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Leishmaniasis is a neglected tropical disease caused by protozoans of the Leishmania genus, against which no effective treatment or control is available. Like other eukaryotes, parasite telomeres are maintained by telomerase, a ribonucleoprotein complex vital for genome stability. Its protein component, TERT (telomerase reverse transcriptase), presents four structural and functional domains, with the TEN (Telomerase N-terminal) and TRBD (Telomerase RNA-binding) located at its N-terminal. The enzyme also contains an RNA component that carries the template copied by the TERT during telomere elongation. Here, we show that the tertiary structure of Leishmania major TERT (LmTERT) is conserved compared to other eukaryotes. However, the LmTERT N-terminal (LmTERT-NT) portion shows structural changes not detected in the entire protein, mainly in the TEN domain. Besides the disordered elements, the TEN gains two long β-sheets but preserves the GQ motif and the residues in β-sheet 5 that interact with the TRAP motif. In both structures, a linker flanks the TEN and TRBD. The TRBD is partially conserved in both structures and contains the canonical QFP and T motifs, invariant residues, and the putative CP and two trypanosomatid-specific motifs (TSM) besides genus-specific amino acid substitutions. Despite the structural changes, the recombinant LmTERT-NT preserves a hydrophobic cavity that binds specifically and in the picomolar range to the telomeric G-rich DNA and the TER 5' end region. Thus, LmTERT-NT shares the canonical structural domains and motifs and is biochemically active. We discuss the importance of the TERT N-terminal region in the parasite's telomerase catalysis., (Copyright © 2025 Elsevier Inc. All rights reserved.)

Eukaryotic chromosomes segregate faithfully prior to nuclear division to ensure genome stability. If segregation becomes defective, the chromosome copy number of the cell may alter leading to aneuploidy and/or polyploidy, both common hallmarks of cancers. In eukaryotes, aurora kinases regulate chromosome segregation during mitosis and meiosis, but their functions in the divergent, single-celled eukaryotic pathogen Trypanosoma brucei are less understood. Here, we focused on one of three aurora kinases in these parasites, TbAUK3, a homologue of the human aurora kinase AURKC, whose functions are primarily restricted to meiosis. We show that RNAi targeted depletion of TbAUK3 correlates with nuclear segregation defects, reduced proliferation, and decreased DNA synthesis, suggestive of a role for TbAUK3 during mitotic, not meiotic, chromosome segregation. Moreover, we uncover a putative role for TbAUK3 during the parasite's response to DNA damage since we show that depletion of TbAUK3 enhances DNA instability and sensitivity to genotoxic agents., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Alexis Iskantar reports a relationship with Qiagen that includes: employment. 1) Corresponding author is an editor for Molecular and Biochemical Parasitology - Prof. Dr. Richard McCulloch 2) Co-author is an employee of Qiagen at present. Qiagen played no role in this study nor was the co-author an employee of Qiagen when the study was underaken- Mr. Alexis Iskandar If there are other authors, they 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 © 2025 The Authors. Published by Elsevier B.V. All rights reserved.)

Giardia duodenalis causes giardiasis in humans, companion, livestock and wild animals. Control of infection involves drugs as benzimidazoles (e.g., albendazole, ABZ) and 5-nitroheterocyclics [5-NHs: metronidazole (MTZ), furazolidone (FZD), nitazoxanide (NTZ)] as first-line agents. During infection, Giardia is exposed to immune and pro-oxidant host responses involving nitric oxide (NO). In Giardia, NO is detoxified by a flavohemoglobin (gFlHb), a heme-containing enzyme which is absent in mammals. gFlHb has NO dioxygenase and NADH oxidase activities converting NO into nitrate and producing a superoxide anion (O 2 •- ) that causes oxidative stress and parasite death. The modulation of gFlHb activities may provide novel approaches for treatment of giardiasis. We investigated the capacity of selected benzimidazole-2-carbamates (BZCs: ABZ, oxibendazole, nocodazole), non-BZCs (thiabendazole), an ehtylphenylcarbamate (LQM-996) and 5-NHs (MTZ, NTZ, FZD and some derivatives) to bind to recombinant gFlHb at the heme group, modifying NADH consumption activity and/or inducing ROS production. Of these, BZCs and NTZ bind to heme and increased O 2 •- production (i.e. caused enzyme subversion), whereas MTZ binds to heme but inhibited NADH consumption. LQM-996 decreased NADH consumption and two out of four NTZ derivatives altered NADH oxidase activity. In silico docking and molecular dynamics studies suggested the interaction of distinct drug moieties in ABZ and NTZ with gFlHb sites involved in NADH and NO catalysis. These findings provide new insights on gFlHb as a novel target of BZCs, MTZ and NTZ, and provides a useful platform to assess the compounds binding capacity to gFlHb prior to experimental and clinical trials in giardiasis., Competing Interests: Conflict of interest statement The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Reversible transformation of bovine leukocytes by the intracellular parasites Theileria annulata and Theileria parva is central to pathogenesis of the diseases they cause, tropical theileriosis and East Coast Fever, respectively. Parasite-dependent constitutive activation of major host transcription factors such as AP-1 (Activating Protein 1) and NF-κB (Nuclear Factor-Kappa B) sustains the transformed state. Although parasite interaction with host cell signaling pathways upstream of AP-1 have been studied, the precise contribution of Theileria encoded factors capable of modulating AP-1 transcriptional activity, and other infection-altered signaling pathways is not fully understood. We previously showed that the Ta9 protein from T. annulata (TA15705) is secreted into the host cell cytoplasm and contributes to infection-induced AP-1 transcriptional activity. The current study employed RNA-seq to investigate the ability of ectopically expressed Ta9 to modulate the gene transcription profile of a bovine macrophage cell line, BoMac. RNA-seq identified 560 (400 upregulated and 160 downregulated) differentially expressed genes. KEGG analysis predicted a high number of upregulated genes associated with carcinogenesis such as CCND1, CDKN1A, ETV4, ETV5, FLI1, FRA1, GLI2, GRO1, HCK, IL7R, MYBL1, MYCN, PIM1 and TAL1. Ta9 introduction also affected genes associated with proinflammatory processes such as cytokines, chemokines, growth factors and metalloproteinases. Enrichment analysis of differentially expressed genes revealed that Ta9 is potentially involved in activating other host cell signaling pathways in addition to those that lead to induction of AP-1. Comparing our data with data on differentially expressed BoMac genes modulated by the secreted TashAT2 factor of T. annulata identified the gene encoding the tyrosine protein kinase hematopoietic cell kinase (HCK) as common to both data sets. HCK is essential for the proliferation of T. parva-transformed B cells and herein, we demonstrate that enzymatic activity of HCK is also essential for T. annulata- and T. lestoquardi-transformed macrophage proliferation., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Shahin Tajeri reports article publishing charges was provided by Free University of Berlin. Shahin Tajeri reports a relationship with Free University of Berlin that includes: employment. If there are other authors, they 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 Ltd.. All rights reserved.)

UBC13 is an orthologue of Homo sapiens ubiquitin-conjugation E2 enzymes described in Leishmania mexicana, a null mutant lacking this gene cannot be produced, suggesting essential functions in this parasite. Leishmania infantum is an etiological agent of visceral leishmaniasis, the most severe type of disease that is potentially fatal if untreated. The ubiquitination process has been targeted for leishmanicidal compounds, indicating its essential function in parasite homeostasis. Therefore, the molecular characterization of the ubiquitination process may provide a better understanding of the molecular and cellular basis of leishmaniasis. Here, we characterized the gene LINF_350017900 in Leishmania infantum, which was named LinfUBC13, an E2 orthologue of UBC13 in Leishmania mexicana and the UBE2D family in Homo sapiens, sharing 72-74 % identity with UBE2D1, UBE2D2, and UBE2D3. LinfUbc13 contains conserved catalytic residues, including Cys86 and the HPN motif, which are essential for ubiquitin-conjugating activity. Structural analysis revealed a high similarity between LinfUbc13 and human UBE2D proteins, with a root-mean-square deviation (RMSD) of 0.4 Å, suggesting conserved functions. Recombinant LinfUbc13 was expressed and shown to accept ubiquitin from E1, forming a thioester intermediate. Functional assays demonstrated that LinfUbc13 transfers ubiquitin to p53 through human HDM2 E3 ligase, confirming its role in ubiquitination. Subcellular localization showed that LinfUbc13 was distributed throughout the parasite cytoplasm. These findings highlight the conserved nature of the ubiquitin-proteasome system between Leishmania infantum and Homo sapiens, showing that LinfUbc13 is an E2 enzyme that plays a crucial role in parasitic development., Competing Interests: Competing interests The authors have declared that no competing interests exist., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Fibrillarin is a key nucleolar S-adenosyl-L-methionine (SAM)-dependent methyltransferase, highly conserved among species in both sequence and function. It is actively involved in numerous cellular processes, particularly in the early stages of pre-ribosomal RNA processing. Although a fibrillarin orthologue has been identified in Giardia, an intestinal protozoan parasite responsible for numerous infections worldwide, its functional and structural features in this parasite remain largely unexplored. In this study, bioinformatics tools were used to analyze the sequence and structure of Giardia fibrillarin to provide insights that could aid future experimental and therapeutic investigations. Taking advantage of the protein sequence of Giardia fibrillarin, multiple bioinformatics tools were employed to estimate its domains, nuclear and nucleolar localization signals (NLS and NoLS), post-translational modifications, phylogeny, and three-dimensional structure. The analysis revealed significant conservation of Giardia fibrillarin, showing close relationships with archaeal and parasitic amoeba orthologs. It retains essential features such as the SAM-dependent methyltransferase domain, a glycine- and arginine-rich domain, and both NLS and NoLS. While the core region of the protein is structurally conserved, the N-terminal region exhibits notable divergence. The potential for inhibition of Giardia fibrillarin by SAM competitors suggests that it could be a promising target for drug development, particularly for strains resistant to current treatments., Competing Interests: Declarations. Ethical approval: Not applicable. Consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The author declares no competing interests., (© 2024. The Author(s).)

