Protozoan Parasite Research Articles

First record of Perkinsus beihaiensis in cultured mussels Mytilus coruscus in the East China Sea.

During the investigation of parasitic pathogens of Mytilus coruscus, infection of a Perkinsus-like protozoan parasite was detected by alternative Ray's Fluid Thioglycolate Medium (ARFTM). The diameter of hypnospores or prezoosporangia was 8–27 (15.6 ± 4.0, n = 111) μm. The prevalence of the Perkinsus-like species in M. coruscus was 25 and 12.5% using ARFTM and PCR, respectively. The ITS1-5.8S-ITS2 fragments amplified by PCR assay had 100% homology to that of P. beihaiensis, suggesting that the protozoan parasite was P. beihaisensis and M. coruscus was its new host in East China Sea (ECS). Histological analysis showed the presence of trophozoites of P. beihaiensis in gill, mantle and visceral mass, and the schizonts only found in visceral mass. Perkinsus beihaiensis infection led to inflammatory reaction of hemocyte and the destruction of digestive tubules in visceral mass, which had negative effect on health of the farmed M. coruscus and it deserves more attention.

Microscopic and molecular detection of Trichomonas vaginalis in outpatients seeking medical care in Upper Egypt.

Trichomoniasis remains one of the most significant sexually transmitted disease (STDs) for public health. The disease is caused by parasitic protozoa, Trichomonas vaginalis (T. vaginalis), which is often underestimated in tropical medicine. Despite its public health importance, the epidemiology and molecular characteristics of trichomoniasis in Egypt remains poorly understood, particularly in the southern part of the country (Upper Egypt). This study targeted exploring the genetic variability of T. vaginalis infections in Egyptian women living in Upper Egypt using restriction fragment length polymorphism (RFLP). This cross-sectional study included 150 female patients, who visited the gynaecology and obstetrics outpatient clinics at Sohag General Hospital between 2019 and 2022, exhibiting symptoms of trichomoniasis. Vaginal washout samples were collected from each patient and analyzed using three diagnostic techniques: direct wet mount microscopy, culture on TYM Diamond's medium, and PCR amplification and Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) targeting the actin gene, which was applied to all 16 samples that tested positive in culture. The PCR-RFLP results were then visualized through agarose gels electrophoresis to detect DNA fragments. Out of 150 vaginal washout samples, 12 cases (8%) tested positive for T. vaginalis trophozoites via direct wet mount microscopy, while 16 samples (10.6%) were positive in culture. Additionally, PCR-RFLP analysis of the 16 culture-positive samples revealed that 13 samples were confirmed positive using this molecular method. The amplified products were digested with the restriction enzyme Hind II, yielding three DNA fragments of 60, 213, and 827 bp, which were then detected by agarose gel electrophoresis. Digestion with RsaI produced five fragments measuring 87, 103/106, 236, and 568 bp, while MseI digestion resulted in three distinct fragments of 204, 315, and 581 bp. This study provides robust baseline data on the prevalence and microscopic characteristics of T. vaginalis in Upper Egypt, while also presenting, for the first time, molecular detection and genotyping and revealed that genotype E is the only prevalent genotype in the region.

Analysis of the Correlation Between Toxoplasma gondii Seropositivity and Alzheimer's Disease.

Alzheimer's disease (AD) is a multifactorial brain disorder and infectious diseases are considered as one of the predisposing factors for AD. Toxoplasma gondii, an obligate intracellular parasitic protozoan, is suspected of being associated with AD. Serum samples were collected from 109 AD patients and 114 age-matched healthy controls. ELISA was performed using recombinant T. gondii cyst wall protein 1 (CST1) to detect T. gondii antibodies. A parallel experiment was performed with Toxoplasma gondii tachyzoites lysate protein. To analyze whether factors associated with the onset of AD included chronic T. gondii infection, a multivariate logistic regression model was applied, further validating the correlation between chronic T. gondii infection and AD. AD patients exhibited significantly higher levels of Toxoplasma-specific antibodies in their serum compared to the control group, with statistically significant differences (p < 0.05). Multivariate logistic regression analysis revealed that Toxoplasma infection is a risk factor for AD (p < 0.01), and the CST1 antigen can significantly improve the model's performance in predicting the occurrence of AD. The results indicate that chronic infection with Toxoplasma gondii could be one of the risk factors for the development of AD, potentially predisposing individuals with underlying health conditions to the disease. This further validates the correlation between Toxoplasma gondii and AD.

