Evaluation of probiotic efficacy of indigenous yeast strain, Saccharomyces cerevisiae Y-89 isolated from a traditional fermented beverage of West Bengal, India having protective effect against DSS-induced colitis in experimental mice.

Increasing awareness regarding health promotion and disease prevention has driven inclusion of fermented foods and beverages in the daily diet. These are the enormous sources of beneficial microbes, probiotics. This study aims to isolate yeast strains having probiotic potential and effectivity against colitis. Initially, ninety-two yeast strains were isolated from Haria, an ethnic fermented beverage of West Bengal, India. Primary screening was done by their acid (pH 4) and bile salt (0.3%) tolerance ability. Four potent isolates were selected and found effective against Entamoeba histolytica, as this human pathogen is responsible to cause colitis. They were identified as Saccharomyces cerevisiae. They showed luxurious growth even at 37 oC, tolerance up to 5% of NaCl, resistance to gastric juice and high bile salt (2.0%) and oro-gastrointestinal transit tolerance. They exhibited good auto-aggregation and co-aggregation ability and strong hydrophobicity. Finally, heat map and principal component analysis revealed that strain Y-89 was the best candidate. It was further characterised and found to have significant protective effects against DSS-induced colitis in experimental mice model. It includes improvement in colon length, body weight and organ indices; reduction in disease activity index; reduction in cholesterol, LDL, SGPT, SGOT, urea and creatinine levels; improvement in HDL, ALP, total protein and albumin levels; decrease in coliform count and restoration of tissue damage. This study demonstrates that the S. cerevisiae strain Y-89 possesses remarkable probiotic traits and can be used as a potential bio-therapeutic candidate for the prevention of colitis.

The macrophage polarization in Entamoeba histolytica infection modulation by the C fragment of the intermediate subunit of Gal/GalNAc-inhibitable lectin.

The protozoan parasite Entamoeba histolytica is the causative agent of amebiasis, with clinical outcomes ranging from asymptomatic infections to severe invasive diseases. The innate immune system, particularly macrophages, is of paramount importance in resisting the invasion of host tissues and organs by the trophozoites of E. histolytica. Parasite-derived pathogenic factors, such as lectins, play a pivotal role in the promotion of macrophage polarization phenotypes that have undergone alteration. Nevertheless, the precise mechanisms by which E. histolytica modulates immune polarization remain largely unknown. The current study focused on the immunomodulatory effects of the Igl-C fragment of E. histolytica Gal/GalNAc lectin on macrophage polarization. These results demonstrated that Igl-C could induce the secretion of IL-1ß, IL-6, and other cytokines, activating a mixed M1/M2 polarization state. M1 polarization of macrophages occurs in the early stages and gradually transitions to M2 polarization in the later stages, which may contribute to the persistence of the infection. Igl-C induces the macrophage M1 phenotype and causes the release of immune effector molecules, including iNOS and cytokines, by activating the NF-κB p65 and JAK-STAT1 transcription factor signaling pathways. Furthermore, Igl-C supports the macrophage M2 phenotype via JAK-STAT3 and IL-4-STAT6 pathways, which activate arginase expression in later stages, contributing to the tissue regeneration and persistence of the parasite. The involvement of distinct signaling pathways in mediating this response highlights the complex interplay between the parasite and the host immune system. These findings enhance our understanding of the Igl-C-mediated pathogenic mechanisms during E. histolytica infection.

Immunogenicity and safety of an Entamoeba histolytica adjuvanted protein vaccine candidate (LecA+GLA-3M-052 liposomes) in rhesus macaques.

Entamoeba histolytica, the causative agent of amebiasis, is one of the top three parasitic causes of mortality worldwide. However, no vaccine exists against amebiasis. Using a lead candidate vaccine containing the LecA fragment of Gal-lectin and GLA-3M-052 liposome adjuvant, we immunized rhesus macaques via intranasal or intramuscular routes. The vaccine elicited high-avidity functional humoral responses as seen by the inhibition of amebic attachment to mammalian target cells by plasma and stool antibodies. Importantly, antigen-specific IFN-γ-secreting peripheral blood mononuclear cells (PBMCs) and IgG/IgA memory B cells (BMEM) were detected in immunized animals. Furthermore, antigen-specific antibody and cellular responses were maintained for at least 8 months after the final immunization as observed by robust LecA-specific BMEM as well as IFN-γ+ PBMC responses. Overall, both intranasal and intramuscular immunizations elicited a durable and functional response in systemic and mucosal compartments, which supports advancing the LecA+GLA-3M-052 liposome vaccine candidate to clinical testing.

