Characterization of Microsporidia-Induced Developmental Arrest and a Transmembrane Leucine-Rich Repeat Protein in Caenorhabditis elegans

Microsporidia comprise a highly diverged phylum of intracellular, eukaryotic pathogens, with some species able to cause life-threatening illnesses in immunocompromised patients. To better understand microsporidian infection in animals, we study infection of the genetic model organism Caenorhabditis elegans and a species of microsporidia, Nematocida parisii, which infects Caenorhabditis nematodes in the wild. We conducted a targeted RNAi screen for host C. elegans genes important for infection and growth of N. parisii, using nematode larval arrest as an assay for infection. Here, we present the results of this RNAi screen, and our analyses on one of the RNAi hits from the screen that was ultimately not corroborated by loss of function mutants. This hit was an RNAi clone against F56A8.3, a conserved gene that encodes a transmembrane protein containing leucine-rich repeats (LRRs), a domain found in numerous pathogen receptors from other systems. This RNAi clone caused C. elegans to be resistant to infection by N. parisii, leading to reduced larval arrest and lower pathogen load. Characterization of the endogenous F56A8.3 protein revealed that it is expressed in the intestine, localized to the membrane around lysosome-related organelles (LROs), and exists in two different protein isoforms in C. elegans. We used the CRISPR-Cas9 system to edit the F56A8.3 locus and created both a frameshift mutant resulting in a truncated protein and a complete knockout mutant. Neither of these mutants was able to recapitulate the infection phenotypes of the RNAi clone, indicating that the RNAi-mediated phenotypes are due to an off-target effect of the RNAi clone. Nevertheless, this study describes microsporidia-induced developmental arrest in C. elegans, presents results from an RNAi screen for host genes important for microsporidian infection, and characterizes aspects of the conserved F56A8.3 gene and its protein product.     

Differential expression of surface glycoconjugates on Entamoeba histolytica and Entamoeba dispar

The human large intestine can harbor two morphologically similar amoebae; the invasive Entamoeba histolytica and the non-invasive Entamoeba dispar. Whereas E. histolytica can produce intestinal and extra-intestinal lesions, E. dispar is present in non-symptomatic carriers. Although biochemical, genetic and proteomic studies have identified clear differences between these Entamoebae, it has become clear that several molecules, once assumed to be involved in tissue destruction, exist in both the virulent and the avirulent species. As surface molecules may play a role in invasion and could therefore determine which amoebae are invasive, we analyzed the glycoconjugate composition of E. histolytica and E. dispar using lectins. There was a significant difference between E. histolytica and E. dispar in the expression of glycoconjugates containing d-mannose and N-acetyl-α-d-galactosamine residues, but not between virulent and avirulent strains of E. histolytica. N-glycoconjugates with terminal α (1–3)-linked mannose residues participate in the adhesion and subsequent cytotoxicity of E. histolytica to cultured hamster hepatocytes. One of them probably is the Gal/GalNAc lectin.     

Identification of asymptomatic Entamoeba histolytica infection by a serological screening test: A cross-sectional study of an HIV-negative men who have sex with men cohort in Japan

BackgroundAmebiasis, caused by Entamoeba histolytica, is spreading in developing countries and in many developed countries as a sexually transmitted infection. Here, we evaluated the efficacy of serological screening to identify asymptomatic E. histolytica infection as a potential epidemiological control measure to limit its spread.Methodology/Principal findingsThis cross-sectional study was carried out between January and March 2021 in an HIV-negative men who have sex with men (MSM) cohort at the National Center for Global Health and Medicine. Serological screening was performed using a commercially available ELISA kit. For seropositive individuals, we performed stool polymerase chain reaction (PCR) to determine current E. histolytica infection. We performed E. histolytica serological screening of 312 participants. None had a history of E. histolytica infection prior to the study. The overall E. histolytica seropositivity was 6.7% (21/312), which was similar to that found by the rapid plasma reagin test (17/312). We identified current infection in 8 of 20 seropositive participants (40.0%) by stool PCR.Conclusions/SignificanceOur serological screening approach constitutes a potentially practical epidemiological strategy. Active epidemiological surveys, in combination with an effective screening strategy for asymptomatically infected individuals, should be applied to help reduce sexually transmitted E. histolytica infections.     