Leishmania spp. are intracellular protozoan parasites causative of visceral and cutaneous leishmaniasis. Recognized as a neglected tropical disease affecting millions of people around the world, this affliction represents a major public health problem. In Brazil, pentavalent antimony (Sb V ), the main therapy used to treat all clinical forms of leishmaniasis, has become increasingly associated with treatment failure. Many factors can influence leishmaniasis treatment outcome, including low expression aquaglyceroporin by the parasite and high activity of the ATP-binding cassette (ABC) transporters, efflux pumps whose activity has been associated with drug resistance in a variety of diseases. Current evidence suggests that some ABC transporters (e.g., MRP1 and MDR1) play a role in drug resistance in leishmaniasis. One way to potentially overcome Sb V resistance may be a combined therapeutic strategy involving anti-Leishmania drugs administered together with ABC transporter inhibitors; however, toxicity poses a major challenge to the adoption of this approach., Competing Interests: Declarations 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 Ltd.. All rights reserved.)

Toxoplasma gondii is an apicomplexan parasite infecting all mammals including humans and causes toxoplasmosis. There is no vaccine available for humans and thus vaccine development efforts continue using novel antigens and/or vaccine platforms. Since our previous microarray screening study showed that ROP6 is a suitable antigen to be used in vaccine studies, in this study, we aimed to design an optimized mRNA construct encoding the ROP6 protein and then demonstrate its efficiency and immunogenicity using in vitro methods. For this, we constructed a pT7CFE1-Chis/ROP6 vector encoding optimized ROP6 mRNA containing EMCV 5'UTR with IRES and a 20 nucleotides fragment from alpha globin 3' UTR. Then, we generated the optimized ROP6 mRNAs with anti-reverse cap analogue (ARCA) and approximately 150 nucleotide long poly-A tail. Next, HEK293T cells were transfected with the optimized ROP6 mRNAs to show recombinant ROP6 protein expression capability. Moreover, we expressed in vitro recombinant ROP6 protein in HeLa cell lysate using the pT7CFE1-Chis/ROP6 vector to reveal the immunogenicity of recombinant ROP6 protein using sera samples collected from mice infected with PRU strain of T. gondii. The IFA and Western blot results showed that the optimized ROP6 mRNAs successfully expressed the recombinant ROP6 protein in HEK293T cells. Moreover the recombinant ROP6 protein expressed in HeLa cell lysate strongly reacted with sera samples collected from mice. The absorbance difference detected among positive and negative mice serum samples analyzed was statistically significant, indicating that the recombinant ROP6 protein was immunogenic (P = 0.0003). In conclusion, this study demonstrated that the optimized ROP6 mRNAs can be used in the development of mRNA vaccines against 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.)

Background: The limited efficacy of the two recently approved malaria vaccines, RTS,S/AS01 and R21/Matrix- M™, highlights the need for alternative vaccine candidate genes. Plasmodium falciparum Reticulocyte Binding Protein Homologue 5 (Pfrh5) is a promising malaria vaccine candidate, given its limited polymorphism, its essential role in parasite survival, a lack of immune selection pressure and higher efficacy against multiple parasites strains. This study evaluated the genetic diversity of Pfrh5 gene among parasites from regions with varying malaria transmission intensities in Mainland Tanzania, to generate baseline data for this potential malaria vaccine candidate., Methods: This study utilized secondary data of 697 whole-genome sequences which were generated by the MalariaGEN Community Network. The samples which were sequenced to generated the data were collected between 2010 and 2015 from five districts within five regions of Mainland Tanzania, with varying endemicities (Morogoro-urban district in Morogoro region, Muheza in Tanga, Kigoma-Ujiji in Kigoma, Muleba in Kagera, and Nachingwea district in Lindi region). Wright's fixation index (F ST ), Wright's inbreeding coefficient (Fws), Principal component analysis (PCA), nucleotide diversity (π), haplotype network, haplotype diversity (Hd), Tajima's D, and Linkage disequilibrium (LD) were used to assess the diversity of the gene., Results: Of the sequences used in this study, 84.5% (n = 589/697) passed quality control and 313 (53.1%) were monoclonal (contained infections from a single strain of P. falciparum) and were used for haplotype diversity and haplotype network analysis. High within-host diversity (Fws < 0.95) was reported in Kigoma-Ujiji (60.7%), Morogoro-urban (53.1%), and Nachingwea (50.8%), while Muleba (53.9%) and Muheza (61.6%) had low within-host diversity (Fws ≥ 0.95). PCA did not show any population structure and the mean F ST value was 0.015. Low nucleotide diversity values were observed across the study sites (mean π = 0.00056). A total of 27 haplotypes were observed among the 313 monoclonal samples and under-fives exhibited higher haplotype counts. The Pf3D7 was detected as Hap&#95;1, which occurred in 16/313 (5.1%) monoclonal sequences. Negative Tajima's D values were observed among the parasite populations in all the study sites., Conclusion: Low levels of polymorphism in the pfrh5 gene were observed based on low nucleotide and haplotype diversity, a lack of population structure and negative Tajima's D values. This study provides essential data on the diversity of the Pfrh5 gene indicating that it can be considered in the development of the next generation malaria vaccines. Robust and intensive studies of this and other candidate genes are crucial to support the prioritization of the Pfrh5 gene for potential inclusion in a broadly cross-protective malaria vaccine., Competing Interests: Declarations. Ethical approval and consent to participat: The study utilized sequence data downloaded from the open-source MalariaGEN database ( https://www.malariagen.net/resource/34 ). The study protocols for the projects that collected the samples were reviewed and approved by the Tanzanian Medical Research Coordinating Committee (MRCC) of NIMR. Permission to conduct the study in Morogoro-urban-Morogoro, Muheza– Tanga, Muleba – Kagera, Nachingwea -Lindi and Kigoma-Ujiji- Kigoma was sought in writing and obtained from the district and regional medical officers. Written informed consent was obtained from patients or parents/guardians in the case of children. Appropriate information (about the study and the protocol/methods) in a language that was understood by the parents/guardians of the study patients was compiled and provided before consent was obtained. Permission to publish was obtained from the Director General of NIMR. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Toxoplasmosis is a significant zoonotic disease that poses a serious threat to both human and animal health. Despite ongoing research, developing an effective vaccine for toxoplasmosis remains a challenge. In this study, we evaluated the vaccine potential of the Toxoplasma Urm1 gene deletion mutant (PruΔUrm1) by assessing its pathogenicity and protective efficacy in mice. Using CRISPR/Cas9 technology, we successfully created a type II Toxoplasma gondii Pru mutant strain with a deleted Urm1 gene. Compared to the wild-type parasite, the PruΔUrm1 strain exhibited significantly reduced invasive and proliferative abilities in vitro. In in vivo studies, mice intraperitoneally infected with the parental Pru strain showed severe symptoms including emaciation, hunching, and high mortality rates. In contrast, mice infected with PruΔUrm1 tachyzoites demonstrated a 100 % survival rate, no overt symptoms, and a markedly reduced parasite burden in the liver, spleen, and lungs, indicating reduced pathogenicity. Notably, PruΔUrm1 vaccination triggered a strong immune response, characterized by significantly elevated cytokine levels, including TNF-α, IFN-γ and IL-10. Furthermore, we assessed the immunoprotective efficacy of PruΔUrm1 vaccination in mice against type I strains. Mice immunized with PruΔUrm1 were able to resist the tachyzoites of type I RH wild-type parasites, achieving a 100 % survival rate and significantly reduced parasite loads in the liver, spleen and lungs. These data demonstrate that PruΔUrm1 immunization provides effective protection against acute Toxoplasma infections and holds promise as a potential vaccine candidate for 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 Ltd. All rights reserved.)

Chagas disease is a chronic infection caused by the protozoan parasite, Trypanosoma cruzi, with limited benefits of the currently available anti-parasitic chemotherapeutic approaches to halt the progression of heart disease. Recombinant TSA-1-C4 and Tc24-C4 proteins have been developed as promising antigen candidates for therapeutic vaccines, leading to propose them in combination as a bivalent recombinant protein strategy. In this study, we evaluated the immunomodulatory effect of the combined TSA-1-C4 and Tc24-C4 recombinant proteins by in vitro assays using murine macrophages. Macrophages from naïve Balb/c mice were isolated and stimulated with TSA-1-C4 plus Tc24-C4 recombinant proteins, hence, supernatants were recovered to measure host NO, H 2 O 2 , as well as, TNF-α, IL-1β, IL-6 and IL-10 cytokine responses. Later, stimulated macrophages were co-cultured with CD8 + T cells from naïve mice, and inflammatory cytokine-profiles were measured from supernatants. We observed that combining both antigens promotes the activation of host macrophages by NO and H 2 O 2 release; moreover, these macrophages also induced considerable pro-inflammatory immune-responses mediated by TNF-, IL-1β and IL-6 cytokines compared to either TSA-1-C4 or Tc24-C4 stimulated macrophages. In addition, naïve CD8 + T cells in presence of TSA-1-C4 plus Tc24-C4 stimulated-macrophages similarly boosted the pro-inflammatory immune profile by significant production of IFN-γ and TNF-α cytokines. These results support immunological advantages for the use of TSA-1-C4 and Tc24-C4 combination as vaccine candidates against T. cruzi., Competing Interests: Declarations. Ethics approval: All studies were approved by the institutional bioethics committee of the “Centro de Investigaciones Regionales Dr. Hideyo Noguchi,”, Universidad Autónoma de Yucatán (Reference #CEI-08-2019) and were maintained in accordance with the principles and guidelines of National Institutes of Health Guide for Treatment and Care for Laboratory Animals and by the Mexican Official Norm for Animal Care and Handing,NOM‐062‐ZOO‐1999. Consent to participate: Not applicable. Conflict of interests: The authors declare no competing interests., (© 2025. The Author(s).)