The 26 S proteasome in Entamoeba histolytica: divergence of the substrate binding pockets from host proteasomes.

OBJECTIVE: Proteasomes are conserved proteases crucial for proteostasis in eukaryotes and are promising drug targets for protozoan parasites. Yet, the proteasomes of Entamoeba histolytica remain understudied. The study's objective was to analyse the differences in the substrate binding pockets of amoeba proteasomes from those of host, and computational modelling of ß5 catalytic subunit, with the goal of finding selective inhibitors. RESULTS: Comparative sequence analysis revealed differences in substrate binding sites of E. histolytica proteasomes, especially in the S1 and S3 pockets of the catalytic beta subunits, implying differences in substrate preference and susceptibility to inhibitors from host proteasomes. This was strongly supported by significantly lower sensitivity to MG132 mediated inhibition of amoebic proteasome ß5 subunit's chymotryptic activity compared to human proteasomes, also reflected in lower sensitivity of E. histolytica to MG132 for inhibition of proliferation. Computational models of ß4 and ß5 subunits, and a docked ß4-ß5 model revealed a binding pocket between ß4-ß5, similar to that of Leishmania tarentolae. Selective inhibitors for visceral leishmaniasis, LXE408 and compound 8, docked well to this pocket. This functional and sequence-based analysis predicts differences between amoebic and host proteasomes that can be utilized to develop rationally designed, selective inhibitors against E. histolytica.

The Entamoeba histolytica Vps26 (EhVps26) retromeric protein is involved in phagocytosis: Bioinformatic and experimental approaches.

The retromer is a cellular structure that recruits and recycles proteins inside the cell. In mammalian and yeast, the retromer components have been widely studied, but very little in parasites. In yeast, it is formed by a SNX-BAR membrane remodeling heterodimer and the cargo selecting complex (CSC), composed by three proteins. One of them, the Vps26 protein, possesses a flexible and intrinsically disordered region (IDR), that facilitates interactions with other proteins and contributes to the retromer binding to the endosomal membrane. In Entamoeba histolytica, the protozoan parasite responsible for human amoebiasis, the retromer actively participates during the high mobility and phagocytosis of trophozoites, but the molecular details in these events, are almost unknown. Here, we studied the EhVps26 role in phagocytosis. Bioinformatic analyses of EhVps26 revealed a typical arrestin folding structure of the protein, and a long and charged IDR, as described in other systems. EhVps26 molecular dynamics simulations (MDS) allowed us to predict binding pockets for EhVps35, EhSNX3, and a PX domain-containing protein; these pockets were disorganized in a EhVps26 truncated version lacking the IDR. The AlphaFold2 software predicted the interaction of EhVps26 with EhVps35, EhVps29 and EhSNX3, in a model similar to the reported mammalian crystals. By confocal and transmission electron microscopy, EhVps26 was found in the trophozoites plasma membrane, cytosol, endosomes, and Golgi-like apparatus. During phagocytosis, it followed the erythrocytes pathway, probably participating in cargoes selection and recycling. Ehvps26 gene knocking down evidenced that the EhVps26 protein is necessary for efficient phagocytosis.

Characterization of Entamoeba fatty acid elongases; validation as targets and provision of promising leads for new drugs against amebiasis.