Proteomic Identification of Oxidized Proteins in Entamoeba histolytica by Resin-Assisted Capture: Insights into the Role of Arginase in Resistance to Oxidative Stress

Entamoeba histolytica is an obligate protozoan parasite of humans, and amebiasis, an infectious disease which targets the intestine and/or liver, is the second most common cause of human death due to a protozoan after malaria. Although amebiasis is usually asymptomatic, E. histolytica has potent pathogenic potential. During host infection, the parasite is exposed to reactive oxygen species that are produced and released by cells of the innate immune system at the site of infection. The ability of the parasite to survive oxidative stress (OS) is essential for a successful invasion of the host. Although the effects of OS on the regulation of gene expression in E. histolytica and the characterization of some proteins whose function in the parasite''s defense against OS have been previously studied, our knowledge of oxidized proteins in E. histolytica is lacking. In order to fill this knowledge gap, we performed a large-scale identification and quantification of the oxidized proteins in oxidatively stressed E. histolytica trophozoites using resin-assisted capture coupled to mass spectrometry. We detected 154 oxidized proteins (OXs) and the functions of some of these proteins were associated with antioxidant activity, maintaining the parasite''s cytoskeleton, translation, catalysis, and transport. We also found that oxidation of the Gal/GalNAc impairs its function and contributes to the inhibition of E. histolytica adherence to host cells. We also provide evidence that arginase, an enzyme which converts L-arginine into L-ornithine and urea, is involved in the protection of the parasite against OS. Collectively, these results emphasize the importance of OS as a critical regulator of E. histolytica''s functions and indicate a new role for arginase in E. histolytica''s resistance to OS.     

Analysis of D-A locus of tRNA-linked short tandem repeats reveals transmission of Entamoeba histolytica and E. dispar among students in the Thai-Myanmar border region of northwest Thailand

Intestinal parasitic infections, including those caused by Entamoeba species, are a persistent problem in rural areas of Thailand. The aims of this study were to identify pathogenic Entamoeba species and to analyze their genotypic diversity. Stool samples were collected from 1,233 students of three schools located in the Thai-Myanmar border region of Tak Province, Thailand. The prevalence of Entamoeba infection was measured by polymerase chain reaction (PCR) using species-specific primers. Thirty-one (2.5%) positive cases were detected for E. histolytica, 55 (4.5%) for E. dispar, and 271 (22.0%) for E. coli. Positive samples for E. histolytica and E. dispar were exclusively obtained from a few school classes, whereas E. coli was detected in all grades. No infections caused by E. moshkovskii, E. nuttalli, E. chattoni, and E. polecki were detected in the students studied. The D-A locus of tRNA-linked short tandem repeats was analyzed in samples of E. histolytica (n = 13) and E. dispar (n = 47) to investigate their diversity and potential modes of transmission. Five genotypes of E. histolytica and 13 genotypes of E. dispar were identified. Sequences of the D-A were divergent, but several unique genotypes were significantly prevalent in limited classes, indicating that intra-classroom transmission has occurred. As it was unlikely that infection would have been limited within school classes if the mode of transmission of E. histolytica and E. dispar had been through the intake of contaminated drinking water or food, these results suggest a direct or indirect person-to-person transmission mode within school classes. Positive rates for three Entamoeba species were 2-fold higher in students who had siblings in the schools than in those without siblings, suggesting that transmission occurred even at home due to heavy contacts among siblings.     