DNA N6-methyladenine (6mA) is a potential epigenetic mark involved in gene transcription in eukaryotes, yet the regulatory mechanism governing its methyltransferase (MTase) activity remains obscure. Here, we exploited the 6mA MTase AMT1 to elucidate its auto-regulation in the unicellular eukaryote Tetrahymena thermophila. The detailed endogenous localization of AMT1 in vegetative and sexual stages revealed a correlation between the 6mA reestablishment in the new MAC and the occurrence of zygotically expressed AMT1. Catalytically inactive AMT1 reduced 6mA level on the AMT1 gene and its expression, suggesting that AMT1 modulated its own transcription via 6mA. Furthermore, AMT1-dependent 6mA regulated the transcription of its target genes, thereby affecting cell fitness. Our findings unveil a positive feedback loop of transcriptional activation on the AMT1 gene and highlight the crucial role of AMT1-dependent 6mA in gene transcription., (© The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Leishmania spp. cause a wide range of human diseases, localized skin lesions, mucocutaneous and visceral infections. In the present study, the aim was to investigate the potential role of sanguinarine as a specific inhibitor of Leishmania PP2C that can induce apoptosis in the parasite. The results demonstrated that sanguinarine inhibits, in a dose-dependent mode at 72 h, the growth and phosphatase activity of both Leishmania major and Leishmania mexicana promastigotes. Therefore, all assays were performed from this time period onwards. TUNEL assay was used to identify apoptosis and indicated apoptosis in L. major and L. mexicana promastigotes. Similarly, Western blot assay showed that PARP, a DNA damage indicator molecule, was present in L. major and L. mexicana promastigotes incubated with the inhibitor. In addition, differential expression of the proapoptotic protein Bax and the antiapoptotic protein Bcl-2 was observed in both Leishmania species. Finally, the protein phosphatase PP2C expression was not affected, whereas p38 MAPK phosphorylation was increased in L. major promastigotes than in L. mexicana promastigotes. Therefore, sanguinarine proved to be an inhibitor of the growth and PP2C enzymatic activity of L. major and L. mexicana promastigotes, and with it, this inhibition induced apoptosis., Competing Interests: Declarations. Competing Interests: The authors declare no competing interests., (© 2024. The Author(s).)

Despite the significant understanding of phase separation in proteins with intrinsically disordered regions, a considerable percentage of proteins without such regions also undergo phase separation, presenting an intriguing area for ongoing research across all kingdoms of life. Using a combination of spectroscopic and microscopic techniques, we report here for the first time that a low temperature and low pH can trigger the liquid-liquid phase separation (LLPS) of a parasitic protein, kinetoplastid membrane protein-11 (KMP-11). Electrostatic and hydrophobic forces are found to be essential for the formation and stability of phase-separated protein assemblies. We show further that the increase in the ionic strength beyond a threshold decreases the interchain electrostatic interactions acting between the alternate charged blocks, altering the propensity for phase separation. More interestingly, the addition of cholesterol inhibits LLPS by engaging the cholesterol recognition amino acid consensus (CRAC)-like domains present in the protein. This was further confirmed using a CRAC-deleted mutant with perturbed cholesterol binding, which did not undergo LLPS.

Background: Malaria remains a leading cause of death worldwide, claiming over 600,000 lives each year. Over 90% of these deaths, mostly among children under 5 years, occur in sub-Saharan Africa and are caused by Plasmodium falciparum. The merozoites stage of the parasite, crucial for asexual development invade erythrocytes through ligand-receptor interactions. Erythrocyte binding antigen (EBA)-175 is one of the key ligands facilitating invasion via interaction with glycoprotein A (GpA) receptors on the erythrocytes. EBA-175 is known to exist in two dimorphic allelic (F and C) forms with each found to infer different virulence. There is paucity of data on the prevalence of these alleles and their epidemiology in the Ghanaian malaria landscape and hence this study., Methods: Parasite gDNA was extracted from archived Dried Blood Spots (DBS) prepared from 700 confirmed malaria-infected individuals and analysed for P. falciparum EBA-175 dimorphism. Selective eba-175 gene amplification via nested PCR and allele scoring using agarose gel electrophoresis for F, C and F/C alleles., Results: Of the total 632 successfully genotyped samples, prevalence of F, C, and F/C allelic forms were 61.2% (n = 387), 20.7% (n = 131), and 18.0% (n = 114), respectively. Seasonality analysis did not reveal a statistically significant difference in the prevalence of dimorphic forms between the wet (n = 475) and dry (n = 157) seasons (p = 0.051). The prevalence ratio (wet/dry) for C, F and F/C were determined to be 1.0, 1.1 and 1.4, respectively. Between 2019 and 2022, the prevalence of the alleles changed significantly (χ 2  = 6.5427, p = 0.03). Geometric mean parasite density for the C, F, and F/C alleles were 21,477.1 [95%CI 15,749.2 - 29,288.1], 18,308.0 [95%CI 15,149.9-22,124.5] and 22,690.4[95% CI 16,891.9-30,479.2], respectively., Conclusion: The F-allele was the most prevalent form across all age groups, followed by the C allele and mixed F/C alleles. No significant difference in allele prevalence was observed between the high malaria season (wet) and low malaria season (dry). However, a statistically significant difference in the temporal prevalence of pure alleles (F & C) between two time points was observed. The current study adds to the existing body of knowledge on eba-175 allelic dimorphism and highlights the co-circulation of alleles in high malaria endemic areas in Ghana., Competing Interests: Declarations. Ethics approval and consent to participate: Scientific and ethical approval for the use of human subjects was obtained from the Ghana Health Service Ethics Review Committee (GHS-ERC 005/12/17). All participants provided informed consent prior to inclusion in the study. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

One of the most extensive forms of natural genome editing occurs in ciliates, a group of microbial eukaryotes. Ciliate germline and somatic genomes are contained in distinct nuclei within the same cell. During the massive reorganization process of somatic genome development, ciliates eliminate tens of thousands of DNA sequences from a germline genome copy. Recently, we showed that the chromatin remodeler ISWI1 is required for somatic genome development in the ciliate Paramecium tetraurelia Here, we describe two high similarity paralogous proteins, ICOPa and ICOPb, essential for their genome editing. ICOPa and ICOPb are highly divergent from known proteins; the only domain detected showed distant homology with the WSD (WHIM2 + WHIM3) motif. We show that both ICOPa and ICOPb interact with the chromatin remodeler ISWI1. Upon ICOP knockdown, changes in alternative DNA excision boundaries and nucleosome densities are similar to those observed for ISWI1 knockdown. We thus propose that a complex comprising ISWI1 and either or both ICOPa and ICOPb are needed for Paramecium 's precise genome editing., (© 2025 Singh et al.; Published by Cold Spring Harbor Laboratory Press.)

Uridine insertion/deletion (U-indel) RNA editing of mitochondrial transcripts is a posttranscriptional modification in kinetoplastid organisms, resulting in the generation of mature mRNAs from cryptic precursors. This RNA editing process involves a multiprotein complex holoenzyme and multiple accessory factors. Recent investigations have highlighted the pivotal involvement of accessory RNA-binding proteins (RBPs) in modulating RNA editing in Trypanosoma brucei , often in a transcript-specific manner. DRBD18 is a multifunctional RBP that reportedly impacts the stability, processing, export, and translation of nuclear-encoded mRNAs. However, mass spectrometry studies report DRBD18-RESC interactions, prompting us to investigate its role in mitochondrial U-indel RNA editing. In this study, we demonstrate the specific and RNase-sensitive interaction of DRBD18 with multiple RESC factors. Depletion of DRBD18 through RNA interference in procyclic form T. brucei leads to a significant reduction in the levels of edited A6 and COIII mitochondrial transcripts, whereas its overexpression causes a notable increase in the abundance of these edited mRNAs. RNA immunoprecipitation/qRT-PCR analysis indicates a direct role for DRBD18 in A6 and COIII mRNA editing. We also examined the impact of arginine methylation of DRBD18 in the editing process, revealing that the hypomethylated form of DRBD18, rather than the arginine-methylated version, is essential for promoting these editing events. In conclusion, our findings demonstrate that DRBD18 directly affects the editing of A6 and COIII mRNAs, with its function being modulated by its arginine methylation status, marking the first report of a mitochondrial function for this protein and identifying it as a newly characterized RNA editing auxiliary factor., (© 2025 Pandey et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