Entamoeba histolytica is a protozoan parasite belonging to the phylum Amoebozoa that causes amebiasis, a global public health problem. E. histolytica alternates its form between a proliferative trophozoite and a dormant cyst. Trophozoite proliferation is closely associated with amebiasis symptoms and pathogenesis whereas cysts transmit the disease. Drugs are available for clinical use; however, they have issues of adverse effects and dual targeting of disease symptoms and transmission remains to be improved. Development of new drugs is therefore urgently needed. An untargeted lipidomics analysis recently revealed structural uniqueness of the Entamoeba lipidome at different stages of the parasite's life cycle involving very long (26-30 carbons) and/or medium (8-12 carbons) acyl chains linked to glycerophospholipids and sphingolipids. Here, we investigated the physiology of this unique acyl chain diversity in Entamoeba, a non-photosynthetic protist. We characterized E. histolytica fatty acid elongases (EhFAEs), which are typically components of the fatty acid elongation cycle of photosynthetic protists and plants. An approach combining genetics and lipidomics revealed that EhFAEs are involved in the production of medium and very long acyl chains in E. histolytica. This approach also showed that the K3 group herbicides, flufenacet, cafenstrole, and fenoxasulfone, inhibited the production of very long acyl chains, thereby impairing Entamoeba trophozoite proliferation and cyst formation. Importantly, none of these three compounds showed toxicity to a human cell line; therefore, EhFAEs are reasonable targets for developing new anti-amebiasis drugs and these compounds are promising leads for such drugs. Interestingly, in the Amoebazoan lineage, gain and loss of the genes encoding two different types of fatty acid elongase have occurred during evolution, which may be relevant to parasite adaptation. Acyl chain diversity in lipids is therefore a unique and indispensable feature for parasitic adaptation of Entamoeba.

Entamoeba histolytica in Different Water Sources of Nigde Province of Turkey.

PURPOSE: This study was carried out to determine the presence of Entamoeba histolytica in water sources of Nigde province in Turkey, between June and November 2021. METHODS: A total of 90 water samples were taken from 15 different water sources (drinking water, well water, spring water, wastewater and dam water) every month and the presence of E. histolytica antigens in the samples was examined by ELISA. RESULTS: The positivity for E. histolytica was determined in 7 (7.7%) of 90 samples. While no antigens were found in any of the samples in June and September, E. histolytica was positive for three samples (20%) in July, one sample (6.6%) in August and October and two samples in November (13.3%). One of 24 dam samples (4.1%), 1 of 12 wastewater samples (8.3%), 1 of 12 well samples (8.3%), and 4 of 24 fountain samples (16.6%) that examined by ELISA were found positive. On the other hand, none of the examined 18 spring samples were positive. In addition, 4 (8.8%) of 45 samples that examined in summer and 3 (6.6%) of 45 samples that examined in autumn were detected positive by using ELISA. Entamoeba histolytica positivity in samples was statistically insignificant in terms of months, water resources and seasons (P > 0.05). CONCLUSION: As a result, the presence of E. histolytica, which is an important public health problem in water sources, was determined for the first time in Nigde province of Türkiye with this study.

Multiplex PCR for gastrointestinal parasites in stool: Benchmarking against direct microscopy and simplex PCR.

PURPOSE: To develop and validate a multiplex conventional PCR assay to simultaneously detect Cryptosporidium spp., Entamoeba histolytica, and Giardia lamblia in diarrheal samples as a rapid, cost-effective, and sensitive diagnostic tool for prevalent co-infections for improved diagnostic accuracy and efficiency in resource-limited settings. METHODS: Stool samples collected from patients with gastrointestinal symptoms after taking written consent, processed via wet mount, iodine mount, and PCR assays. Cohen's kappa statistical analysis was done to test agreement. RESULT: Among 240 patients, 28.75% showed intestinal protozoa via Microscopy; Single-plex and multiplex PCR demonstrated 100% concordance, detecting 27.9%; confirmed by sequencing. Highest parasite positivity was observed in transplant and immunocompromised patients, with moderate to almost perfect agreement between microscopy and molecular methods. CONCLUSION: Multiplex-conventional PCR offers superior sensitivity and specificity over microscopy and 100% concordance with single-plex PCR, enabling rapid, cost-effective diagnosis of multiple parasites from single stool sample. Its adoption could revolutionize parasitic infection management in routine diagnostics.

Endocytosis in anaerobic parasitic protists.

The incorporation of different molecules by eukaryotic cells occurs through endocytosis, which is critical to the cell's survival and ability to reproduce. Although this process has been studied in greater detail in mammalian and yeast cells, several groups working with pathogenic protists have made relevant contributions. This review analysed the most relevant data on the endocytic process in anaerobic protists (Entamoeba histolytica, Giardia intestinalis, Trichomonas vaginalis, and Tritrichomonas foetus). Many protozoa can exert endocytic activity across their entire surface and do so with great intensity, as with E. histolytica. The available data on the endocytic pathway and the participation of PI-3 kinase, Rab, and Rho molecular complexes is reviewed from a historical perspective.

The epidemiology of intestinal protozoa in the Israeli population based on molecular stool test: a nationwide study.