Epidemiology and diagnostics of intestinal parasitic infections in Italy: a multicentric survey

Background and Aims: parasitic infections are becoming more common in non-endemic countries because of intensified immigration and international travels; however, the parasitic disease burden is often underestimated because of underdiagnosis and lack of surveillance in industrialized countries. Also, in Italy, epidemiological data on intestinal parasites affecting humans are scarce and scattered in different areas of the country. The aim of this retrospective, observational study was to evaluate the prevalence of intestinal parasitic infections employing Parasitological Stool Examination (PSE) and to verify the quality of the diagnostic methodologies adopted to identify intestinal parasites in Italian laboratories. Materials and Methods: the study involved 28 Italian diagnostic laboratories and 36389 patients, from June 2015 to May 2016. Results: our data showed that 3173 out of 36389 subjects (8.7%) were carriers of one or more pathogenic or non-pathogenic protozoa or helminths. When only organisms known to cause intestinal disease were considered, an overall positive rate of 3.8% was found (1400/36389). Among the 1400 patients carrying one or more pathogenic species, protozoan infections predominated and were identified in 1138 subjects (3.1%); Dientamoeba fragilis and Giardia duodenalis represent the main intestinal pathogens. Concerning parasitic worms, only 262 individuals (0.7%) exhibited helminth infection; the most frequent finding was represented by Enterobius vermicularis, followed by Taenia spp. Conclusions: this survey also indicated that not all diagnostic laboratories can guarantee proper performance for parasitological diagnosis. In this context, close collaboration between physicians and clinical microbiologists is warranted to improve the diagnostics of intestinal parasitic infections, thus ensuring good quality service.

Enhancing Antileishmanial Activity of Amidoxime-Based Compounds Bearing a 4,5-Dihydrofuran Scaffold: In Vitro Screening Against Leishmania amazonensis.

Leishmaniasis, a protozoan disease affecting humans, exposes significant shortcomings in current treatments. In continuation to our previous findings on amidoxime-based antileishmanial compounds bearing a 4,5-dihydrofuran scaffold, twelve new amidoxime derivatives substituted at position 3 with an amide bearing a nitrogen heterocycle were synthesized. This series was designed to replace the sulfone and aryl group on a previously reported HIT. The synthesis of these compounds involved the following three-step pathway: manganese (III) acetate-based cyclization of a β-ketoester, followed by amidation with LiHMDS and a final reaction with hydroxylamine. Three of them, containing either bromine, chlorine, or methyl substitutions and featuring a pyridine moiety, showed an interesting toxicity-activity relationship in vitro. They exhibited IC50 values of 15.0 µM, 16.0 µM, and 17.0 µM against the promastigote form of the parasite and IC50 values of 0.5 µM, 0.6 µM, and 0.3 µM against the intracellular amastigote form, respectively. A selectivity index (SI) greater than 300 was established between the cytotoxic concentrations (in murine macrophages) and the effective concentrations (against the intracellular form of Leishmania amazonensis). This SI is at least seventy times higher than that observed for Pentamidine and twenty-five times higher than that observed for the reference HIT, as previously reported.

AGC family kinase of Entamoeba histolytica: Decoding the members biochemically.

Entamoeba histolytica, a protozoan parasite, is the causative agent of amoebiasis, which is a significant global health concern. The virulence mechanisms underlying its pathogenicity are multifaceted and complex. However, endocytic processes and motility are well accepted virulence determinants. As previously reported, an AGCK family kinase, EhAGCK1 to be involved in trogocytosis exclusively while another one from same family named EhAGCK2 participates in all actin dependent endocytic processes. As the kinase dead mutants of EhAGCK1 showed significant defect in destruction of live host cells and also the localisation pattern of same is distinguishable from EhAGCK2. From observations so far, it appears that former initiates a distinguishable signaling cascade. In this work, we have demonstrated distinct biochemical properties of kinases involved in related yet distinguishable endocytic processes for the first time. Our biochemical characterization highlights distinct ion dependency of EhAGCK1 along with substrate specificity. We also show upstream activator of these kinases, 3-phosphoinositide dependent kinase 1 (PDK1) activity and its role in activating the kinase activity. The kinases exhibit property of autophosphorylation, and which may regulate the kinase activity subsequently. Summarily, these studies show that EhAGCK1 and EhAGCK2 show distinct biochemical properties which further confirm their unique role in related endocytic processes of trogocytosis and phagocytosis.