Antineoplastic kinase inhibitors: A new class of potent anti-amoebic compounds

Entamoeba histolytica is a protozoan parasite which infects approximately 50 million people worldwide, resulting in an estimated 70,000 deaths every year. Since the 1960s E. histolytica infection has been successfully treated with metronidazole. However, drawbacks to metronidazole therapy exist, including adverse effects, a long treatment course, and the need for an additional drug to prevent cyst-mediated transmission. E. histolytica possesses a kinome with approximately 300–400 members, some of which have been previously studied as potential targets for the development of amoebicidal drug candidates. However, while these efforts have uncovered novel potent inhibitors of E. histolytica kinases, none have resulted in approved drugs. In this study we took the alternative approach of testing a set of twelve previously FDA-approved antineoplastic kinase inhibitors against E. histolytica trophozoites in vitro. This resulted in the identification of dasatinib, bosutinib, and ibrutinib as amoebicidal agents at low-micromolar concentrations. Next, we utilized a recently developed computational tool to identify twelve additional drugs with human protein target profiles similar to the three initial hits. Testing of these additional twelve drugs led to the identification of ponatinib, neratinib, and olmutinib were identified as highly potent, with EC₅₀ values in the sub-micromolar range. All of these six drugs were found to kill E. histolytica trophozoites as rapidly as metronidazole. Furthermore, ibrutinib was found to kill the transmissible cyst stage of the model organism E. invadens. Ibrutinib thus possesses both amoebicidal and cysticidal properties, in contrast to all drugs used in the current therapeutic strategy. These findings together reveal antineoplastic kinase inhibitors as a highly promising class of potent drugs against this widespread and devastating disease.     

PI Kinase-EhGEF2-EhRho5 axis contributes to LPA stimulated macropinocytosis in Entamoeba histolytica

Entamoeba histolytica is a protozoan responsible for several pathologies in humans. Trophozoites breach the intestinal site to enter the bloodstream and thus traverse to a secondary site. Macropinocytosis and phagocytosis, collectively accounting for heterophagy, are the two major processes responsible for sustenance of Entamoeba histolytica within the host. Both of these processes require significant rearrangements in the structure to entrap the target. Rho GTPases play an indispensable role in mustering proteins that regulate cytoskeletal remodelling. Unlike phagocytosis which has been studied in extensive detail, information on machinery of macropinocytosis in E. histolytica is still limited. In the current study, using site directed mutagenesis and RNAi based silencing, coupled with functional studies, we have demonstrated the involvement of EhRho5 in constitutive and LPA stimulated macropinocytosis. We also report that LPA, a bioactive phospholipid present in the bloodstream of the host, activates EhRho5 and translocates it from cytosol to plasma membrane and endomembrane compartments. Using biochemical and FRAP studies, we established that a PI Kinase acts upstream of EhRho5 in LPA mediated signalling. We further identified EhGEF2 as a guanine nucleotide exchange factor of EhRho5. In the amoebic trophozoites, EhGEF2 depletion leads to reduced macropinocytic efficiency of trophozoites, thus phenocopying its substrate. Upon LPA stimulation, EhGEF2 is found to sequester near the plasma membrane in a wortmannin sensitive fashion, explaining a possible mode for activation of EhRho5 in the amoebic trophozoites. Collectively, we propose that LPA stimulated macropinocytosis in E. histolytica is driven by the PI Kinase-EhGEF2-EhRho5 axis.     

An atypical EhGEF regulates phagocytosis in Entamoeba histolytica through EhRho1

The parasite Entamoeba histolytica is the etiological agent of amoebiasis, a major cause of morbidity and mortality due to parasitic diseases in developing countries. Phagocytosis is an essential mode of obtaining nutrition and has been associated with the virulence behaviour of E. histolytica. Signalling pathways involved in activation of cytoskeletal dynamics required for phagocytosis remains to be elucidated in this parasite. Our group has been studying initiation of phagocytosis and formation of phagosomes in E. histolytica and have described some of the molecules that play key roles in the process. Here we showed the involvement of non-Dbl Rho Guanine Nucleotide Exchange Factor, EhGEF in regulation of amoebic phagocytosis by regulating activation of EhRho1. EhGEF was found in the phagocytic cups during the progression of cups, until closure of phagosomes, but not in the phagosomes themselves. Our observation from imaging, pull down experiments and down regulating expression of different molecules suggest that EhGEF interacts with EhRho1 and it is required during initiation of phagocytosis and phagosome formation. Also, biophysical, and computational analysis reveals that EhGEF mediates GTP exchange on EhRho1 via an unconventional pathway. In conclusion, we describe a non-Dbl EhGEF of EhRho1 which is involved in endocytic processes of E. histolytica.     

