Parasite Development Research Articles

Ectopic overexpression of Plasmodium falciparum DNA-/RNA-binding Alba proteins misregulates virulence gene homeostasis during asexual blood development.

Alba domain-containing proteins are ubiquitously found in archaea and eukaryotes. By binding to either DNA, RNA, or DNA:RNA hybrids, these proteins function in genome stabilization, chromatin organization, gene regulation, and/or translational modulation. In the malaria parasite Plasmodium falciparum, six Alba domain proteins PfAlba1-6 have been described, of which PfAlba1 has emerged as a "master regulator" of translation during parasite intra-erythrocytic development (IED). Given that a tight control of gene expression is especially important during IED, when malaria pathogenesis manifests, in this study, we focus on three other P. falciparum Albas, PfAlba2-4. Because genetic manipulation of the genomic loci of PfAlba2-4 was unsuccessful, we overexpressed each of these proteins from an episome under a strong constitutive promoter. We observed that PfAlba2 or PfAlba3 overexpression strongly reduced parasite growth and impacted IED stage transitions. In contrast, elevated levels of PfAlba4 were well-tolerated by the parasite. In keeping with this, differential gene expression analysis using RNA-seq of PfAlba2 or PfAlba3 overexpressing strains revealed a significant misregulation of mRNAs encoding virulence factors, such as those related to erythrocyte invasion; a general repression of var gene expression was also apparent. PfAlba4 overexpression, on the other hand, did not significantly perturb the steady-state transcriptome of IED stages and appeared to enhance var mRNA levels. Moreover, distinct sets of genes were targeted by each PfAlba for regulation. Taken together, this study highlights the nonredundant roles of PfAlba proteins in the P. falciparum IED, emphasizing their importance in subtelomeric chromatin biology and RNA regulation.IMPORTANCEThe malaria parasite Plasmodium falciparum tightly controls the expression of its genes at the epigenetic, transcriptional, post-transcriptional, and translational levels to synthesize essential proteins, including virulence factors, in a timely and spatially coordinated manner. A family of six proteins implicated in this process is called PfAlba, characterized by the presence of the DNA-, RNA- or DNA:RNA hybrid-binding Alba domain. To better understand the cellular pathways regulated by this protein family, we overexpressed three PfAlbas during P. falciparum intra-erythrocytic growth and found that high levels of PfAlba2 and PfAlba3 were detrimental to parasite development. This was accompanied by significant changes in the parasite's transcriptome, either with regards to mRNA steady-state levels or expression timing. PfAlba4 overexpression, on the other hand, was well-tolerated by the parasite. Overall, our results delineate specific pathways targeted by individual PfAlbas for regulation and link PfAlba2/PfAlba3 to mutually exclusive expression of the virulence-promoting surface antigen PfEMP1.

Analysis of Possible Coexistence of Microsporidia, Plasmodium falciparum and Wuchereria bancrofti in Anopheles gambiae s.l within Ahmadu Bello University, Zaria, Nigeria.

Anopheles gambiae is a vector of Plasmodium falciparum and Wuchereria bancrofti. Endosymbionts are reported to block development of various parasites in mosquitoes. Microsporidia was reported to affect the development of P. falciparum in mosquitoes. Data on such observation is limited in Nigeria. Therefore, the prevalence of Microsporidia and its coinfection with W. bancrofti and P. falciparum in An. gambiae s.l was studied within Ahmadu Bello University, Zaria. Of the 912 mosquitoes sampled, 124 were An. gambiae s.l The midgut assessment of the Anopheles mosquitoes using light microscopy and polymerase chain reaction (PCR) showed a 12% prevalence of a mono microsporidia infection with no coinfection with either P. falciparum or W. bancrofti. Only 4.03% of the An. gambiae s.l. were found to be coinfected with P. falciparum and W. bancrofti while no mosquito harboured all the microorganisms CONCLUSION: This data further supports the potential of Microsporidia as an antagonist for the development of pathogens in mosquitoes.