Diversity and prevalence of Leucocytozoon in black flies (Diptera: Simuliidae) of Thailand.

Leucocytozoonosis, a parasitic disease of birds, is caused by haemosporidian protozoan parasites of the genus Leucocytozoon, which infect diverse avian species, including poultry. These parasites are transmitted by several black fly species, but knowledge of the factors determining the diversity and prevalence in these vectors, which is crucial for fully understanding disease epidemiology, is largely unexplored. In this study, we investigated factors associated with the prevalence and diversity of Leucocytozoon species in black flies from Thailand. Adults of two black fly taxa (Simulium asakoae Takaoka and Davies complex and S. khelangense Takaoka, Srisuka and Saeung) were collected using sweep nets at nine locations in northern and northeastern regions of Thailand. Specimens were identified morphologically and the results corroborated by DNA barcoding. Molecular methods using specific primers for amplification of the mitochondrial cytochrome b (cyt b) gene of Leucocytozoon were used to detect the parasite in black flies. Species and lineages of Leucocytozoon were determined using the MalAvi database of malaria parasites and related haemosporidians in avian hosts. Regression analysis was used to examine relationships between Leucocytozoon diversity and prevalence, black fly abundance and habitat characteristics. A total of 11,718 adult black flies were collected, of which 4367 were members of the S. asakoae complex and 7351 were S. khelangense. For molecular detection of Leucocytozoon, we randomly selected 300 individual female black flies of the S. asakoae complex and 850 females of S. khelangense pooled into groups of five individuals (= 170 pools). A total of 34 of the 300 specimens of the S. asakoae complex and 118 of the 170 pools of S. khelangense were positive for Leucocytozoon. Fifty-four lineages (haplotypes) were identified, all of which belonged to those reported in domestic chickens, Gallus gallus, with one exception that was identified in S. khelangense and found to be closely related to the Leucocytozoon lineages reported in owls; this is the first record of the latter lineage in Asian black flies. Among these haplotypes, nine and 45 were exclusively found in the S. asakoae complex and S. khelangense, respectively. No lineage was shared between these black fly taxa. Analysis of similarity (ANOSIM) revealed significant Leucocytozoon lineage composition between the two black flies. Phylogenetic analysis found that Leucocytozoon lineages in the S. asakoae complex and S. khelangense are largely isolated, agreeing with the ANOSIM result. The overall prevalence of Leucocytozoon in the S. asakoae complex was 11.3% and ranged from 9% to 13% in each collection. Leucocytozoon prevalence in S. khelangense was 21%, varying from 13% to 37% in each collection. The Shannon H' index indicated greater Leucocytozoon diversity in S. khelangense (H' = 3.044) than in the S. asakoae complex (H' = 1.920). Regression analysis revealed that Leucocytozoon diversity was positively related to black fly abundance and negatively related to maximum air temperature. The results of this study show that the prevalence and diversity of Leucocytozoon lineages in the S. asakoae complex and S. khelangense from Thailand were associated with the abundance of these black flies and with air temperature. The Leucocytozoon lineages identified also showed some degree of black fly taxon specificity, possibly related to different abundance peaks of these vectors. The environmental conditions that favor the development of black flies are possibly a driver of Leucocytozoon prevalence, diversity and vector-parasite co-evolution.