Stool examination using microscopy was the traditional method for the diagnosis of intestinal parasites. Recently, the use of molecular tests to identify stool protozoa has become the main tool used in most clinical laboratories in Israel. This study aimed to evaluate the prevalence of intestinal parasites in Israel and to compare this prevalence in laboratories that use molecular tests vs a laboratory that uses microscopy. Samples collected from January to October 2021 at seven laboratories were analyzed by real-time PCR (RT-PCR) or by microscopy. The multiplex panel included the following pathogens: Giardia lamblia, Entamoeba histolytica, Cryptosporidium spp., Cyclospora, Dientamoeba fragilis, and Blastocystis spp. Overall, 138,415 stool samples were tested by RT-PCR and 6,444 by microscopy. At least one protozoa species was identified in 28.4% of the PCR-tested samples compared to 4.6% of the microscopy-tested samples. D. fragilis was the most common PCR-identified species (29%). D. fragilis, G. lamblia, and Cryptosporidium spp. were mainly found in pediatric population, while Blastocystis spp. was most prevalent among adults (P < 0.001). In a sub-cohort of 21,480 samples, co-infection was found in 4,113 (19.15%) samples, with Blastocystis spp. and D. fragilis being the most common (14.9%) pair. Molecular stool testing proved more sensitive compared to microscopy. D. fragilis was the most commonly detected pathogen. The above profile was identified during the COVID pandemic when traveling was highly restricted and most likely represents the locally circulating protozoa. IMPORTANCE: This study sheds light on the prevalence of stool parasites in Israel. Additionally, this study indicates that the shift from microscope analysis to molecular tests improved protozoa diagnosis.

Evidence of γ-secretase complex involved in the regulation of intramembrane proteolysis in Entamoeba histolytica.

Presenilins (PSNs) are multifunctional membrane proteins involved in signal transduction, lysosomal acidification, and certain physiological processes related to mitochondria. The aspartic protease activity of PSN and the formation of a γ-secretase complex with other subunits such as nicastrin (NCT) are required for the biological functions. Although PSN is widely conserved in eukaryotes, most studies on PSN were conducted in metazoans. Homologous genes for PSN and NCT (EhPSN and EhNCT, respectively) are encoded in the genome of Entamoeba histolytica, however, their functions remain unknown. In this study, we showed that EhPSN and EhNCT form a complex on the cell membrane, demonstrating that the parasite possesses γ-secretase. The predicted structure of EhPSN was similar to the human homolog, demonstrated by the crystal structure, and phylogenetic analysis indicated good conservation between EhPSN and human PSN, supporting the premise that EhPSN functions as a subunit of γ-secretase. By contrast, EhNCT appears to have undergone remarkable structural changes during its evolution. Blue native-polyacrylamide gel electrophoresis combined with western blotting indicated that a 150-kDa single band contains both EhPSN (estimated molecular size: 47-kDa) and EhNCT (64-kDa), suggesting that the complex also contains other unknown components or post-translational modifications. Coimmunoprecipitation from amebic lysates also confirmed that EhPSN and EhNCT formed a complex. Indirect immunofluorescence analysis revealed that the complex localized to the plasma membrane. Moreover, EhPSN exhibited protease activity, which was suppressed by a γ-secretase inhibitor. This is the first report of a γ-secretase complex in protozoan parasites.

Fibronectin induces a transition from amoeboid to a fan morphology and modifies migration in Entamoeba histolytica.

Cell migration modes can vary, depending on a number of environmental and intracellular factors. The high motility of the pathogenic amoeba Entamoeba histolytica is a decisive factor in its ability to cross the human colonic barrier. We used quantitative live imaging techniques to study the migration of this parasite on fibronectin, a key tissue component. Entamoeba histolytica amoebae on fibronectin contain abundant podosome-like structures. By using a laminar flow chamber, we determined that the adhesion forces generated on fibronectin were twice those on non-coated glass. When migrating on fibronectin, elongated amoeboid cells converted into fan-shaped cells characterized by the presence of a dorsal column of F-actin and a broad cytoplasmic extension at the front. The fan shape depended on the Arp2/3 complex, and the amoebae moved laterally and more slowly. Intracellular measurements of physical variables related to fluid dynamics revealed that cytoplasmic pressure gradients were weaker within fan-shaped cells; hence, actomyosin motors might be less involved in driving the cell body forward. We also found that the Rho-associated coiled-coil containing protein kinase regulated podosome dynamics. We conclude that E. histolytica spontaneously changes its migration mode as a function of the substrate composition. This adaptive ability might favour E. histolytica's invasion of human colonic tissue. By combining microfluidic experiments, mechanical modelling, and image analysis, our work also introduces a computational pipeline for the study of cell migration.

