that TgMIF that interacts with CD74, antizyme inhibitor 1 (AZIN1), and signal transducer and activator of transcription 1 (STAT1) modulates endocytosis, restoration of mitochondrial function, and macrophage polarization, respectively. These interactions promote therapeutic efficacy in mice infected with MTB, thereby presenting a potential route to host-directed therapy development. Furthermore, TgMIF, in combination with first-line TB drugs, significantly inhibited drug-resistant MTB strains, including multidrug-resistant TB. These results demonstrate that TgMIF is potentially a multifaceted therapeutic agent against TB, acting through immune modulation, enhancement of mitochondrial function, and dependent on STAT1 and AZIN1 pathways.

Neutrophils and macrophages confine pathogens by entrapping them in extracellular traps (ETs) through activating TLR9 function. However, plasmodial parasites secreted TatD-like DNases (TatD) to counteract ETs-mediated immune clearance. We found that TLR9 mutant mice increased susceptibility to rodent malaria, suggesting TLR9 is a key protein for host defense. We found that the proportion of neutrophils and macrophages in response to plasmodial parasite infection in the TLR9 mutant mice was significantly reduced compared to that of the WT mice. Importantly, PbTatD can directly bind to the surface TLR9 (sTLR9) on macrophages, which blocking the phosphorylation of mitogen-activated protein kinase and nuclear factor-κB, negatively regulated the signaling of ETs formation by both macrophages and neutrophils. Such, P. berghei TatD is a parasite virulence factor that can inhibit the proliferation of macrophages and neutrophils through directly binding to TLR9 receptors on the cell surface, thereby blocking the activation of the downstream MyD88-NF-kB pathways., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Chinese1 is the predominant Toxoplasma gondii lineage in China, and significant phenotypic differences are observed within the lineage. WH3 and WH6 are two representative strains of Chinese 1, which exhibit divergent virulence and pathogenicity in mice. However, virulence determinants and their modulating mechanisms remain elusive. A global genome expression analysis of the WH3 and WH6 transcriptional profiles identified microneme secretory protein 6 (MIC6), which may be associated with the phenotypic difference observed in WH3. In the present study, the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome-editing technique was used to generate a T. gondii microneme secretory protein (TgMIC6) knockout in WH3. Wild-type mice and different mouse and human cell lines were infected with the WH3, WH3-Δmic6, and WH6 strains. The survival rate of mice, related cytokine levels in serum, and the proliferation of parasites were observed. These results suggested that TgMIC6 is an important effector molecule that determines the differential virulence of WH3 in vivo and in vitro. Furthermore, MIC6 may enhance WH3 virulence via inhibition of host cell autophagy and activation of key molecules in the epidermal growth factor receptor (EGFR)-Akt-mammalian target of rapamycin (mTOR) classical autophagy pathway. CD40L was cleared in vivo by i.p injection of CD40L monoclonal antibody, and it was found that the virulence of WH3-Δmic6 to mice was restored to a certain extent in the absence of CD40L. This study elucidates the virulence determinants and immune escape strategies of Toxoplasma gondii in China. Moreover, these data will aid the development of effective strategies for the prevention and control of toxoplasmosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Inhibition of the lactate transporter PfFNT is a valid novel mode of action against malaria parasites. Current pyridine-substituted pentafluoro-3-hydroxy-pent-2-en-1-ones act as substrate analogs with submicromolar EC 50 in vitro, and >99.7% activity in mice. The recently solved structure of a PfFNT-inhibitor complex visualized the binding mode. Here, we extended the inhibitor layout by series of amine- and anilide-linked pyridine p-substituents to generate additional interactions in the cytoplasmic vestibule. Virtual docking indicated hydrogen bonding to Tyr31 and Ser102. Fluorescence cross-correlation spectroscopy yielded respectively enhanced target affinity. Strikingly, the in vitro activity increased by 1 order of magnitude to 14.8 nM at negligible cytotoxicity. While p -amine substitutions were rapidly metabolized, the more stable p -acetanilide cleared 99.7% of parasites at 4 × 50 mg kg -1 in a mouse malaria model. Future stabilization of the p-substitution against metabolism may translate the gain in in vitro potency to the in vivo situation.