Fine structural features of the free‐living stages of Amyloodinium ocellatum (Dinoflagellata, Thoracosphaeraceae): A marine fish ectoparasite

AbstractAmyloodinium ocellatum is a protozoan parasite that causes amyloodiniosis in marine and brackish water fish, threatening global aquaculture. The present study investigates the morphology and ultrastructure of the free‐living stages of A. ocellatum (tomont and dinospore) using light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Dinospores measured 13.03–19.66 μm in length, 12.32–18.71 μm in width, and were laterally flattened. Dinospores had a transverse flagellum for propulsion and a longitudinal flagellum for direction control. The cyst wall had three distinct layers and included cellulose. The outer wall was coated with numerous bacteria. The orange‐red speckled eyespot was observed all tomont developmental stages and in the dinospore of A. ocellatum. Tomonts proliferation required successive nuclear division, the formation of new cyst walls, and cytoplasmic segregation. The cytoplasm comprises mainly the matrix, organelles, and inclusions. The matrix was grainy and evenly distributed. In addition to organelles, including mitochondria with tubular cristae, Golgi apparatus, and endoplasmic reticulum, the cytoplasm had starch grains and lipid droplets as inclusions. The A. ocellatum cells lacked chloroplasts. This study provides the first ultrastructural view of the cytoplasmic structure of the free‐living stages of A. ocellatum.

Ichthyobodo necator infection as a first record in cultured Dicentrarchus labrax: morphomolecular characterization and infestation patterns in correlation to water quality parameters

Ichthyobodo necator is a damaging and economically important protozoan parasite; however, its clinical patterns and incidence, correlated to environmental factors, are still poorly understood. This study provides the first report of Ichthyobodo necator infestation among Dicentrarchus labrax. We recorded significant mortalities in the cultured D. labrax of average size (5.92 g weight, and 11.55 cm length) in El-Max Station, National Institute of Oceanography and Fisheries, NIOF, Egypt, during the period extending from early summer to early fall (June to October) 2023. Behavioral and gross lesions of the infested fish were recorded; besides, dead and moribund fish were collected. Wet smears of the skin and gill of the affected fish were examined by compound light microscopy. Moreover, water samples from the affected ponds were collected and examined for their physicochemical parameters. The bodonid biflagellated Ichthyobodo necator (Henneguy) C. Pinto, 1928 was isolated from the skin and gills and identified based on specific morphological characteristics. DNA sequencing of PCR products and phylogenetic analysis confirmed identification, with clustering of the current isolates with I. necator isolates from different fish species. The multivariate analysis revealed a positive correlation between the prevalence and infestation intensity of Ichthyobodo, increased temperature levels, decreased dissolved oxygen DO, and also correlated with higher ammonia, and pH values. Additionally, the elevated concentrations of iron, calcium, magnesium, zinc, and copper in pond water were positively correlated with infestation and mortalities. These water parameter levels promote respiratory distress, accelerated prevalence, and infestation intensities of I. necator, particularly during the infestation peak in August and September. This study bridges the knowledge gap regarding I. necator parasite infestations and their correlation with water quality parameters.

NuSAP4 regulates chromosome segregation in Trypanosoma brucei by promoting bipolar spindle assembly

Faithful chromosome segregation in eukaryotes requires the assembly of a bipolar spindle and the faithful attachment of kinetochores to spindle microtubules, which are regulated by various spindle-associated proteins (SAPs) that play distinct functions in regulating spindle dynamics and microtubule-kinetochore attachment. The protozoan parasite Trypanosoma brucei employs evolutionarily conserved and kinetoplastid-specific proteins, including some kinetoplastid-specific nucleus- and spindle-associated proteins (NuSAPs), to regulate chromosome segregation. Here, we characterized NuSAP4 and its functional interplay with diverse SAPs in promoting chromosome segregation in T. brucei. NuSAP4 associates with the spindle during mitosis and concentrates at spindle poles where it interacts with SPB1 and MAP103. Knockdown of NuSAP4 impairs chromosome segregation by disrupting bipolar spindle assembly and spindle pole protein localization. These results uncover the mechanistic role of NuSAP4 in regulating chromosome segregation by promoting bipolar spindle assembly, and highlight the unusual features of mitotic regulation by spindle-associated proteins in this early divergent microbial eukaryote.