Entamoeba histolytica: EhADH, an Alix Protein, Participates in Several Virulence Events through Its Different Domains.

Entamoeba histolytica is the protozoan causative of human amoebiasis. The EhADH adhesin (687 aa) is a protein involved in tissue invasion, phagocytosis and host-cell lysis. EhADH adheres to the prey and follows its arrival to the multivesicular bodies. It is an accessory protein of the endosomal sorting complexes required for transport (ESCRT) machinery. Here, to study the role of different parts of EhADH during virulence events, we produced trophozoites overexpressing the three domains of EhADH, Bro1 (1-400 aa), Linker (246-446 aa) and Adh (444-687 aa) to evaluate their role in virulence. The TrophozBro11-400 slightly increased adherence and phagocytosis, but these trophozoites showed a higher ability to destroy cell monolayers, augment the permeability of cultured epithelial cells and mouse colon, and produce more damage to hamster livers. The TrophozLinker226-446 also increased the virulence properties, but with lower effect than the TrophozBro11-400. In addition, this fragment participates in cholesterol transport and GTPase binding. Interestingly, the TrophozAdh444-687 produced the highest effect on adherence and phagocytosis, but it poorly influenced the monolayers destruction; nevertheless, they augmented the colon and liver damage. To identify the protein partners of each domain, we used recombinant peptides. Pull-down assays and mass spectrometry showed that Bro1 domain interplays with EhADH, Gal/GalNAc lectin, EhCPs, ESCRT machinery components and cytoskeleton proteins. While EhADH, ubiquitin, EhRabB, EhNPC1 and EhHSP70 were associated to the Linker domain, and EhADH, EhHSP70, EhPrx and metabolic enzymes interacted to the Adh domain. The diverse protein association confirms that EhADH is a versatile molecule with multiple functions probably given by its capacity to form distinct molecular complexes.

A high-dimensional platform for observing neutrophil-parasite interactions.

Diarrheal diseases with infectious etiology remain a major cause of death globally, particularly in low-income countries. Entamoeba histolytica is a pathogenic protozoan parasite that is the causative agent of amebiasis. Amebiasis has a wide presentation in clinical severity with many factors, including the bacterial microbiota, contributing to this variation. The innate immune response also plays a critical role in regulating the severity of E. histolytica infection, with neutrophils reported to have a protective role. Despite this, the precise mechanism of how neutrophils mediate amebic killing is poorly understood. Thus, modern platforms that allow for inquiry of granulocyte-ameba interactions will increase our understanding of this disease. Herein, we describe an assay for neutrophil killing of E. histolytica by utilizing high-dimensional spectral flow cytometry. Neutrophils were isolated from wild-type 5-week-old C57BL/6 mice and co-cultured with E. histolytica at various multiplicity of infections (MOIs). After co-culture, neutrophils and E. histolytica were stained for spectral flow cytometry. Cell populations were identified using surface markers and fluorescence minus one (FMO) controls. We have previously shown that animals colonized with a component of the human microbiota, Clostridium scindens, were protected from E. histolytica. This protection was associated with elevated neutrophil count. Here, we explored amebic killing capacity and observed that neutrophils from animals with C. scindens possessed heightened amebic killing compared with controls. Thus, this study establishes a novel platform that can provide an in-depth analysis of granulocyte-parasite interactions in various contexts, including during alteration of the intestinal microbiota.IMPORTANCEThe tools for studying host immune cell-E. histolytica interactions are limited. Factors, such as parasite heterogeneity, infectivity, and difficulties with culture systems and animal models, make interrogation of these interactions challenging. Thus, Entamoeba researchers can benefit from next-generation models that allow for the analysis of both host and parasite cells. Here, we demonstrate the use of a novel platform that allows for the determination of parasite-host cell interactions and customizable high-dimensional phenotyping of both populations. Indeed, spectral flow cytometry can approach >40 markers on a single panel and can be paired with custom-developed parasite antibodies that can be conjugated to fluorochromes via commercially available kits. This platform affords researchers the capability to test highly precise hypotheses regarding host-parasite interactions.