The evolution of genetic diversity and population structure of Plasmodium vivax as malaria elimination approaches remains unclear. This study analyzed the genetic variation and molecular epidemiology of P. vivax from Yala Province in southern Thailand, an area in the pre-elimination phase. Seventy P. vivax isolates, collected between 2017 and 2020, were genotyped for domain II of pvdbp and the 42-kDa region of pvmsp1 using amplicon deep sequencing. Data from Yala province were compared to published data from Tak province, where transmission was higher. Key analyses included nucleotide diversity (π), haplotype diversity (Hd), natural selection, recombination rates, and complexity of infection (COI). Genetic diversity in Yala was relatively low (π = 0.008 dbp and 0.014 msp1 ; Hd = 0.774 dbp and 0.407 msp1 ) compared to Tak (π = 0.012 dbp and 0.027 msp1 ; Hd = 0.849 dbp and 0.962 msp1 ). In Yala, polyclonal infections were found in 53.7% of pvdbp II and 47.8% of pvmsp1 42 isolates, with average COI of 1.6 and 1.7. Both genes were under balancing selection. Distinct genetic differences were found between Yala and Tak in pvmsp1 42 , providing a local genotypic profile useful for tracing parasite origins., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Tubulin detyrosination has been implicated in various human disorders and is important for regulating microtubule dynamics. While in most organisms this modification is restricted to α-tubulin, in trypanosomatid parasites, it occurs on both α- and β-tubulin. Here, we show that in Leishmania , a single vasohibin (LmVASH) enzyme is responsible for differential kinetics of α- and β-tubulin detyrosination. LmVASH knockout parasites, which are completely devoid of detyrosination, show decreased levels of glutamylation and exhibit a strongly diminished pathogenicity in mice, correlating with decreased proliferation in macrophages. Reduced virulence is associated with altered morphogenesis and flagellum remodeling in detyrosination-deficient amastigotes. Flagellum shortening in the absence of detyrosination is caused by hyperactivity of a microtubule-depolymerizing Kinesin-13 homolog, demonstrating its function as a key reader of the trypanosomatid-tubulin code. Taken together, our work establishes the importance of tubulin detyrosination in remodeling the microtubule-based cytoskeleton required for efficient proliferation in the mammalian host. This highlights tubulin detyrosination as a potential target for therapeutic action against leishmaniasis., Competing Interests: Competing interests statement:The authors declare no competing interest.

Diverse eukaryotic cells assemble microtubule networks that vary in structure and composition. While we understand how cells build microtubule networks with specialized functions, we do not know how microtubule networks diversify across deep evolutionary timescales. This problem has remained unresolved because most organisms use shared pools of tubulins for multiple networks, making it difficult to trace the evolution of any single network. In contrast, the amoeboflagellate Naegleria expresses distinct tubulin genes to build distinct microtubule networks: while Naegleria builds flagella from conserved tubulins during differentiation, it uses divergent tubulins to build its mitotic spindle. This genetic separation makes for an internally controlled system to study independent microtubule networks in a single organismal and genomic context. To explore the evolution of these microtubule networks, we identified conserved microtubule-binding proteins and used transcriptional profiling of mitosis and differentiation to determine which are upregulated during the assembly of each network. Surprisingly, most microtubule-binding proteins are upregulated during only one process, suggesting that Naegleria uses distinct component pools to specialize its microtubule networks. Furthermore, the divergent residues of mitotic tubulins tend to fall within the binding sites of differentiation-specific microtubule regulators, suggesting that interactions between microtubules and their binding proteins constrain tubulin sequence diversification. We therefore propose a model for cytoskeletal evolution in which pools of microtubule network components constrain and guide the diversification of the entire network, so that the evolution of tubulin is inextricably linked to that of its binding partners., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mitochondrial ribosomes (mitoribosomes) are essential, and their function of synthesising mitochondrial proteins is universal. The core of almost all mitoribosomes is formed from a small number of long and self-folding rRNA molecules. In contrast, the mitoribosome of the apicomplexan parasite Toxoplasma gondii assembles from over 50 extremely short rRNA molecules. Here, we use cryo-EM to discover the features that enable this unusual mitoribosome to perform its function. We reveal that poly-A tails added to rRNA molecules are integrated into the ribosome, and we demonstrate their essentiality for mitoribosome formation and for parasite survival. This is a distinct function for poly-A tails, which are otherwise known primarily as stabilisers of messenger RNAs. Furthermore, while ribosomes typically consist of unique rRNA sequences, here nine sequences are used twice, each copy integrated in a different mitoribosome domain, revealing one of the mechanisms enabling the extreme mitochondrial genome reduction characteristic to Apicomplexa and to a large group of related microbial eukaryotes. Finally, several transcription factor-like proteins are repurposed to compensate for reduced or lost critical ribosomal domains, including members of the ApiAP2 family thus far considered to be DNA-binding transcription factors., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Background: To understand the emergence and spread of drug-resistant parasites in malaria-endemic areas, accurate assessment and monitoring of antimalarial drug resistance markers is critical. Recent advances in next-generation sequencing (NGS) technologies have enabled the tracking of drug-resistant malaria parasites., Methods: In this study, we used Targeted Amplicon Deep Sequencing (TADS) to characterise the genetic diversity of the Pfk13, Pfdhfr, Pfdhps, and Pfmdr1 genes among primary school-going children in 15 counties in Kenya (Bungoma, Busia, Homa Bay, Migori, Kakamega, Kilifi, Kirinyaga, Kisii, Kisumu, Kwale, Siaya, Tana River, Turkana, Vihiga and West Pokot). A total of 920 dried blood spot (DBS) samples collected from 121 selected primary schools within the country were used to extract genomic DNA. A nested polymerase chain reaction (PCR) was used to generate amplicons that were sequenced to determine the prevalence of known and novel polymorphisms., Results: Pfk13 mutations associated with artemisinin resistance were present as mixed genotype infections for the C469Y mutation in 23 samples (4%), the A675V mutation in 2 samples (1.7%), and the P553L mutation in 7 samples (1.2%). The A578S mutation, was also identified in mixed infections, appearing in 15.2% of the 87 samples analysed. The Pfdhfr 51I and 108 N pyrimethamine-resistance mutations were at fixation (100% frequency), and the Pfmdr1 Y184F mutation, which is linked to reduced susceptibility to several antimalarial drugs, especially those used in combination therapies for malaria treatment, was detected in 97.5% of the samples as mixed-genotype infections., Conclusion: The genomic surveillance of asymptomatic school children in Kenya provides an early warning signal of at least 1 of the 3 validated artemisinin resistance mutations circulating in all regions in Western Kenya sampled except Homa Bay and Kisii Counties. These signals in asymptomatic and mixed infections would have been missed without deep sequencing., Competing Interests: Declarations. Ethics approval and consent to participate: This study was conducted in accordance with the ethical principles of the Declaration of Helsinki. Ethical approval was sourced from the Kenya Medical Research Institute (KEMRI) and National Ethics Review Committee (number KEMRI/SERU/ESACIPAC/11/3822). The student’s parents or guardians provided informed consent while each child assented. Additional approval was provided by the county health and education authorities. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests. Clinical trial number: Not applicable. Underlying data: This project contains the following data: • Kenyansa, Victor, 2024, “FASTQ Files for the dhps and dhfr genes”, https://doi.org/10.7910/DVN/TRUDPB , Harvard Dataverse, V1. • Kenyansa, Victor, 2024, “FASTQ Files for the k13-675 fragment gene”, https://doi.org/10.7910/DVN/Q7PD5P , Harvard Dataverse, V1. • Kenyansa, Victor, 2024, “FASTQ files for the mdr1, ama1 and k13-469 fragment genes”, https://doi.org/10.7910/DVN/2TJH9F , Harvard Dataverse, V1. • Kenyansa, Victor, 2024, “FASTA files for the k13-675 fragment gene”, https://doi.org/10.7910/DVN/O82JXI , Harvard Dataverse, V1., (© 2025. The Author(s).)

The autophagy-related ubiquitin-like conjugation systems, the ATG8 and ATG12 systems, are universally conserved in eukaryotes. However, the covalent bond in the ATG12 system has recently been shown to be evolutionarily lost in Apicomplexa. Here, we show that all genes associated with the ATG12 system are absent in piroplasmida, a lineage within Apicomplexa. Comparative genomics of ATGs further shows that piroplasm ATG3 has lost the region necessary for ATG12 binding. However, our in vitro functional analysis using recombinant proteins demonstrated that ATG3 retained the ability to interact with ATG8 in Babesia bovis , a model species in piroplasmida. These findings provide evidence that the ATG8 system is functional, while the ATG12 system is completely lost in the common ancestor of piroplasmida and highlight the evolutionary flexibility of the ATG12 system in Apicomplexa.