Evaluation of the transmission-blocking potential of Plasmodium vivax antigen Pvg37 using transgenic rodent parasites and clinical isolates

BackgroundPlasmodium vivax is a major cause of malaria, particularly outside Africa, necessitating effective strategies for public health management. Transmission-blocking vaccines (TBVs) have shown the potential to inhibit malaria transmission by targeting antigens expressed in sexual-stage parasites. Pbg37, a conserved protein expressed in sexual stages from gametocyte to ookinete in the rodent parasite P. berghei, is a viable target for TBV development.Methods and findingsIn this study, we constructed a transgenic strain, TrPvg37Pb, expressing Pvg37 using the P. berghei ΔPbg37 strain. Initial findings demonstrated that the replacement of Pbg37 with the exogenous Pvg37 did not impact parasite growth or development. Notably, Pvg37 was expressed during the gametocyte to ookinete development and was associated with the plasmic membrane, similar to Pbg37. To evaluate the potential of Pvg37 as a TBV candidate, we synthesized two Pvg37 polypeptides and immunized rabbits to generate antibodies. In vitro experiments demonstrated that anti-Pvg37-P2 antibodies significantly inhibited the formation of male gametes and ookinetes in the transgenic TrPvg37Pb parasite. Additionally, in mosquito feeding assays, mosquitos feeding on TrPvg37Pb-infected mice passively transferred with anti-Pvg37-P2 antibodies showed a significant 80.2% decrease in oocyst density compared to the control group. Furthermore, in direct membrane feeding experiments using four clinical P. vivax isolates, the anti-Pvg37 antibodies significantly reduced oocyst density by 28.6–50.4%.ConclusionPvg37 is a promising candidate for P. vivax TBV development, deserving further research and optimization to enhance its immunogenicity and transmission-blocking activity.

Variations in extracellular vesicle shedding of Cystoisospora suis stages (Apicomplexa: Coccidia).

Cystoisospora suis, a porcine enteral parasite of the order Coccidia, is characterized by a complex life cycle, with asexual and sexual development in the epithelium of the host gut and an environmental phase as an oocyst. All developmental stages vary greatly in their morphology and function, and therefore excrete different bioactive molecules for intercellular communication. Due to their complex development, we hypothesized that the extracellular vesicles (EVs) cargo is highly dependent on the life cycle stages from which they are released. This study aimed to characterize and compare EVs of all developmental stages of C. suis. Nanoparticle tracking analysis and microscopy were used to determine particle numbers and size distributions of stage-specific parasite EVs. Furthermore, Fourier-transform infrared spectral analysis was employed for the metabolic fingerprinting of EVs, and the lipid and protein profiles of all parasite stages were determined. Overall, the study revealed that asexual, sexual and transmissible stages of C. suis release different EVs during the parasite's life cycle. EVs of endogenous asexual and sexual stages were found to be more similar to each other than to those of the transmissible environmental stage, the oocyst. Furthermore, the ratio of fatty acids to polysaccharides and proteins changed during parasite development. In particular, proteins associated with the Apicomplexa and those involved in vesicle shedding showed changes in expression in all parasite stages. Lipid analysis showed that fatty acids were found in the same concentration through all parasite stages, whereas the amount of stereolipids, sphingolipids and glycerolipids changed between the parasite stages. In conclusion, this study, which presents the first known characterization of C. suis EVs, demonstrates a link between EVs and the respective developmental stages of the parasite, and putative functions in the parasite-parasite and host-parasite interplays.

Climate Change and Malaria: A Call for Robust Analytics.

Mosquito ecology and behavior and malaria parasite development display marked sensitivity to weather, in particular to temperature and precipitation. Therefore, climate change is expected to profoundly affect malaria epidemiology in its transmission, spatiotemporal distribution and consequent disease burden. However, malaria transmission is also complicated by other factors (e.g. urbanization, socioeconomic development, genetics, drug resistance) which together constitute a highly complex, dynamical system, where the influence of any single factor can be masked by others. In this study, we therefore aim to re-evaluate the evidence underlying the widespread belief that climate change will increase worldwide malaria transmission. We review two broad types of study that have contributed to this evidence-base: i) studies that project changes in transmission due to inferred relationships between environmental and mosquito entomology, and ii) regression-based studies that look for associations between environmental variables and malaria prevalence. We then employ a simple statistical model to show that environmental variables alone do not account for the observed spatiotemporal variation in malaria prevalence. Our review raises several concerns about the robustness of the analyses used for advocacy around climate change and malaria. We find that, while climate change's effect on malaria is highly plausible, empirical evidence is much less certain. Future research on climate change and malaria must become integrated into malaria control programs, and understood in context as one factor among many. Our work outlines gaps in modelling that we believe are priorities for future research.