Giardia duodenalis, an important zoonotic protozoan parasite, adheres to host intestinal epithelial cells (IECs) via the ventral disc and causes giardiasis characterized mainly by diarrhea. To date, it remains elusive how excretory-secretory products (ESPs) of Giardia enter IECs and how the cells respond to the entry. Herein, we initially demonstrated that ESPs evoked IEC endocytosis in vitro. We indicated that ESPs contributed vitally in triggering intrinsic apoptosis, pro-inflammatory responses, tight junction (TJ) protein expressional changes, and autophagy in IECs. Endocytosis was further proven to be implicated in those ESPs-triggered IEC responses. Ten predicted virulent excretory-secretory proteins of G. duodenalis were investigated for their capability to activate clathrin/caveolin-mediated endocytosis (CME/CavME) in IECs. Pyridoxamine 5'-phosphate oxidase (PNPO) was confirmed to be an important contributor. PNPO was subsequently verified as a vital promoter in the induction of giardiasis-related IEC apoptosis, inflammation, and TJ protein downregulation. Most importantly, this process seemed to be involved majorly in PNPO-evoked CME pathway, rather than CavME. Collectively, this study identified Giardia ESPs, notably PNPO, as potentially important pathogenic factors during noninvasive infection. It was also noteworthy that ESPs-evoked endocytosis might play a role in triggering giardiasis-inducing cellular regulation. These findings would deepen our understanding about the role of ESPs, notably PNPO, in the pathogenesis of giardiasis and the potential attributed endocytosis mechanism., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Myeloid leukemia factor 1 (Mlf1) was identified as a proto-oncoprotein that affects hematopoietic differentiation in humans. However, its cellular function remains elusive, spanning roles from cell cycle regulation to modulation of protein aggregate formation and participation in ciliogenesis. Given that structurally conserved homologs of Mlf1 can be found across the eukaryotic tree of life, we decided to characterize its cellular role underlying this phenotypic pleiotropy. Using a model of the unicellular eukaryote Giardia intestinalis, we demonstrate that its Mlf1 homolog (GiMlf) mainly localizes to two types of cytosolic foci: microtubular structures, where it interacts with Hsp40, and ubiquitin-rich, membraneless compartments, found adjacent to mitochondrion-related organelles known as mitosomes, containing the 26S proteasome regulatory subunit 4. Upon cellular stress, GiMlf either relocates to the affected compartment or disperses across the cytoplasm, subsequently accumulating into enlarged foci during the recovery phase. In vitro assays suggest that GiMlf can be recruited to membranes through its affinity for signaling phospholipids. Importantly, cytosolic foci diminish in the gimlf knockout strain, which exhibits extensive proteomic changes indicative of compromised proteostasis. Consistent with data from other cellular systems, we propose that Mlf acts in the response to proteotoxic stress by mediating the formation of function-specific foci for protein folding and degradation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Vinopalová et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Naegleria fowleri is the etiological agent of primary amebic meningoencephalitis (PAM), a rapidly progressive acute and fulminant infection that affects the central nervous system, particularly of children and young adults, which has a mortality rate greater than 95%, and its symptomatologic similarity with other meningitis caused by virus or bacteria makes it difficult to make a quick and timely diagnosis that prevents the progression of the infection. It is necessary to know the antigenic determinants as well as the pathogenicity mechanisms of this amoeba to implement strategies that allow for better antiamoebic therapeutic and diagnostic targets that directly impact the health sector. Therefore, the aim of this work was to analyze some virulence factors as part of extracellular vesicle (EV) cargo secreted by N. fowleri. The EV secretion to the extracellular medium was evaluated in trophozoites fixed and incubated with anti-N. fowleri antibody while molecular identification of EV cargo was performed by SDS-PAGE, Western blot, and RT-PCR. Our results showed that N. fowleri secretes a wide variety of vesicle sizes ranging from 0.2 to > 2 μm, and these EVs were recognized by antibodies anti-Naegleropore B, anti-19 kDa polypeptide band, anti-membrane protein Mp2CL5, anti-protease cathepsin B, and anti-actin. Furthermore, these vesicles were localized in the trophozoites cytoplasm or secreted into the extracellular medium. Specifically in relation to small vesicles, our purified exosomes were recognized by CD63 and Hsp70 markers, along with the previously mentioned proteins. RT-PCR analysis was made through the isolation of EVs from N. fowleri trophozoite culture by concentration, filtration, and ultracentrifugation. Interestingly, we obtained PCR products for Nfa1, NPB, Mp2CL5, and CatB genes as part of exosomes cargo. This suggests that the molecules identified in this work could play an important role in communication as well as in infectious processes caused by this amoeba. Therefore, the study and characterization of the pathogenicity mechanisms, as well as the virulence factors released by N. fowleri remains a key point to provide valuable information for the development of therapeutic treatments, vaccine design, or biomarkers for a timely diagnosis against infections caused by protozoa., (© 2024. The Author(s).)