A novel role of peroxiredoxin 2 in diabetic kidney disease progression by activating the classically activated macrophages

Diabetic kidney disease (DKD) is the main cause of deaths due to diabetes mellitus (DM). Due to the complexity of its onset, it is difficult to achieve accurate prevention and treatment. The classically activated macrophage (M1) polarization is a crucial proinflammatory mechanism of DKD, while the interaction and cascade effects of oxidative stress and inflammatory response remain to be elucidated. A urine proteomic analysis of patients with DM indicated that peroxiredoxin 2 (PRDX2) had the higher abundance in DKD. We recently found that PRDX of parasitic protozoa Entamoeba histolytica, which was similar to human PRDX2 in amino acid sequence and spatial structure, could activate the inflammatory response of macrophages through toll-like receptor 4 (TLR4). Hence, our study was designed to explore the role of PRDX2 in chronic inflammation during DKD. Combined with in vivo and in vitro experiments, results showed that the PRDX2 was positively correlated with DKD progression and upregulated by high glucose or recombinant tumor necrosis factor-α in renal tubular epithelial cells; Besides, recombinant PRDX2 could promote M1 polarization of macrophages, and enhance the migration as well as phagocytic ability of macrophages through TLR4. In summary, our study has explored the novel role of PRDX2 in DKD to provide a basis for further research on the diagnosis and treatment of DKD.

Evaluation of different inflammatory markers during the infection of domestic cats (Felis catus) by Cystoisospora felis (Coccidia: Apicomplexa)

BackgroundCystoisospora felis or Isospora felis is a ubiquitous apicomplexan protozoon parasite infecting domestic cats worldwide.Objectives of the studythis study aims to identify the causative agent of diarrhea in cats by determining several elevating stressors caused by these coccidian protozoans with molecular characterization. So, from January 2023 to April 2023, a total of 370 domestic cats were hospitalized at various clinics in the Cairo and Giza Governorates. Fecal samples were taken from these animals and examined by concentration floatation techniques using a saturated salt solution. The positive samples were sporulated to identify the collected oocyst. Venous blood was taken from the infected cats to evaluate the associated oxidative stress marker (lipid peroxidation products (MDA).ResultsOut of 370 examined domestic cats, 27(7.29%) were positive for C. felis. The MDA levels increased with age, and females were higher than males. DNA was extracted from fecal samples for amplification of the ITS1 gene, followed by sequencing. The ITS1 gene was amplified and showed bands at 224 bp. The partial nucleotide sequence of the ITS1 gene was aligned with the reference sequences.In a conclusionC. felis increases the free radicals, which in turn means the animals have stress and need a schedule to treat these animals with new, safe protocol drugs that give no resistance and are highly efficient.

Occurrence and assemblage distribution of Giardia Duodenalis in symptomatic and asymptomatic patients in southeastern Iran (2019–2022)

BackgroundThe ubiquitous protozoan parasite Giardia duodenalis is a major contributor to the global burden of diarrhoea, particularly in young children living in poor-resource regions. Although rarely mortal, giardiasis is associated with growth retardation and cognitive impairment in early childhood. Here we investigate the epidemiology of human giardiasis in Iranshahr (south-eastern Iran), a region where this information was previously lacking.MethodsStool samples were collected from 17,455 outpatients and inpatients attended at three major hospital settings during April 2020 and March 2022. Microscopy was used as a screening method for the presence of Giardia cysts, and the identification of G. duodenalis assemblages was carried out using PCR and Sanger sequencing.ResultsThe overall prevalence of giardiasis was 1.87 (326/17,455; 95% CI: 1.7–2.1). Being female was positively associated with higher odds of giardiasis (p = 0.014). Individuals without diarrhoea were less likely to have giardiasis (p = 0.022). Individuals attending the Iran Hospital were more likely to harbour G. duodenalis infections compared to those attending at the Khatam Hospital and the Clinical Reference Laboratory (p = 0.001). Our sequence analyses revealed the presence of assemblages A (56.5%, 13/23), B (39.1%, 9/23), and A + B (4.4%, 1/23). No association was observed between the occurrence of a given assemblage and the occurrence of diarhroea.ConclusionsGiardia infections were found at relatively low prevalence rates in both symptomatic and asymptomatic individuals seeking medical attention. Being female, having diarrhoea, and being sampled during 2020–21 were predictors of giardiasis. Although limited, our molecular data indicate that some Giardia infections may be zoonotic in nature. These data should be corroborated and expanded in future epidemiological studies targeting simultaneously human, animal, and environmental (water) samples to improve our understanding of the epidemiology of giardiasis in Iran.