Characterization of the Sodium Multi-Vitamin Transporter in the Mosquito Anopheles stephensi and Its Capacity to Mobilize Pantothenate and Biotin.

Pantothenate (Pan), or vitamin B5, is essential for the synthesis of co-enzyme A (CoA), acetyl-CoA, and numerous downstream physiological processes. We previously demonstrated that Pan is not only essential for mosquito survival, but also for the development of malaria parasites within the mosquito, suggesting that targeting Pan and CoA biosynthesis may be a novel approach for malaria control. However, little is known about how Pan is acquired and mobilized within the mosquito. In this work, we examined Pan levels in the important human malaria vector Anopheles stephensi, including the abundance of Pan during immature development and adulthood. We also assessed the distribution of Pan in various adult tissues and examined the impact of provisioning Pan to the mosquito via a sugar or blood meal on mosquito survival and reproduction. Furthermore, we examined how Pan was mobilized in the mosquito via a putative Pan transporter, the A. stephensi sodium multi-vitamin transporter. We demonstrated that this transporter is capable of mobilizing both Pan and biotin (vitamin B7) in a dose dependent manner. We also assessed the distribution of A. stephensi sodium multi-vitamin transporter in the mosquito and its capacity to transport vitamins. This work establishes the basic physiology of Pan uptake and mobilization in the mosquito, providing essential information for Pan based malaria control strategies.

Molecular and biological characterization of transforming growth factor-β homolog derived from Trichinella spiralis

The cytokine homologs, particularly transforming growth factor (TGF)-β, is a crucial immunomodulatory molecule and involved in growth and developmental processes in several helminths. In this study, the basic properties and functions of T. spiralis TGF-β homolog 2 (TsTGH2) were characterized using bioinformatics and molecular biology approaches. Bioinformatics analyses indicated that TsTGH2 belongs to the TGF-β subfamily. Recombinant TsTGH2 (rTsTGH2) expressed in Escherichia coli was used to produce a polyclonal antibody (pAb) in mice. Western blot and immunolocalization using pAb detected native TsTGH2 in crude worm antigens from muscle larvae and adults, showing it was mainly localized in the body wall muscles and the epithelia of the ovary and uterus. To assess the interplay between TsTGH2 and the human TGF-β signaling pathway, rTsTGH2 produced in a HEK293T cell was incubated with the SBE luciferase-HEK293 cell. The result indicated a significant increase in luciferase activity after treatment with rTsTGH2 compared to untreated control (p < 0.05). In conclusion, these findings are the first to characterize the basic properties and functions of TGF-β homologs in T. spiralis, demonstrating their interaction with the human TGF-β receptor. Further investigation is required to identify and optimize an appropriate expression system or conditions for TsTGH2. Additionally, studies are needed to clarify the specific role of native TsTGH2 in parasite development and host immunomodulation.

Ultrastructural expansion microscopy (U-ExM) visualization of malaria parasite dense granules using RESA as a representative marker protein.

Dense granules (DG) are understudied apical organelles in merozoites, the malaria parasite stage that invades erythrocytes. Only six proteins have been identified which localize to DGs, despite that DG proteins play crucial roles in multiple steps of intraerythrocytic parasite development. To develop a tool for investigating DG structure and function, this study applied ultrastructural expansion microscopy (U-ExM) to visualize the ring-infected erythrocyte surface antigen (RESA) in Plasmodium falciparum merozoites. Merozoites were expanded to approximately four times their original size, allowing the identification of DGs without the need for electron microscopy. RESA localization in merozoite DGs was confirmed by staining with a combination of anti-RESA mAb and protein staining by NHS-ester. The translocation of RESA to the infected erythrocyte membrane was also observed in early ring-stage parasites. These results are in good agreement with the RESA localization reported using immunoelectron microscopy (IEM). By using U-ExM, the identification of novel DG proteins will be facilitated without time-consuming IEM, thereby enhancing our understanding of erythrocyte parasitism by P. falciparum.

Autophagy protein Atg7 is essential for maintaining malaria parasite cellular homeostasis and organelle biogenesis.