Kinetoplastids are a clade of eukaryotic protozoans that include human parasitic pathogens like trypanosomes and Leishmania species. In these organisms, protein-coding genes are transcribed as polycistronic pre-mRNAs, which need to be processed by the coupled action of trans-splicing and polyadenylation to yield monogenic mature mRNAs. During trans-splicing, a universal RNA sequence, the spliced leader RNA (SL RNA) mini-exon, is added to the 5'-end of each mRNA. The 5'-end of this mini-exon carries a hypermethylated cap structure and is bound by a trypanosomatid-specific cap-binding complex (CBC). The function of three of the kinetoplastid CBC subunits is unknown, but an essential role in cap-binding and trans-splicing has been suggested. Here, we report cryo-EM structures that reveal the molecular architecture of the Trypanosoma brucei CBC (TbCBC) complex. We find that TbCBC interacts with two distinct features of the SL RNA. The TbCBP20 subunit interacts with the m 7 G cap while TbCBP66 recognizes double-stranded portions of the SL RNA. Our findings pave the way for future research on mRNA maturation in kinetoplastids. Moreover, the observed structural similarities and differences between TbCBC and the mammalian cap-binding complex will be crucial for considering the potential of TbCBC as a target for anti-trypanosomatid drug development., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Programmed-cell death is an antimicrobial defense mechanism that promotes clearance of intracellular pathogens. Toxoplasma counteracts host immune defenses by secreting effector proteins into host cells; however, how the parasite evades lytic cell death and the effectors involved remain poorly characterized. We identified ROP55, a rhoptry protein that promotes parasite survival by preventing lytic cell death in absence of IFN-γ stimulation. RNA-Seq analysis revealed that ROP55 acts as a repressor of host pro-inflammatory responses. In THP-1 monocytes ΔROP55 infection increased NF-κB p65 nuclear translocation, IL-1β production, and GSDMD cleavage compared to wild type or complemented parasites. ΔROP55 infection also induced RIPK3-dependent necroptosis in human and mouse primary macrophages. Moreover, ΔROP55 parasites were significantly impaired in virulence in female mice and prevented NF-κB activation and parasite clearance in mBMDM. These findings place ROP55 as a major virulence factor, dampening lytic cell death and enabling Toxoplasma to evade clearance from infected cells., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Toxoplasma gondii is an obligate intracellular parasite with sexual reproduction in the intestinal epithelium of felines. The depletion of two gene repressors, AP2XI-2 and AP2XII-1, induces merozoite formation and gene expression towards sexual commitment. Based on RNA-seq datasets of AP2XI-2 and AP2XII-1 knock downs we identified subtelomeric (ST) TgB12 and hypothetical (HP) genes upregulated. Some of the differentially expressed genes (DEGs) are arranged in ST clusters. These DEG products are characterized by high isoelectric points (pI) and may encode small proteins. The potential roles of these clusters of DEG ST genes in environmental resistance or parasite sexual development of T. gondii is discussed., 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.)

A sustained blood-stage infection of the human malaria parasite P. falciparum relies on the active exit of merozoites from their host erythrocytes. During this process, named egress, the infected red blood cell undergoes sequential morphological events: the rounding-up of the surrounding parasitophorous vacuole, the disruption of the vacuole membrane and finally the rupture of the red blood cell membrane. These events are coordinated by two intracellular second messengers, cGMP and calcium ions (Ca2+), that control the activation of their dedicated kinases, PKG and CDPKs respectively, and thus the secretion of parasitic factors that assist membranes rupture. We had previously identified the serine-threonine phosphatase PP1 as an essential enzyme required for the rupture of the surrounding vacuole. Here, we address its precise positioning and function within the egress signaling pathway by combining chemical genetics and live-microscopy. Fluorescent reporters of the parasitophorous vacuole morphology were expressed in the conditional PfPP1-iKO line which allowed to monitor the kinetics of natural and induced egress, as well as the rescue capacity of known egress inducers. Our results underscore a dual function for PP1 in the egress cascade. First, we provide further evidence that PP1 controls the homeostasis of the second messenger cGMP by modulating the basal activity of guanylyl cyclase alpha and consequently the PKG-dependent downstream Ca2+ signaling. Second, we demonstrate that PP1 also regulates the rounding-up of the parasitophorous vacuole, as this step is almost completely abolished in PfPP1-null schizonts. Strikingly, our data show that rounding-up is the step triggered by egress inducers, and support its reliance on Ca2+, as the calcium ionophore A23187 bypasses the egress defect of PfPP1-null schizonts, restores proper egress kinetics and promotes the initiation of the rounding-up step. Therefore, this study places the phosphatase PP1 upstream of the cGMP-PKG signaling pathway, and sheds new light on the regulation of rounding-up, the first step in P. falciparum blood stage egress cascade., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2025 Seveno 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.)

Among new antimalarials discovered over the past decade are multiple chemical scaffolds that target Plasmodium falciparum P-type ATPase ( Pf ATP4). This essential protein is a Na + pump responsible for the maintenance of Na + homeostasis. Pf ATP4 belongs to the type two-dimensional (2D) subfamily of P-type ATPases, for which no structures have been determined. To gain better insight into the structure/function relationship of this validated drug target, we generated a homology model of Pf ATP4 based on sarco/endoplasmic reticulum Ca 2+ ATPase, a P2A-type ATPase, and refined the model using molecular dynamics in its explicit membrane environment. This model predicted several residues in Pf ATP4 critical for its function, as well as those that impart resistance to various Pf ATP4 inhibitors. To validate our model, we developed a genetic system involving merodiploid states of Pf ATP4 in which the endogenous gene was conditionally expressed, and the second allele was mutated to assess its effect on the parasite. Our model predicted residues involved in Na + coordination as well as the phosphorylation cycle of Pf ATP4. Phenotypic characterization of these mutants involved assessment of parasite growth, localization of mutated Pf ATP4, response to treatment with known Pf ATP4 inhibitors, and evaluation of the downstream consequences of Na + influx. Our results were consistent with modeled predictions of the essentiality of the critical residues. Additionally, our approach confirmed the phenotypic consequences of resistance-associated mutations as well as a potential structural basis for the fitness cost associated with some mutations. Taken together, our approach provides a means to explore the structure/function relationship of essential genes in haploid organisms., Competing Interests: Competing interests statement:The authors declare no competing interest.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a diverse family of variant surface antigens, encoded by var genes, that mediates binding of infected erythrocytes to human cells and plays a key role in parasite immune evasion and malaria pathology. The increased availability of parasite genome sequence data has revolutionised the study of PfEMP1 diversity across multiple P. falciparum isolates. However, making functional sense of genomic data relies on the ability to infer binding phenotype from var gene sequence. For P. falciparum rosetting, the binding of infected erythrocytes to uninfected erythrocytes, the analysis of var gene/PfEMP1 sequences encoding the phenotype is limited, with only eight rosette-mediating PfEMP1 variants described to date. These known rosetting PfEMP1 variants fall into two types, characterised by N-terminal domains known as "domain cassette" 11 (DC11) and DC16. Here we test the hypothesis that DC11 and DC16 are the only PfEMP1 types in the P. falciparum genome that mediate rosetting, by examining a set of thirteen recent culture-adapted Kenyan parasite lines. We first analysed the var gene/PfEMP1 repertoires of the Kenyan lines and identified an average of three DC11 or DC16 PfEMP1 variants per genotype. In vitro rosette selection of the parasite lines yielded four with a high rosette frequency, and analysis of their var gene transcription, infected erythrocyte PfEMP1 surface expression, rosette disruption and erythrocyte binding function identified four novel rosette-mediating PfEMP1 variants. Two of these were of the predicted DC11 type (one showing the dual rosetting/IgM-Fc-binding phenotype), whereas two contained DC15 (DBLα1.2-CIDRα1.5b) a PfEMP1 type not previously associated with rosetting. We also showed that a Thai parasite line expressing a DC8-like PfEMP1 binds to erythrocytes to form rosettes. Hence, these data expand current knowledge of rosetting mechanisms and emphasize that the PfEMP1 types mediating rosetting are more diverse than previously recognised., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2025 McLean 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.)

Background: The determinants of differences in host infectivity among Cryptosporidium species and subtypes are poorly understood. Results from recent comparative genomic studies suggest that gains and losses of multicopy subtelomeric genes encoding insulinase-like proteases (INS-19 and INS-20 in Cryptosporidium parvum and their orthologs in closely related species) may potentially contribute to these differences., Methodology/principal Findings: In this study, we investigated the expression and biological function of the INS-19 and INS-20 of C. parvum. CRISPR/Cas9 was used to endogenously tag both genes with the hemagglutinin epitope. Immunofluorescence analysis revealed that INS-19 and INS-20 are expressed at different developmental stages of the pathogen. Although knockout of either had no detectable effect on the in vitro growth of C. parvum, knockout of INS-20, deletion of its multiple domains, or mutation of the active motif in the functional domain reduced the intensity of C. parvum infection in IFN-γ knockout mice. Consistent with this, mice infected with the INS-20-deleted mutant had reduced intestinal damage and parasite burden., Conclusions/significance: These results suggest that INS-19 and INS-20 have stage-specific expression with distinct biological functions, and that the presence of the INS-20 in zoonotic C. parvum contributes to its infectivity and fitness in mice., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2025 Huang 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.)

Trypanosoma cruzi is the causative agent of Chagas disease, a neglected tropical disease, and one of the most important parasitic diseases worldwide. The first genome of T. cruzi was sequenced in 2005, and its complexity made assembly and annotation challenging. Nowadays, new sequencing methods have improved some strains' genome sequence and annotation, revealing this parasite's extensive genetic diversity and complexity. In this review, we examine the genetic diversity, the genomic structure, and the principal multi-gene families involved in the pathogenicity of T. cruzi . The T. cruzi genome sequence is divided into two compartments: the core (conserved) and the disruptive (variable in length and multicopy gene families among strains). The disruptive region has also been described as genome plasticity and plays a key role in the parasite survival and infection process. This region comprises several multi-gene families, including trans-sialidases, mucins, and mucin-associated surface proteins (MASPs). Trans-sialidases are the most prevalent genes in the genome with a key role in the infection process, while mucins and MASPs are also significant glycosylated proteins expressed on the parasite surface, essential for its biological functions, as host-parasite interaction, host cell invasion or protection against the host immune system, in both insect and mammalian stages. Collectively, in this review, some of the most recent advances in the structure and composition of the T. cruzi genome are reviewed.