Entamoeba histolytica Dmc1 Catalyzes Homologous DNA Pairing and Strand Exchange That Is Stimulated by Calcium and Hop2-Mnd1

Meiosis depends on homologous recombination (HR) in most sexually reproducing organisms. Efficient meiotic HR requires the activity of the meiosis-specific recombinase, Dmc1. Previous work shows Dmc1 is expressed in Entamoeba histolytica, a eukaryotic parasite responsible for amoebiasis throughout the world, suggesting this organism undergoes meiosis. Here, we demonstrate Dmc1 protein is expressed in E. histolytica. We show that purified ehDmc1 forms presynaptic filaments and catalyzes ATP-dependent homologous DNA pairing and DNA strand exchange over at least several thousand base pairs. The DNA pairing and strand exchange activities are enhanced by the presence of calcium and the meiosis-specific recombination accessory factor, Hop2-Mnd1. In combination, calcium and Hop2-Mnd1 dramatically increase the rate of DNA strand exchange activity of ehDmc1. The biochemical system described herein provides a basis on which to better understand the role of ehDmc1 and other HR proteins in E. histolytica.     

Anaerobic peroxisomes in Entamoeba histolytica metabolize myo-inositol

Entamoeba histolytica is believed to be devoid of peroxisomes, like most anaerobic protists. In this work, we provided the first evidence that peroxisomes are present in E. histolytica, although only seven proteins responsible for peroxisome biogenesis (peroxins) were identified (Pex1, Pex6, Pex5, Pex11, Pex14, Pex16, and Pex19). Targeting matrix proteins to peroxisomes is reduced to the PTS1-dependent pathway mediated via the soluble Pex5 receptor, while the PTS2 receptor Pex7 is absent. Immunofluorescence microscopy showed that peroxisomal markers (Pex5, Pex14, Pex16, Pex19) are present in vesicles distinct from mitosomes, the endoplasmic reticulum, and the endosome/phagosome system, except Pex11, which has dual localization in peroxisomes and mitosomes. Immunoelectron microscopy revealed that Pex14 localized to vesicles of approximately 90–100 nm in diameter. Proteomic analyses of affinity-purified peroxisomes and in silico PTS1 predictions provided datasets of 655 and 56 peroxisomal candidates, respectively; however, only six proteins were shared by both datasets, including myo-inositol dehydrogenase (myo-IDH). Peroxisomal NAD-dependent myo-IDH appeared to be a dimeric enzyme with high affinity to myo-inositol (Km 0.044 mM) and can utilize also scyllo-inositol, D-glucose and D-xylose as substrates. Phylogenetic analyses revealed that orthologs of myo-IDH with PTS1 are present in E. dispar, E. nutalli and E. moshkovskii but not in E. invadens, and form a monophyletic clade of mostly peroxisomal orthologs with free-living Mastigamoeba balamuthi and Pelomyxa schiedti. The presence of peroxisomes in E. histolytica and other archamoebae breaks the paradigm of peroxisome absence in anaerobes and provides a new potential target for the development of antiparasitic drugs.     

In Vivo RNAi Rescue in Drosophila melanogaster with Genomic Transgenes from Drosophila pseudoobscura

Background

Systematic, large-scale RNA interference (RNAi) approaches are very valuable to systematically investigate biological processes in cell culture or in tissues of organisms such as Drosophila. A notorious pitfall of all RNAi technologies are potential false positives caused by unspecific knock-down of genes other than the intended target gene. The ultimate proof for RNAi specificity is a rescue by a construct immune to RNAi, typically originating from a related species.