Plasmodium parasites have a complex life cycle that transitions between mosquito and mammalian hosts, and undergo continuous cellular remodeling to adapt to various drastic environments. Following hepatocyte invasion, the parasite discards superfluous organelles for intracellular replication, and the remnant organelles undergo extensive branching and mature into hepatic merozoites. Autophagy is a ubiquitous eukaryotic process that permits the recycling of intracellular components. Here, we show that the Plasmodium berghei autophagy-related E1-like enzyme Atg7 is expressed in the blood, sporozoites, and liver stages, localized to the parasite cytosol, and is essential for the localization of Atg8 on the membrane and the development of parasite blood and liver forms. We found that depleting Atg7 abolishes Atg8 lipidation, exocytosis of micronemes, organelle biogenesis, and the formation of merozoites during liver-stage development. Overall, this study establishes the essential functions of Atg7 in Plasmodium blood and liver stages, and highlights its role in maintaining the parasite's cellular homeostasis and organelle biogenesis.IMPORTANCEThe malaria life cycle involves two hosts, mosquitoes and vertebrates. Plasmodium parasites undergo complex intracellular and extracellular stages during this transition. Here, we report that an autophagy-related E1-like enzyme Atg7 is required to conjugate Atg8 on the apicoplast membrane. Atg7 depletion in Plasmodium berghei resulted in the loss of Atg8 lipidation and multiple defects like clearance of micronemes, organelle biogenesis, and maturation of hepatic schizonts during liver-stage development. The essentiality of Plasmodium Atg7 in blood and liver stages suggests it is a prospective target for developing autophagy-specific inhibitors. These results highlight the importance of autophagy in malaria parasite development.

Characterization of differentially regulated carboxypeptidase (metallopeptidase M32) protein in Miltefosine resistant Leishmania donovani parasites.

Carboxypeptidase, a member of the metallopeptidase M32 family, catalyses the C-terminal hydrolysis of a variety of peptides and proteins in the presence of metal ions. To characterize Leishmania donovani carboxypeptidase (LdCP) in miltefosine (MIL) drug-resistant parasites. We performed the MTT assay and cell cycle analysis to confirm the MIL resistance of clinical isolates. LdCP gene was cloned and expressed in E. coli Artic strain. The purified LdCP protein was used for antibody generation and biochemical characterization. MTT assay and cell cycle analysis revealed that all three isolates exhibit MIL resistance. LdCP constitutively expressed in both promastigote and amastigote stages of parasites, and its activity increased 2-3 fold in MIL-resistant parasites. LdCP has high α-helical content at physiological pH and temperature. The protein is quite thermostable with a Tm of 63°C and susceptible to chemical denaturation, with 50% unfolding induced by 3.59M urea or 0.31M guanidine hydrochloride (GdmCl). LC-MS/MS study reveals that LdCP interacts with membrane-associated proteins that have ATP binding sites and involved in protein phosphorylation. To our knowledge, this is the first study to characterize the carboxypeptidase of L. donovani that appears to contribute to the development of MIL resistance parasites.

Inhibitors of malaria parasite cyclic nucleotide phosphodiesterases block asexual blood-stage development and mosquito transmission.

Cyclic nucleotide-dependent phosphodiesterases (PDEs) play essential roles in regulating the malaria parasite life cycle, suggesting that they may be promising antimalarial drug targets. PDE inhibitors are used safely to treat a range of noninfectious human disorders. Here, we report three subseries of fast-acting and potent Plasmodium falciparum PDEβ inhibitors that block asexual blood-stage parasite development and that are also active against human clinical isolates. Two of the inhibitor subseries also have potent transmission-blocking activity by targeting PDEs expressed during sexual parasite development. In vitro drug selection experiments generated parasites with moderately reduced susceptibility to the inhibitors. Whole-genome sequencing of these parasites detected no mutations in PDEβ but rather mutations in downstream effectors: either the catalytic or regulatory subunits of cyclic adenosine monophosphate-dependent protein kinase (PKA) or in the 3-phosphoinositide-dependent protein kinase that is required for PKA activation. Several properties of these P. falciparum PDE inhibitor series make them attractive for further progression through the antimalarial drug discovery pipeline.

Influence of Household Roof Types on the Development of Plasmodium vivax in Anopheles stephensi Mosquitoes.