Uridine insertion/deletion editing of mitochondrial messenger RNAs (mRNAs) in kinetoplastids entails the coordinated action of three complexes. RNA Editing Catalytic Complexes (RECCs) catalyze the enzymatic reactions, while the RNA Editing Substrate Binding Complex (RESC) and RNA Editing Helicase 2 Complex (REH2C) coordinate interactions between RECCs, mRNAs and hundreds of guide RNAs that direct edited sequences. Additionally, numerous auxiliary factors are required for productive editing of specific mRNAs. Here, we elucidate the role of KRBP72, an editing auxiliary factor of the ABC adenosine triphosphatase (ATPase) family that exhibits RNA-binding activity. In procyclic form Trypanosoma brucei, KRBP72 knockdown leads to a pause in editing at the base of a predicted stem loop structure in adenosine triphosphate synthase subunit 6 (A6) mRNA. Enhanced cross-linking and affinity purification revealed KRBP72 binding sites both within and upstream of this stem loop. KRBP72 ATPase activity is essential for its A6 mRNA editing function; however, its RNA-binding activity is dispensable. KRBP72 interacts with most RESC proteins in an RNase-sensitive manner. By contrast, RESC12A associates with KRBP72 in an RNase-insensitive fashion, and RESC12A promotes KRBP72's interaction with RNA. Hence, KRBP72 ATPase activity facilitates progression of editing through a challenging secondary structure, highlighting this protein's crucial role in A6 mRNA editing., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Many eIF4F and poly(A)-binding protein (PABP) paralogues are found in trypanosomes: six eIF4E, five eIF4G, one eIF4A and two PABPs. They are expressed simultaneously and assemble into different complexes, contrasting the situation in metazoans that use distinct complexes in different cell types/developmental stages. Each eIF4F complex has its own proteins, messenger RNAs (mRNAs) and, consequently, a distinct function. We set out to study the function and regulation of the two eIF4F complexes of the parasite Trypanosoma cruzi and identified the associated proteins and mRNAs of eIF4E3 and eIF4E4 in cells in exponential growth and in nutritional stress, an inducer of differentiation to an infective stage. Upon stress, eIF4G and PABP remain associated with the eIF4E, but the associations with other 43S pre-initiation factors decrease, indicating ribosome attachment is impaired. Most eIF4E3-associated mRNAs encode for proteins involved in anabolic metabolism, while eIF4E4 associate with mRNAs encoding ribosomal proteins as in Trypanosoma brucei. Interestingly, for both eIF4E3/4, more mRNAs were associated in stressed cells than in non-stressed cells, even though these have lower translational efficiencies in stress. In summary, trypanosomes have two co-existing eIF4F complexes associating to different mRNAs, but not stress/differentiation-associated mRNAs. Under stress, both complexes exit translation but remain bound to their mRNA targets., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Malaria is the parasitic disease with the highest global morbidity and mortality. According to estimates from the World Health Organization (WHO), there were around 249 million cases in 2022, with 3.4% occurring in Angola. The emergence and spread of drug-resistant Plasmodium falciparum have compromised anti-malarial efficacy and threatens malaria elimination campaigns using artemisinin-based combination therapy (ACT). Increased copy number (CNV) of the P. falciparum gene plasmepsin 2 (pfpm2) have been reported to confer parasite tolerance to piperaquine (PPQ) and the multidrug resistance-1 (pfmdr1), resistance to mefloquine (MEF) and decreased susceptibility to lumefantrine (LUM). PPQ, MEF and LUM are ACT partner drugs. Therefore, CNV detection is a useful tool to track ACT resistance risk. The potential for future treatment failure of artemisinin-based combinations (that include PPQ, LUM and AMQ), due to parasite resistance in the region, emphasizes the need for continued molecular surveillance., Methods: One hundred and nine clinically derived samples were collected at Hospital Central Dr. António Agostinho Neto (HCL) in Lubango, Angola. qPCR targeting the small-subunit 18S rRNA gene was used to confirm P. falciparum infection. Copy number estimates were determined using a SYBR green-based quantitative PCR assay., Results: Overall, this study revealed a low number of resistance CNVs present in the parasite population at Lubango, for the genes pfmdr1 and pfpm2. Of the 102 samples successfully analysed for pfpm2 10 (9.8%) carried increased CNV and 9/101 (8.9%) carried increased CNV of pfmdr1., Conclusions: This study provides, for the first time, evidence for the presence of CNVs in the pfpm2 and pfmdr1 genes in P. falciparum isolates from southern Angola., Competing Interests: Declarations. Ethics approval and consent to participate: DNA samples were collected following individual oral consent and signed informed written consent from all participants or their guardians. Ethical clearance for the work was obtained from: Comité de Ética Independente da Faculdade de Medicina da Universidade Agostinho Neto and by the Hospital Central Dr. António Agostinho Neto (HCL) institutional ethics review committee (deliberation Nº08/2020, on 14 November 2020). Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Falciparum malaria relies extensively on cell-to-cell communication, and earlier research on the function of exosomal proteins derived from infected red blood cells (iRBCs) has been classified into numerous important roles. In this study, the exosomes were derived from Pf3D7 -iRBCs cultured in vitro during synchronized trophozoite stages. The isolated exosomes were assessed using NTA, FE-SEM, and flow cytometry. Our study reported heterogeneous populations of exosomes during the infection. Additionally, label-free quantification based on LC/MS-MS for protein profiling revealed the presence of both parasitic and host (RBC) proteins; out of a total of 124 proteins detected, 20 Pf3D7 proteins and 80 RBC proteins were identified. Exosomal RBC protein expression is different in cRBCs-Exo and iRBCs-Exo, which shows how the parasite and RBCs interact with each other. Functional classification reported that the majority of these Pf3D7 proteins are uncharacterized with unknown functions, few of which are involved in biological processes such as regulation of complement activation, response to external stimuli, immune system-mediated signaling pathway, protein processing, etc. Hence, studying these exosomal proteins and comparing them to previous research has helped us understand how exosomes help cells to communicate in malaria. It may also reveal new potential biomarkers for diagnostic methods or therapies for malaria.

Proper adhesion of cells to their environment is essential for the normal functioning of single cells and multicellular organisms. To attach to the extracellular matrix (ECM), mammalian cells form integrin adhesion complexes consisting of many proteins that together link the ECM and the actin cytoskeleton. Similar to mammalian cells, the amoeboid cells of the protist Dictyostelium discoideum also use multiprotein adhesion complexes to control their attachment to the underlying surface. However, the exact composition of the multiprotein complexes and the signaling pathways involved in the regulation of adhesion in D. discoideum have not yet been elucidated. Here, we show that the IQGAP-related protein IqgC is important for normal attachment of D. discoideum cells to the substratum. Mutant iqgC-null cells have impaired adhesion, whereas overexpression of IqgC promotes directional migration. A RasGAP C-terminal (RGCt) domain of IqgC is sufficient for its localization in the ventral adhesion focal complexes, while RasGAP activity of a GAP-related domain (GRD) is additionally required for the proper function of IqgC in adhesion. We identify the small GTPase RapA as a novel direct IqgC interactor and show that IqgC participates in a RapA-regulated signaling pathway targeting the adhesion complexes that include talin A, myosin VII, and paxillin B. On the basis of our results, we propose that IqgC is a positive regulator of adhesion, responsible for the strengthening of ventral adhesion structures and for the temporal control of their subsequent degradation., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors have no relevant financial or nonfinancial interests to disclose., (© 2025. The Author(s).)

Malaria parasites must respond quickly to environmental changes, including during their transmission between mammalian and mosquito hosts. Therefore, female gametocytes proactively produce and translationally repress mRNAs that encode essential proteins that the zygote requires to establish a new infection. While the release of translational repression of individual mRNAs has been documented, the details of the global release of translational repression have not. Moreover, changes in the spatial arrangement and composition of the DOZI/CITH/ALBA complex that contribute to translational control are also not known. Therefore, we have conducted the first quantitative, comparative transcriptomics and DIA-MS proteomics of Plasmodium parasites across the host-to-vector transmission event to document the global release of translational repression. Using female gametocytes and zygotes of P. yoelii, we found that ~200 transcripts are released for translation soon after fertilization, including those encoding essential functions. Moreover, we identified that many transcripts remain repressed beyond this point. TurboID-based proximity proteomics of the DOZI/CITH/ALBA regulatory complex revealed substantial spatial and/or compositional changes across this transmission event, which are consistent with recent, paradigm-shifting models of translational control. Together, these data provide a model for the essential translational control mechanisms that promote Plasmodium's efficient transmission from mammalian host to mosquito vector., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2025 Rios 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.)