Methodology/Principal Findings

We show that primary sequence divergence in areas targeted by Drosophila melanogaster RNAi hairpins in five non-melanogaster species is sufficient to identify orthologs for 81% of the genes that are predicted to be RNAi refractory. We use clones from a genomic fosmid library of Drosophila pseudoobscura to demonstrate the rescue of RNAi phenotypes in Drosophila melanogaster muscles. Four out of five fosmid clones we tested harbour cross-species functionality for the gene assayed, and three out of the four rescue a RNAi phenotype in Drosophila melanogaster.

Conclusions/Significance

The Drosophila pseudoobscura fosmid library is designed for seamless cross-species transgenesis and can be readily used to demonstrate specificity of RNAi phenotypes in a systematic manner.     

Conservation and function of Rab small GTPases in Entamoeba: Annotation of E. invadens Rab and its use for the understanding of Entamoeba biology

Entamoeba invadens is a reptilian enteric protozoan parasite closely related to the human pathogen Entamoeba histolytica and a good model organism of encystation. To understand the molecular mechanism of vesicular trafficking involved in the encystation of Entamoeba, we examined the conservation of Rab small GTPases between the two species. E. invadens has over 100 Rab genes, similar to E. histolytica. Most of the Rab subfamilies are conserved between the two species, while a number of species-specific Rabs are also present. We annotated all E. invadens Rabs according to the previous nomenclature [Saito-Nakano, Y., Loftus, B.J., Hall, N., Nozaki, T., 2005. The diversity of Rab GTPases in Entamoeba histolytica. Experimental Parasitology 110, 244-252]. Comparative genomic analysis suggested that the fundamental vesicular traffic machinery is well conserved, while there are species-specific protein transport mechanisms. We also reviewed the function of Rabs in Entamoeba, and proposed the use of the annotation of E. invadens Rab genes to understand the ubiquitous importance of Rab-mediated membrane trafficking during important biological processes including differentiation in Entamoeba.     

Longevity and resistance to stress correlate with DNA repair capacity in Caenorhabditis elegans

DNA repair is an important mechanism by which cells maintain genomic integrity. Decline in DNA repair capacity or defects in repair factors are thought to contribute to premature aging in mammals. The nematode Caenorhabditis elegans is a good model for studying longevity and DNA repair because of key advances in understanding the genetics of aging in this organism. Long-lived C. elegans mutants have been identified and shown to be resistant to oxidizing agents and UV irradiation, suggesting a genetically determined correlation between DNA repair capacity and life span. In this report, gene-specific DNA repair is compared in wild-type C. elegans and stress-resistant C. elegans mutants for the first time. DNA repair capacity is higher in long-lived C. elegans mutants than in wild-type animals. In addition, RNAi knockdown of the nucleotide excision repair gene xpa-1 increased sensitivity to UV and reduced the life span of long-lived C. elegans mutants. These findings support that DNA repair capacity correlates with longevity in C. elegans.     

Evaluation of the C-Terminal Fragment of Entamoeba histolytica Gal/GalNAc Lectin Intermediate Subunit as a Vaccine Candidate against Amebic Liver Abscess

Background

Entamoeba histolytica is an intestinal protozoan parasite that causes amoebiasis, including amebic dysentery and liver abscesses. E. histolytica invades host tissues by adhering onto cells and phagocytosing them depending on the adaptation and expression of pathogenic factors, including Gal/GalNAc lectin. We have previously reported that E. histolytica possesses multiple CXXC sequence motifs, with the intermediate subunit of Gal/GalNAc lectin (i.e., Igl) as a key factor affecting the amoeba''s pathogenicity. The present work showed the effect of immunization with recombinant Igl on amebic liver abscess formation and the corresponding immunological properties.