Urbanization and microclimate variation in cities can influence mosquito behavior and parasite development, thus affecting malaria transmission. This study investigates how the impact of microclimate variations due to household roof types can aid in the survival of Anopheles stephensi and the development of Plasmodium vivax in an urban slum setting. Understanding these vital environmental interactions is essential for devising effective control strategies to achieve malaria elimination. Anopheles stephensi (F1) mosquitoes were membrane-fed with blood collected from P. vivax-infected patients before (day 0) and during (day 1) antimalarial treatment. The parasite development and mosquito survival were monitored in simulated microclimatic conditions of a variety of household roof types (thatched, asbestos, tiled) against standard laboratory conditions. Mosquito dissections were undertaken to detect oocysts and sporozoites in An. stephensi mosquitoes (oocyst: day 3-5, sporozoites: day 7-11). The maximum number of oocysts were detected in infected mosquitoes in thatched-roof conditions, whereas the largest oocyst was in the asbestos roof type. Circumsporozoite-ELISA results indicated the presence of sporozoites in infected mosquitoes for up to 29 days under standard conditions, 18 days in thatched-roof and asbestos roof conditions, and 14 days in tiled conditions. The univariate binary logistic regression model indicated a significant influence of microclimatic conditions of thatched roofs on parasite development. The Kaplan-Meier survival analysis revealed that the median survival of P. vivax-infected An. stephensi in thatched-roof conditions was 14 days, followed by asbestos (11 days) and tiled (10 days) roof conditions. In conclusion, thatched-roof houses were favorable for the development and survival of P. vivax-infected An. stephensi.

Echinococcus multilocularis delta/notch signalling components are expressed in post-mitotic cells.

Pluripotent somatic stem cells are the drivers of unlimited growth of Echinococcus multilocularis metacestode tissue within the organs of the intermediate host. To understand the dynamics of parasite proliferation within the host, it is therefore important to delineate basic mechanisms of Echinococcus stem cell maintenance and differentiation. We herein undertake the first step towards characterizing the role of an evolutionarily old metazoan cell-cell communication system, delta/notch signalling, in Echinococcus cell fate decisions. Bioinformatic analyses revealed that all central components of this pathway are encoded by the Echinococcus genome and are expressed in parasite larval stages. By in situ hybridisation, we analyzed the expression patterns of clearly identified delta-like ligands, delta1 and delta2, as well as two notch receptors, notch1 and notch2, in metacestode tissue. Except for delta1, which is not expressed in the metacestode, all other components are expressed in distinct cells throughout the parasite's germinal layer. Combined in situ hybridisation and EdU incorporation experiments together with pulse-chase assays further indicate that delta2, notch1, and notch2 are exclusively expressed in post-mitotic cells. Echinococcus asymmetric stem cell division, leading to the progeny of different fates, therefore most probably not involves delta/notch signalling components. Our analyses are relevant for understanding the interplay of fate-determining signalling pathways in Echinococcus cell differentiation and form a basis for further experiments into the role of delta/notch signalling in parasite development.

Mapping of Nuclear Localization Signal in Secreted Liver-Specific Protein 2 of Plasmodium falciparum.

The secretory proteome of Plasmodium exhibits differential spatial and functional activity within host cells. Plasmodium secretes proteins that translocate into the human host cell nucleus. Liver-specific protein 2 of Plasmodium falciparum (Pf-LISP2) shows nuclear accumulation in human hepatocytes during the late liver stage of malaria parasite development. However, the nuclear translocation mechanism for Pf-LISP2 remains largely uncharacterized. Here, we identified a classical bipartite nuclear localization signal (NLS) located in the C-terminal region of Pf-LISP2. Phylogenetic analysis revealed that this NLS is unique to Plasmodium falciparum and its close relative Plasmodium reichenowi, suggesting an evolutionary adaptation linked to their shared primate hosts. Functional assays confirmed the NLS's nuclear import activity, as fusion constructs of the Pf-LISP2 NLS with Pf-aldolase (Pf-aldolase-NLS-EGFP) localized exclusively to the nucleus of HepG2 cells. Mutation analysis of key lysine and arginine residues in the bipartite NLS demonstrated that the basic amino acid clusters are essential for nuclear localization. Importin-α/β interaction was found to be crucial for Pf-LISP2 nuclear transport, as coexpression of the NLS constructs with the importin-α/β inhibitor mCherry-Bimax2 significantly blocked nuclear translocation. Specific interactions between the lysine and arginine residues of Pf-LISP2's NLS and the conserved tryptophan and asparagine residues of human importin-α1 facilitate the cytosol-to-nuclear translocation of Pf-LISP2. Additionally, LISP2 lacks any nuclear export signal. These results provide new insights into the mechanisms of nuclear transport in Plasmodium falciparum, potentially contributing to the understanding of its pathogenicity and host-cell interactions during liver-stage infection.