Dynamins, or dynamin-related proteins (DRPs), are large mechano-sensitive GTPases that mediate membrane dynamics or organellar fission/fusion events. Plasmodium falciparum encodes three dynamin-like proteins whose functions are poorly understood. Here, we demonstrate that one of these dynamin-related proteins, PfDyn2, is required to divide both the apicoplast and the mitochondrion, a striking divergence from the biology of related parasites. Using super-resolution and ultrastructure expansion microscopy (U-ExM), we show that PfDyn2 is expressed in dividing schizonts, and that it localizes to both the apicoplast and the mitochondrion. Our use of long-term, live-cell microscopy allows for the visualization of apicoplast and mitochondrial division in live parasites at super resolution for the first time, and demonstrates that in PfDyn2-deficient parasites, while the apicoplast and mitochondrion increase in size and complexity, they do not undergo fission. We also show that these organellar fission defects prevent successful individualization of the schizont mass and the formation of new daughter cells, or merozoites because the basal complex, the cytokinetic ring of Plasmodium , cannot fully contract in PfDyn2-deficient parasites, a phenotype secondary to physical blockage by undivided organelles occluding the ring. PfDyn2's singular role in mediating both apicoplast and mitochondrial fission has not been observed in other organisms possessing two endosymbiotic organelles, including other Apicomplexans, thus reflecting a unique, potentially exploitable method of organellar division in P. falciparum .IMPORTANCE Plasmodium falciparum remains a significant global pathogen, causing over 200 million infections and over 600,000 deaths per year. One significant obstacle to the control of malaria is increasing resistance to first-line artemisinin-based antimalarials. Another is a lack of basic knowledge about the cell biology of the parasite. Along with the mitochondrion, Plasmodium contains a second organelle descended from an endosymbiotic event, the apicoplast. Both organelles are common targets for antimalarials, but because many proteins involved in organellar fission are not conserved in Plasmodium , until now, the mechanisms underlying apicoplast and mitochondrial division have been unknown. In this study, we demonstrate that PfDyn2, a dynamin-related protein (DRP), is required for the division of both organelles. We also show that defects in organellar division hinder segmentation of the schizont and formation of invasive merozoites by preventing full contraction of the basal complex. By demonstrating its necessity for the proper division of both the apicoplast and the mitochondria, this study highlights PfDyn2 as a potential target for new antimalarials., Competing Interests: The authors declare no conflict of interest.

The inner membrane complex (IMC), a double-membrane organelle underneath the plasma membrane in apicomplexan parasites, plays a significant role in motility and invasion and confers shape to the cell. We characterized the function of PbIMC1g, a component of the IMC1 family member in Plasmodium berghei . PbIMC1g is recruited to the IMC in late schizonts, activated gametocytes, and ookinetes. Pairwise yeast two-hybrid assays demonstrate that PbIMC1g interacts with IMC1c, a component of the PHIL1 complex, and the core sub-repeat motif "EKI(V)V(I)EVP" in PbIMC1g is essential for this interaction. Localization of PbIMC1g to the IMC was dependent on its IMCp domain, while its C-terminus and palmitoylation sites were required for the full efficiency of proper IMC targeting. PbIMC1g is required for asexual stage development, and its conditional knockdown resulted in a defect in schizogony. Additionally, PbIMC1g was also important for male gametogenesis and ookinete development. As an IMC component that assists in anchoring the glideosome to the subpellicular network, PbIMC1g was also involved in ookinete motility and mosquito midgut invasion. IMC1g from the human parasite Plasmodium vivax could functionally replace PbIMC1g in P. berghei , confirming the evolutionary conservation of IMC1g proteins in Plasmodium spp. Together, this work reveals an essential role of IMC1g in the parasite life cycle and suggests that IMC1 family members likely contribute to parasite gliding and invasion., Importance: The malaria parasite's inner membrane complex is critical to maintain its structural integrity and motility. Here, we identified the function of the IMC1g protein, a member of the IMC1 family, in invasive and proliferative stages of P. berghei . We found that the IMCp domain of PbIMC1g is critical for proper IMC targeting, and PbIMC1g interacts with PbIMC1c. Conditional knockdown of PbIMC1g expression affects schizogony, gametogenesis, and ookinete conversion. PbIMC1g interacts with IMC1c to firmly anchor the glideosome to the subpellicular network. Additionally, we confirmed that IMC1g is functionally conserved in Plasmodium spp. These data reveal the function of IMC1g protein in anchoring the glideosome, providing further insight into the mechanism of the glideosome function., Competing Interests: The authors declare no conflict of interest.

Ciliates undergo developmentally programmed genome elimination, in which small RNAs direct the removal of transposable elements (TEs) during the development of the somatic nucleus. Twenty-five nucleotide scanRNAs (scnRNAs) are produced from the entire germline genome and transported to the maternal somatic nucleus, where selection of scnRNAs corresponding to germline-specific sequences is thought to take place. Selected scnRNAs then guide the elimination of TEs in the developing somatic nucleus. How germline-specific scnRNAs are selected remains to be determined. Here, we provide important mechanistic insights into the scnRNA selection pathway by identifying a Paramecium homolog of Gtsf1 as essential for the selective degradation of scnRNAs corresponding to retained somatic sequences. Consistently, we also show that Gtsf1 is localized in the maternal somatic nucleus where it associates with the scnRNA-binding protein Ptiwi09. Furthermore, we demonstrate that the scnRNA selection process is critical for genome elimination. We propose that Gtsf1 is required for the coordinated degradation of Ptiwi09-scnRNA complexes that pair with target RNA via the ubiquitin pathway, similarly to the mechanism suggested for microRNA target-directed degradation in metazoans., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Despite being the most relevant and critical option for managing Chagas disease, pharmacological therapy is currently limited by the availability of only two drugs, benznidazole and nifurtimox. Their effectiveness is further restricted in the chronic phase of the infection, as they induce severe side effects and require prolonged treatment. Additionally, the use of these drugs can lead to the emergence of substantial resistance problems, compounded by the potential natural resistance of some parasite isolates. This study analyzes the expression of 13 genes by digital PCR in four Mexican T. cruzi isolates treated with NFX and BZN. Each isolate exhibited a unique combination of enzyme expression in response to the oxidative stress induced by the antichagasic agents. Notably, we observed the overexpression of cruzipain (CZP), L-threonine dehydrogenase (TDH), and detoxification-related enzymes such as Glutathionyl spermidine synthetase (GST) and Superoxide dismutase-A (SOD). These findings highlight the need for further studies to elucidate the molecular mechanisms underlying this resistance, which pose both unexpected challenges for Chagas disease therapy and a biological barrier to the action of these drugs. These findings highlight the need for further studies to understand how these resistance mechanisms contribute to treatment failure and constitute a biological barrier to drug action., Competing Interests: Declarations. Competing Interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

The ability of cells to sense and respond to mechanical forces is crucial for navigating their environment and interacting with neighboring cells. Myosin II and cortexillin I form complexes known as contractility kits (CKs) in the cytosol, which facilitate a cytoskeletal response by accumulating locally at the site of inflicted stress. Here, we present a computational model for mechanoresponsiveness in Dictyostelium, analyzing the role of CKs within the mechanoresponsive mechanism grounded in experimentally measured parameters. Our model further elaborates on the established distributions and channeling of contractile proteins before and after mechanical force application. We rigorously validate our computational findings by comparing the responses of wild-type cells, null mutants, overexpression mutants, and cells deficient in CK formation to mechanical stresses. Parallel in vivo experiments measuring myosin II cortical distributions at equilibrium provide additional validation. Our results highlight the essential functions of CKs in cellular mechanosensitivity and suggest new insights into the regulatory dynamics of mechanoresponsiveness., Competing Interests: Declaration of interests The authors declare no competing financial interest., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Introduction: A continuing challenge for malaria control is the ability of Plasmodium falciparum to develop resistance to antimalarial drugs. Members within the Plasmodium transcription factor family AP2 regulate the growth and development of the parasite, and are also thought to be involved in unclear aspects of drug resistance. Here we screened for single nucleotide polymorphisms (SNPs) within the AP2 family and identified 6 non-synonymous mutations within AP2-06B (PF3D7&#95;0613800), with allele frequencies greater than 0.05. One mutation, K3124R, was located in a PfAP2-06B AP2 domain., Methods: To investigate transcriptional regulation by PfAP2-06B, ChIP-seq assays were performed on 3D7/PfAP2-06B-GFP schizonts using antibodies against GFP. The DNA sequences of the artemisinin-resistant CWX and the quinoline-resistant strains PfDd2 and Pf7G8 were analyzed for the genetic diversity of AP2-06B, compared with the Pf3D7 strain as a reference sequence. To determine whether AP2-06B can alter the expression of pfk13 and pfcrt , as well as cause artemisinin and quinoline resistance in Plasmodium , we generated both a K3124R mutation and conditional knockdown of AP2-06B in Pf3D7 using CRISPR/Cas9-mediated genome editing., Results: ChIP-Seq analysis showed that AP2-06B can bind to the loci of the Plasmodium genes pfk13 and pfcrt . The AP2-06B K3124R mutation was also found in the artemisinin-resistant parasite strain CWX and the chloroquine-resistant strains Dd2 and 7G8. Contrary to expectation, Pf3D7 Plasmodium lines modified by either K3124R mutation of AP2-06B or conditional knockdown of AP2-06B did not have altered sensitivity to artemisinin or quinolines by modulating pfk13 or pfcrt expression., Discussion: AP2-06B was predicted to be associated with artemisinin and quinoline resistance, but no change in resistance was observed after mutation or conditional knockdown. Given the multigenic nature of resistance, it might be difficult to recreate a resistance phenotype. In conclusion, whether AP2-06B regulates the development of artemisinin or quinoline resistance remains to be studied., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2025 Shi, Wang, Yang, Ma, Tang, Zhang, Zhu, Wang, Liu and He.)