Animal models for exploring Chagas disease pathogenesis and supporting drug discovery.

SUMMARYInfections with the parasitic protozoan Trypanosoma cruzi cause Chagas disease, which results in serious cardiac and/or digestive pathology in 30%-40% of individuals. However, symptomatic disease can take decades to become apparent, and there is a broad spectrum of possible outcomes. The complex and long-term nature of this infection places a major constraint on the scope for experimental studies in humans. Accordingly, predictive animal models have been a mainstay of Chagas disease research. The resulting data have made major contributions to our understanding of parasite biology, immune responses, and disease pathogenesis and have provided a platform that informs and facilitates the global drug discovery effort. Here, we provide an overview of available animal models and illustrate how they have had a key impact across the field.

Food and Waterborne Cryptosporidiosis from a One Health Perspective: A Comprehensive Review.

A sharp rise in the global population and improved lifestyles has led to questions about the quality of both food and water. Among protozoan parasites, Cryptosporidium is of great importance in this regard. Hence, Cryptosporidium's associated risk factors, its unique characteristics compared to other protozoan parasites, its zoonotic transmission, and associated economic losses in the public health and livestock sectors need to be focused on from a One Health perspective, including collaboration by experts from all three sectors. Cryptosporidium, being the fifth largest food threat, and the second largest cause of mortality in children under five years of age, is of great significance. The contamination of vegetables, fresh fruits, juices, unpasteurized raw milk, uncooked meat, and fish by Cryptosporidium oocysts occurs through infected food handlers, sewage-based contamination, agricultural effluents, infected animal manure being used as biofertilizer, etc., leading to severe foodborne outbreaks. The only Food and Drug Administration (FDA)-approved drug, Nitazoxanide (NTZ), provides inconsistent results in all groups of patients, and currently, there is no vaccine against it. The prime concerns of this review are to provide a deep insight into the Cryptosporidium's global burden, associated water- and foodborne outbreaks, and some future perspectives in an attempt to effectively manage this protozoal disease. A thorough literature search was performed to organize the most relevant, latest, and quantified data, justifying the title. The estimation of its true burden, strategies to break the transmission pathways and life cycle of Cryptosporidium, and the search for vaccine targets through genome editing technology represent some future research perspectives.

Overview Of The Main Viral And Protozoan Infections In Humans

Both viral and protozoan infections pose a serious health risk worldwide, e.g. due to their varied intensity of symptoms - from mild to even lethal. This overview focuses on some of the most important infections caused by these pathogens that still remain a huge concern in some parts of the world. Viral and protozoan infections contribute significantly not only to morbidity, but also mortality, for the most part in regions with limited access to healthcare with low vaccination coverage. Diseases on their own sometimes are mild or even asymptomatic, but the complications or the infections during pregnancy are the reasons why those diseases still remain a huge concern because they may result in serious conditions. Information searches were carried out in online databases, scientific and educational editions, including PubMed, Elsevier, Research Gate, Springer, CDC, WHO. Understanding the biology, transmission routes and severity of those infections as well as pathogenesis of agents that cause them are essential for effective treatment and decline in severe cases and complications.

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.

When more sugar is better-a GPI side chain modification results in a less virulent phenotype during a protozoan infection.