An Early-Life Disruption of Gut Microbiota Has Opposing Effects on Parasite Resistance in Two Host Species.

Gut microbiota regulate multiple aspects of host health, including metabolism and the development of the immune system. However, we still know relatively little about how the gut microbiota influences host responses to parasitism in wild organisms, particularly whether host-microbiota interactions contribute to variation in parasitism across host species. The goal of this study was to determine the role of gut microbiota in shaping how birds respond to nest parasites and investigate whether this relationship varies between host species. Both eastern bluebirds (Sialia sialis) and tree swallows (Tachycineta bicolor) are parasitized by blow flies (Protocalliphora sialia), for which larval flies feed on nestlings' blood. We experimentally manipulated the gut microbiota of nestling bluebirds and tree swallows by dosing nestlings with an oral antibiotic or sterile water as a control. We then quantified nestling physiology (haemoglobin, glucose, parasite-specific IgY antibody levels), body morphometrics, and survival until fledging, as well as blow fly abundance and size. An experimental disruption of nestling gut microbiota increased parasite abundance in tree swallows, but decreased parasite abundance in bluebirds, which suggests that the disruption has opposing effects on resistance across host species. Furthermore, experimental treatment delayed parasite development and had variable effects on nestling body morphometrics and physiology across the two host species. Together, these results suggest that gut microbiota contribute to host differences in resistance to blow flies and can influence host-parasite interactions.

Host genotype and infection status interact to shape microbiomes in Daphnia magna.

Host–bacterial communities (microbiomes) are influenced by a wide range of factors including host genotype and parasite exposure. However, few studies disentangle temporal and host-genotype-specific variation in microbiome response to infection across several host tissues. We experimentally exposed the freshwater crustacean Daphnia magna to its fungal parasite Metschnikowia bicuspidata and characterized changes in host–bacterial communities associated with the parasite's development within the host. We used 16S rRNA gene sequencing to assess bacterial communities of the host (a) 24 h (‘initial parasite exposure’) and (b) 10 days (‘successful infection’) after exposure to a standard dose of M. bicuspidata spores, in host guts, body tissue (excluding guts) and whole individuals. We also investigated whether bacterial community responses to parasite exposure varied by host genotype.Parasite exposure did not immediately alter host gut bacterial communities, but drove host-genotype-specific changes in the bacterial community composition of whole individuals. We validated that these changes were not driven by shifts in bacterial communities of the culturing medium, due to the addition of the parasite spore solution. Successful infection (i.e. the proliferation of M. bicuspidata spores in the host body) reduced alpha diversity and shifted abundance of dominant bacterial orders in the gut. Moreover, it induced a host-genotype-specific changes in body bacterial community composition. Overall, bacterial community responses to parasite exposure and subsequent infection are complex: they occur in a host-genotype-dependent manner, differentially at distinct timepoints after parasite exposure, and in specific host tissue.

Identification of two transcription factors that work coordinately to regulate early development in Entamoeba.

The protozoan parasite Entamoeba has a life cycle that switches between infective cysts and invasive trophozoites. Encystation, a crucial process in parasite biology, is controlled by different mechanisms including transcriptional control. We identified two nuclear proteins in Entamoeba invadens, EIN_066100 and EIN_085620, that regulate parasite development by binding to a DNA motif (TCACTTTC) in the promoter regions of genes upregulated in the first 8 h of stage conversion. Overexpression of EIN_066100, a homolog of MAK16 protein, resulted in reduced amoebic proliferation without affecting encystation efficiency. Overexpression of EIN_085620, a protein with an RNA-recognition motif (RRM), led to increased encystation efficiency. Glutathione S-transferase (GST) pull down assays revealed that EIN_066100 interacts with EIN_085620 both in vivo and in vitro, and this interaction is mediated by the EIN_085620 RRM domain. By evaluating truncated proteins with deletions at either the N-terminal or C-terminal regions of EIN_066100, we elucidated the importance of its N-terminal region in proper protein localization, proliferation, encystation, and interaction with EIN_085620. Taken together, these results indicate a coordinated role of EIN_066100 and EIN_085620 in regulating Entamoeba development. This work sheds light on the molecular mechanisms in the earliest stages of Entamoeba encystation.IMPORTANCEAn important biological process in the biology of Entamoeba is stage conversion, which plays a crucial role in disease propagation, facilitating parasite survival outside the host and spreading to new hosts. Multiple mechanisms contribute to controlling the expression of amebic stage-specific genes such as epigenetic and transcriptional control. Identification of early transcriptional control regulators is crucial to understanding the initiation of the encystation cascade. We identified two nuclear proteins, EIN_066100 and EIN_085620, involved in the proliferation and developmental regulation of E. invadens. These proteins work by direct binding to each other and mediating encystation efficiency. Study of new regulators involved in Entamoeba development represents an important advance in a critical aspect of parasite biology.