This study aims to explore the coding sequence (CDS) of the putative DUS gene in Eimeria media and assess its potential biological functions during the parasite's lifecycle. Initially, oocysts were isolated from fecal samples of rabbits infected with E. media, from which DNA and RNA were extracted. These extractions were used as templates for PCR to successfully amplify the CDS of the DUS gene, confirming its presence within the E. media genome. Further analysis using quantitative PCR (qPCR) demonstrated significantly higher expression of the DUS gene in the precocious line (PL) compared to the wild type (WT). This differential expression highlights a potential functional role for the DUS gene in influencing the development and sporulation processes of E. media, which may contribute to the precocious phenotype. Additionally, bioinformatics analysis provided insights into the evolutionary trends and structural characteristics of the DUS gene across different species, suggesting a broader biological significance. The elevated expression of the DUS gene in the PL suggests its critical involvement in the growth and reproductive processes of the parasite. This finding opens new avenues for research aimed at controlling E. media infection through targeted interventions in the DUS gene expression pathways., Competing Interests: Declarations. Ethics approval and consent to participate: The China Agricultural University's Committee for Laboratory Animal Welfare and Ethical Examination of Animal Experiments approved the experiments. All animal tests complied with the Chinese National Laboratory Animal Guidelines (GB 14925–2010/XG1-2011). Consent for publication: All animal procedures were performed in accordance with the ethical standards of the institution at which the studies were conducted (China Agricultural University). This study did not involve any animal owners as all animals were bred and maintained under the control of the university. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Purpose: Cryptosporidium spp. and Giardia duodenalis are zoonotic protozoan parasites that are widely seen in domestic and wild animals worldwide. While these pathogens, which affect the digestive system of the hosts, cause high economic losses in animal breeding, they are also considered an important public health problem. In recent years, molecular-based studies revealed that 120 genotypes belonging to 44 Cryptosporidium species and eight G. duodenalis assemblages (G. duodenalis A-H) circulate among hosts. The aim of the study was to determine the presence and prevalence of cryptosporidiosis and giardiosis in buffaloes, for which there was only one previous study on the subject in Türkiye., Methods: In this study, Cryptosporidium spp. and Giardia duodenalis were researched in water buffaloes using polymerase chain reaction (PCR) and DNA sequencing. A total of 510 water buffalo stool samples were obtained from Sivas province, an important water buffalo breeding center in Türkiye., Results: Cryptosporidium spp. were detected in 20 samples (3.92%), whereas five samples (0.98%) were found to be infected with G. duodenalis. DNA sequence analyses of 18S rRNA and β-giardin genes revealed that five Cryptosporidium species, C. occultus (n = 1), C. andersoni (n = 1), C. ryanae (n = 16), C. parvum (n = 1), and C. bovis (n = 1), and G. duodenalis assemblages E were circulated in water buffaloes in Türkiye, respectively. In this work, C. ryanae was the most prevalent Cryptosporidium species, and DNA sequence analyses of these samples showed that 100% nucleotide identities were present between them. Cryptosporidium occultus (PP754270), C. andersoni (PP754271), C. ryanae (PP754272-PP754279, PP754281-PP754285, PP754287-PP754289), C. parvum (PP754280), and C. bovis (PP754286) obtained from water buffaloes in this study shared 98.59-100%, 99.88-100%, 99.49-100%, 99.62-100%, and 99.87-100% nucleotide similarity with isolates present in GeneBank, respectively. In addition, G. duodenalis (PP798352-PP798356) isolates had 99.56-100% (β-giardin) nucleotide identities with G. duodenalis isolates., Conclusion: The existence of cryptosporidiosis (the five species) in water buffaloes was reported for the first time in the country. Moreover, one species (C. occultus) has been reported for the first time in Türkiye., Competing Interests: Declarations. Conflict of Interest: The authors declare no competing interests. Ethical Approval: Ethical approval to conduct the study was obtained from the Sivas Cumhuriyet University Local Animal Experiments Ethics Committee (Approval date: 09.02.2021, Approval number: 65202830–050.04.04–496). Consent to Participate: The consent of all animal owners was sorted before this study was carried out. Consent for Publication: All authors have seen and approved the final version of the manuscript being submitted. They warrant that the article is the authors’ original work, has not received prior publication, and is not under consideration for publication elsewhere., (© 2025. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

The path to survival for pathogenic organisms is not straightforward. Pathogens require a set of enzymes for tissue damage generation and to obtain nourishment, as well as a toolbox full of alternatives to bypass host defense mechanisms. Our group has shown that the parasitic protist Entamoeba histolytica encodes for 14 sphingomyelinases (SMases); one of them (acid sphingomyelinase 6, aSMase6) is involved in repairing membrane damage and exhibits hemolytic activity. The enzymatic characterization of aSMase6 has been shown to be activated by magnesium ions but not by zinc, as shown for the human aSMase, and is strongly inhibited by cobalt. However, no structural data are available for the aSMase6 enzyme. In this work, bioinformatic analyses showed that the protist aSMases are diverse enzymes, are evolutionarily related to hemolysins derived from bacteria, and showed a similar overall structure as parasitic, free-living protists and mammalian enzymes. AlphaFold3 models predicted the occupancy of cobalt ions in the active site of the aSMase6 enzyme. Cavity blind docking showed that the substrate is pushed outward of the active site when cobalt is bound instead of magnesium ions. Additionally, the structural models of the aSMase6 of E. histolytica showed a loop that is absent from the rest of the aSMases, suggesting that it may be involved in hemolytic activity, as demonstrated experimentally using the recombinant proteins of aSMase4 and aSMase6. Trichomonas vaginalis enzymes show a putative transmembrane domain and seem functionally different from E. histolytica . This work provides insight into the future biochemical analyses that can show mechanistic features of parasitic protists sphingomyelinases, ultimately rendering these enzymes potential therapeutic targets.

The kinetochore is the macromolecular protein machine that drives chromosome segregation in eukaryotes. In an evolutionarily divergent group of organisms called kinetoplastids, kinetochores are built using a unique set of proteins (KKT1-25 and KKIP1-12). KKT23 is a constitutively localized kinetochore protein containing a C-terminal acetyltransferase domain of unknown function. Here, using X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, we have determined the structure and dynamics of the KKT23 acetyltransferase domain from Trypanosoma brucei and found that it is structurally similar to the GCN5 histone acetyltransferase domain. We find that KKT23 can acetylate the C-terminal tail of histone H2A and that knockdown of KKT23 results in decreased H2A acetylation levels in T. brucei. Finally, we have determined the crystal structure of the N-terminal region of KKT23 and shown that it interacts with KKT22. Our study provides important insights into the structure and function of the unique kinetochore acetyltransferase in trypanosomes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

For the protozoan parasite Leishmania, completion of its life cycle requires sequential adaptation of cellular physiology and nutrient scavenging mechanisms to the different environments of a sand fly alimentary tract and the acidic mammalian host cell phagolysosome. Transmembrane transporters are the gatekeepers of intracellular environments, controlling the flux of solutes and ions across membranes. To discover which transporters are vital for survival as intracellular amastigote forms, we carried out a systematic loss-of-function screen of the L. mexicana transportome. A total of 312 protein components of small molecule carriers, ion channels and pumps were identified and targeted in a CRISPR-Cas9 gene deletion screen in the promastigote form, yielding 188 viable null mutants. Forty transporter deletions caused significant loss of fitness in macrophage and mouse infections. A striking example is the Vacuolar H + ATPase (V-ATPase), which, unexpectedly, was dispensable for promastigote growth in vitro but essential for survival of the disease-causing amastigotes., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Excitable systems of eukaryotic chemotaxis can generate asymmetric signals of Ras-GTP-enriched domains spontaneously to drive random cell migration without guidance cues. However, the molecules responsible for the spontaneous signal generation remain elusive. Here, we characterized RasGEFs encoded in Dictyostelium discoideum by live-cell imaging of the spatiotemporal dynamics of Ras-GTP and hierarchical clustering, finding that RasGEFX is primarily required for the spontaneous generation of Ras-GTP-enriched domains and is essential for random migration in combination with RasGEFB/M/U in starved cells, and they are dispensable for chemotaxis to chemoattractant cAMP. RasGEFX and RasGEFB that co-localize with Ras-GTP regulate the temporal periods and spatial sizes of the oscillatory Ras-GTP waves propagating along the membrane, respectively, and thus control the protrusions of motile cells differently, while RasGEFU and RasGEFM regulate adhesion and migration speed, respectively. Remarkably, RasGEFX is also important for Ras/PIP3-driven macropinocytosis in proliferating cells, but RasGEFB/M/U are not. These findings illustrate a specific and coordinated control of the cytoskeletal dynamics by multiple RasGEFs for spontaneous motility and macropinocytosis., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Capybaras (Hydrochoerus hydrochaeris) are hosts for several parasites of public health importance, including Cryptosporidium spp. Therefore, this study aimed to perform the molecular characterization of Cryptosporidium spp. in fecal samples from capybaras inhabiting urban areas. We analyzed 401 fecal samples from capybaras in two municipalities of the state of Mato Grosso do Sul, Brazil. Fecal samples were purified using centrifugal sedimentation with ethyl acetate. They were then screened for Cryptosporidium spp. by malachite green negative staining and a nested PCR protocol targeting the 18S rRNA gene. Samples positive by microscopy or PCR were examined by PCR protocols targeting the actin, HSP-70, and GP60 genes. Amplicons from all PCR protocols were subjected to genetic sequencing. Microscopic screening and 18S rRNA gene-targeted PCR identified 0.25 % (1/401) and 0.5 % (2/401) of samples, respectively, as positive for Cryptosporidium spp. The genetic sequences of the 18S rRNA, HSP-70, actin, and GP60 genes showed genetic similarity to Cryptosporidium wrairi sequences of 99.07 %, 99.69 %, 99.57 %, and 91.51 %, respectively. Genetic sequencing and phylogenetic analyses identified the novel GP60 subtype family VIIbA13 of C. wrairi. In conclusion, we report in this study a low prevalence of the novel GP60 subtype family VIIbA13 of C. wrairi in free-living capybaras from urban areas of the state of Mato Grosso do Sul, Brazil. We propose that capybaras act as a novel host for C. wrairi., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)