The assembly and function of side chain modifications of glycosylphosphatidylinositol (GPI) units (anchors or free forms) are poorly defined. In a recent study, two enzymes, PIGJ and PIGE, of the protozoan parasite Toxoplasma gondii were identified and shown to be involved in the assembly of such GPI side chains (J. A. Alvarez, E. Gas-Pascual, S. Malhi, J. C. Sánchez-Arcila, et al., mBio 15:e00527-24, 2024, https://doi.org/10.1128/mbio.00527-24). PIGJ adds N-acetylgalactosamine to the GPI core structure, while PIGE subsequently adds a terminal glucose. Deletion of PIGJ resulted in the loss of the side chain and, strikingly, increased mortality in infected mice, in contrast to PIGE knockouts. Absence of the side chain led to increased binding of the scavenger receptor CD36 to mutant parasites. In galectin-3 knockout mice, the virulent phenotype of side-chain-deficient parasites was largely lost. While the exact mechanisms remain to be elucidated by more experiments, these findings provide the first evidence for the importance of GPI side chains in parasite-host interactions in vivo.

EhRacM differentially regulates macropinocytosis and motility in the enteric protozoan parasite Entamoeba histolytica.

Macropinocytosis is an evolutionarily conserved endocytic process that plays a vital role in internalizing extracellular fluids and particles in cells. This non-selective endocytic pathway is crucial for various physiological functions such as nutrient uptake, sensing, signaling, antigen presentation, and cell migration. While macropinocytosis has been extensively studied in macrophages and cancer cells, the molecular mechanisms of macropinocytosis in pathogens are less understood. It has been known that Entamoeba histolytica, the causative agent of amebiasis, exploits macropinocytosis for survival and pathogenesis. Since macropinocytosis is initiated by actin polymerization, leading to the formation of membrane ruffles and the subsequent trapping of solutes in macropinosomes, actin cytoskeleton regulation is crucial. Thus, this study focuses on unraveling the role of well-conserved actin cytoskeleton regulators, Rho small GTPase family proteins, in macropinocytosis in E. histolytica. Through gene silencing of highly transcribed Ehrho/Ehrac genes and following flow cytometry analysis, we identified that silencing EhracM enhances dextran macropinocytosis and affects cellular migration persistence. Live imaging and interactome analysis unveiled the cytosolic and vesicular localization of EhRacM, along with its interaction with signaling and membrane traffic-related proteins, shedding light on EhRacM's multiple roles. Our findings provide insights into the specific regulatory mechanisms of macropinocytosis among endocytic pathways in E. histolytica, highlighting the significance of EhRacM in both macropinocytosis and cellular migration.

Critical role of Babesia bovis spherical body protein 3 in ridge formation on infected red blood cells.

Babesia bovis, an apicomplexan intraerythrocytic protozoan parasite, causes serious economic loss to cattle industries around the world. Infection with this parasite leads to accumulation of infected red blood cells (iRBCs) in the brain microvasculature that results in severe clinical complications known as cerebral babesiosis. Throughout its growth within iRBCs, the parasite exports various proteins to the iRBCs that lead to the formation of protrusions known as "ridges" on the surface of iRBCs, which serve as sites for cytoadhesion to endothelial cells. Spherical body proteins (SBPs; proteins secreted from spherical bodies, which are organelles specific to Piroplasmida) are exported into iRBCs, and four proteins (SBP1-4) have been reported to date. In this study, we elucidated the function of SBP3 using an inducible gene knockdown (KD) system. Localization of SBP3 was assessed by immunofluorescence assay, and only partial colocalization was detected between SBP3 and SBP4 inside the iRBCs. In contrast, colocalization was observed with VESA-1, which is a major parasite ligand responsible for the cytoadhesion. Immunoelectron microscopy confirmed localization of SBP3 at the ridges. SBP3 KD was performed using the glmS system, and effective KD was confirmed by Western blotting, immunofluorescence assay, and RNA-seq analysis. The SBP3 KD parasites showed severe growth defect suggesting its importance for parasite survival in the iRBCs. VESA-1 on the surface of iRBCs was scarcely detected in SBP3 KD parasites, whereas SBP4 was still detected in the iRBCs. Moreover, abolition of ridges on the iRBCs and reduction of iRBCs cytoadhesion to the bovine brain endothelial cells were observed in SBP3 KD parasites. Immunoprecipitation followed by mass spectrometry analysis detected the host Band 3 multiprotein complex, suggesting an association of SBP3 with iRBC cytoskeleton proteins. Taken together, this study revealed the vital role of SBP3 in ridge formation and its significance in the pathogenesis of cerebral babesiosis.