Entamoeba moshkovskii as a potential model organism for Gal/GalNAc lectin intermediate subunit exhibition and functional identification.

In humans, Entamoeba histolytica is the main pathogen causing various amoebiases, while E. moshkovskii falls between being a pathogen and non-pathogen. The two species have similar behavior patterns but differ significantly in pathogenicity, with previous studies and clinical data indicating that E. moshkovskii has a low level of pathogenicity. Meaningfully, the biological characteristics of E. moshkovskii make it a potential model organism and a protein display platform for studying the functions of important Entamoeba proteins. Here, an Amoeba-pcDNA3.1 vector capable of overexpressing E. histolytica-sourced Igl-C protein was constructed and successfully transfected into E. moshkovskii. High levels of expression of the Igl-C, EGFP, and NeoR genes were identified in Igl-C-transfected trophozoites using qRT-PCR, and they were subsequently confirmed using immunoblotting. Transfection of Igl-C protein improved the adherence and phagocytosis of E. moshkovskii, demonstrating that E. histolytica Igl mediated amoebic adhesion. Moreover, as a manifestation of protein virulence, the ability of post-transfected trophozoites to induce inflammation in host macrophages was also enhanced. In conclusion, this study utilizing the characteristics of E. moshkovskii confirmed its potential to serve as a model organism. E. moshkovskii could replace E. histolytica as the target of gene editing, allowing more efficient study of amoebic pathogenicity.

Entamoeba histolytica: In Silico and In Vitro Oligomerization of EhHSTF5 Enhances Its Binding to the HSE of the EhPgp5 Gene Promoter.

Throughout its lifecycle, Entamoeba histolytica encounters a variety of stressful conditions. This parasite possesses Heat Shock Response Elements (HSEs) which are crucial for regulating the expression of various genes, aiding in its adaptation and survival. These HSEs are regulated by Heat Shock Transcription Factors (EhHSTFs). Our research has identified seven such factors in the parasite, designated as EhHSTF1 through to EhHSTF7. Significantly, under heat shock conditions and in the presence of the antiamoebic compound emetine, EhHSTF5, EhHSTF6, and EhHSTF7 show overexpression, highlighting their essential role in gene response to these stressors. Currently, only EhHSTF7 has been confirmed to recognize the HSE as a promoter of the EhPgp5 gene (HSE_EhPgp5), leaving the binding potential of the other EhHSTFs to HSEs yet to be explored. Consequently, our study aimed to examine, both in vitro and in silico, the oligomerization, and binding capabilities of the recombinant EhHSTF5 protein (rEhHSTF5) to HSE_EhPgp5. The in vitro results indicate that the oligomerization of rEhHSTF5 is concentration-dependent, with its dimeric conformation showing a higher affinity for HSE_EhPgp5 than its monomeric state. In silico analysis suggests that the alpha 3 α-helix (α3-helix) of the DNA-binding domain (DBD5) of EhHSTF5 is crucial in binding to the major groove of HSE, primarily through hydrogen bonding and salt-bridge interactions. In summary, our results highlight the importance of oligomerization in enhancing the affinity of rEhHSTF5 for HSE_EhPgp5 and demonstrate its ability to specifically recognize structural motifs within HSE_EhPgp5. These insights significantly contribute to our understanding of one of the potential molecular mechanisms employed by this parasite to efficiently respond to various stressors, thereby enabling successful adaptation and survival within its host environment.

Development of nucleic acid lateral flow immunoassay for molecular detection of Entamoeba moshkovskii and Entamoeba dispar in stool samples.