Plasmodium falciparum protein phosphatase PP7 is required for early ring-stage development.

Plasmodium falciparum causes malaria and is responsible for more than 600,000 deaths each year. Although effective drugs are available to treat disease, the spread of drug-resistant parasites endangers their future efficacy. It is hoped that a better understanding of the biology of malaria parasites will help us to discover new drugs to tackle the resistance problem. Our work is focused on the cell signaling mechanisms that control the development of the parasite throughout its complex life cycle. All signal transduction pathways are ultimately regulated by reversible protein phosphorylation by protein kinase and protein phosphatase enzymes. In this study, we investigate the function of calcium-dependent protein phosphatase PP7 and show that it is essential for the development of ring-stage parasites following the invasion of human erythrocytes. Our results contribute to the understanding of the erythrocytic stages of the parasite life cycle that cause malaria pathology.

Temperature Adaptation in the Postembryonic Development of Acanthocephalus tenuirostris (Palaeacanthocephala: Echinorhynchidae)

Under experimental conditions, the larval development of a fish parasite, Acanthocephalus tenuirostris was studied. This species is common in the eastern regions of Russia from Primorye Territory to the upper reaches of the Kolyma River. The material for the experiments was collected in different areas of the Magadan region which is located mainly in the subarctic climate zone. In several series of experiments, the infection and maintenance of intermediate hosts, the isopod Asellus hilgendorfii, took place at a constant temperature 15°C. In all cases, no more than 45 days were required for the formation of the final cystacanth stage. At the same time, in the hemocoel of isopods a large number of other larval stages were present simultaneously with cystacants. An additional experiment was conducted for the first 45 days at 15°C and the next 207 days at 4–6°C, showed the possibility of further development of larvae at low temperatures. Based on the comparison with the data available in the literature on the development times of European Acanthocephalus species living in temperate climates, it is concluded that the A. tenuirostris has anadaptation to a cold climate, whichprovides a twofold acceleration of the rate ofits development relative to larval A. lucii in Europe.

A bioinformatic approach for the prediction and functional classification of Toxoplasma gondii long non-coding RNAs

Long non-coding RNAs (lncRNAs) have emerged as significant players in diverse cellular processes, including cell differentiation. Advancements in computational methodologies have facilitated the prediction of lncRNA functions, enabling insights even in non-model organisms like pathogenic parasites, in roles such as parasite development, antigenic variation, and epigenetics. In this work, we focus on the apicomplexan Toxoplasma gondii differentiation process, where the infective stage, tachyzoite, can develop into the cysted stage, bradyzoite, under stress conditions. Using a publicly available transcriptome dataset, we predicted putative lncRNA sequences associated with this differentiation process. Notably, a substantial proportion of these putative lncRNAs exhibited stage-specific expression, particularly at the bradyzoite stage. Furthermore, co-expression patterns between coding transcripts and putative TglncRNAs suggest their involvement in shared processes, such as bradyzoite development. Putative TglncRNA loci analysis revealed their potential influence on the expression of nearby coding genes, including subtelomeric genes unique to the T. gondii genome. Finally we propose a k-mer analysis approach to predict putative functional relationships between characterized lncRNAs from model organisms like Homo sapiens and the putative T. gondii lncRNAs. Our perspective led to predict putative T. gondii lncRNA that potentially could act mediating DNA damage repair pathways, opening a new study field to validate this kind of adaptive mechanisms of T. gondii in response to stress conditions.