Entamoeba moshkovskii, recently known as a possible pathogenic amoeba, and the non-pathogenic Entamoeba dispar are morphologically indistinguishable by microscopy. Although PCR was used for differential diagnosis, gel electrophoresis is labor-intensive, time-consuming, and exposed to hazardous elements. In this study, nucleic acid lateral flow immunoassay (NALFIA) was developed to detect E. moshkovskii and E. dispar by post-PCR amplicon analysis. E. moshkovskii primers were labeled with digoxigenin and biotin whereas primers of E. dispar were lebeled with FITC and digoxigenin. The gold nanoparticles were labeled with antibodies corresponding to particular labeling. Based on the established assay, NALFIA could detect as low as 975 fg of E. moshkovskii target DNA (982 parasites or 196 parasites/microliter), and 487.5 fg of E. dispar target DNA (444 parasites or 89 parasites/microliter) without cross-reactivity to other tested intestinal organisms. After testing 91 stool samples, NALFIA was able to detect seven E. moshkovskii (87.5% sensitivity and 100% specificity) and eight E. dispar samples (66.7% sensitivity and 100% specificity) compared to real-time PCR. Interestingly, it detected three mixed infections as real-time PCR. Therefore, it can be a rapid, safe, and effective method for the detection of the emerging pathogens E. moshkovskii and E. dispar in stool samples.

Revisiting the isolation and characterisation of Entamoeba histolytica lipopeptidophosphoglycan.

The parasite Entamoeba histolytica is the cause of amoebic dysentery and liver abscess in humans. On the protozoan cell surface, a variety of glycosylated molecules are involved in the interaction with the environment, such as attachment to the colonic mucus. One of these molecules is the lipopeptidophosphoglycan (LPPG), a complex surface component with antigenic properties. Its structure is only partly known, it is a glycosylphosphatidylinositol (GPI)-linked glycoprotein with a large amount of O-glycosylation. To date, the sequence of a core protein has not been identified. In this study, we further investigated this complex surface molecule aided by the availability of the monoclonal antibody EH5, which had been raised in our laboratory. We studied the extraction of LPPG in various solvent mixtures and discovered that 2-butanol saturated water was simple and superior to other solvents used in the past. The isolated LPPG was subjected to treatment with several proteases and the Ser/Thr specific cleavage agent scandium (III) trifluoromethanesulfonate (scandium triflate). The products were probed with antibody EH5 and the blots showed that the LPPG preparation was largely resistant to standard proteases, but could be cleaved by the scandium compound. These observations could point to the existence of a Ser- or Thr-rich core protein structure.

Modified trichrome stain for faster and improved detection of intestinal protozoan parasites.

Permanent stains such as trichrome have better sensitivity but are time-consuming and the fixative includes toxic mercuric chloride. Thus, a newer modification was tested and found to be a superior, faster and safer staining technique for intestinal parasitic detection. Our study lasted 9 months and a single stool sample was collected from each enrolled patient. We evaluated classical trichrome (T1 - using Schaudinn fixative) with newer modifications, which involved different fixatives with mordant combinations (T2 - acetic acid + hydrated aluminium sulphate, T3 - citric acid + copper sulphate hydrate). Conventional PCR targeting Entamoeba histolytica, Giardia lamblia and Cryptosporidium spp. was taken as the reference. Out of 175 stool samples, 25.1% protozoa were identified by wet mount, 24% by each T1 and T2, 25.7% by T3. Statistically, T3 and T2 had higher sensitivity as compared to T1 and wet mount when PCR was used as reference.

Genome-Wide Classification of Myb Domain-Containing Protein Families in Entamoeba invadens.

Entamoeba histolytica, the causative agent of amebiasis, is the third leading cause of death among parasitic diseases globally. Its life cycle includes encystation, which has been mostly studied in Entamoeba invadens, responsible for reptilian amebiasis. However, the molecular mechanisms underlying this process are not fully understood. Therefore, we focused on the identification and characterization of Myb proteins, which regulate the expression of encystation-related genes in various protozoan parasites. Through bioinformatic analysis, we identified 48 genes in E. invadens encoding MYB-domain-containing proteins. These were classified into single-repeat 1R (20), 2R-MYB proteins (27), and one 4R-MYB protein. The in-silico analysis suggests that these proteins are multifunctional, participating in transcriptional regulation, chromatin remodeling, telomere maintenance, and splicing. Transcriptomic data analysis revealed expression signatures of eimyb genes, suggesting a potential orchestration in the regulation of early and late encystation-excystation genes. Furthermore, we identified probable target genes associated with reproduction, the meiotic cell cycle, ubiquitin-dependent protein catabolism, and endosomal transport. In conclusion, our findings suggest that E. invadens Myb proteins regulate stage-specific proteins and a wide array of cellular processes. This study provides a foundation for further exploration of the molecular mechanisms governing encystation and unveils potential targets for therapeutic intervention in amebiasis.