Methodology/Principal Findings

A prokaryotic expression system was used to prepare the full-length Igl and the N-terminal, middle, and C-terminal fragments (C-Igl) of Igl. Vaccine efficacy was assessed by challenging hamsters with an intrahepatic injection of E. histolytica trophozoites. Hamsters intramuscularly immunized with full-length Igl and C-Igl were found to be 92% and 96% immune to liver abscess formation, respectively. Immune-response evaluation revealed that C-Igl can generate significant humoral immune responses, with high levels of antibodies in sera from immunized hamsters inhibiting 80% of trophozoites adherence to mammalian cells and inducing 80% more complement-mediated lysis of trophozoites compared with the control. C-Igl was further assessed for its cellular response by cytokine-gene qPCR analysis. The productions of IL-4 (8.4-fold) and IL-10 (2-fold) in the spleen cells of immunized hamsters were enhanced after in vitro stimulation. IL-4 expression was also supported by increased programmed cell death 1 ligand 1 gene.

Conclusions/Significance

Immunobiochemical characterization strongly suggests the potential of recombinant Igl, especially the C-terminal fragment, as a vaccine candidate against amoebiasis. Moreover, protection through Th2-cell participation enabled effective humoral immunity against amebic liver abscesses.     

SNAP-Tag Technology Optimized for Use in Entamoeba histolytica

Entamoeba histolytica is a protozoan parasite responsible for invasive intestinal and extraintestinal amebiasis. The pathology of amebiasis is still poorly understood, which can be largely attributed to lack of molecular tools. Here we present the optimization of SNAP-tag technology via codon optimization specific for E. histolytica. The resultant SNAP protein is highly expressed in amebic trophozoites, and shows proper localization when tagged with an endoplasmic reticulum retention signal. We further demonstrate the capabilities of this system using super resolution microscopy, done for the first time in E. histolytica.     

Use of Bacterially Expressed dsRNA to Downregulate Entamoeba histolytica Gene Expression

Background

Modern RNA interference (RNAi) methodologies using small interfering RNA (siRNA) oligonucleotide duplexes or episomally synthesized hairpin RNA are valuable tools for the analysis of gene function in the protozoan parasite Entamoeba histolytica. However, these approaches still require time-consuming procedures including transfection and drug selection, or costly synthetic molecules.

Principal Findings

Here we report an efficient and handy alternative for E. histolytica gene down-regulation mediated by bacterial double-stranded RNA (dsRNA) targeting parasite genes. The Escherichia coli strain HT115 which is unable to degrade dsRNA, was genetically engineered to produce high quantities of long dsRNA segments targeting the genes that encode E. histolytica β-tubulin and virulence factor KERP1. Trophozoites cultured in vitro were directly fed with dsRNA-expressing bacteria or soaked with purified dsRNA. Both dsRNA delivery methods resulted in significant reduction of protein expression. In vitro host cell-parasite assays showed that efficient downregulation of kerp1 gene expression mediated by bacterial dsRNA resulted in significant reduction of parasite adhesion and lytic capabilities, thus supporting a major role for KERP1 in the pathogenic process. Furthermore, treatment of trophozoites cultured in microtiter plates, with a repertoire of eighty-five distinct bacterial dsRNA segments targeting E. histolytica genes with unknown function, led to the identification of three genes potentially involved in the growth of the parasite.

Conclusions

Our results showed that the use of bacterial dsRNA is a powerful method for the study of gene function in E. histolytica. This dsRNA delivery method is also technically suitable for the study of a large number of genes, thus opening interesting perspectives for the identification of novel drug and vaccine targets.     

Improving Transformation of Staphylococcus aureus Belonging to the CC1, CC5 and CC8 Clonal Complexes

Methicillin resistant Staphylococcus aureus (MRSA) is an opportunistic pathogen found in hospital and community environments that can cause serious infections. A major barrier to genetic manipulations of clinical isolates has been the considerable difficulty in transforming these strains with foreign plasmids, such as those from E. coli, in part due to the type I and IV Restriction Modification (R-M) barriers. Here we combine a Plasmid Artificial Modification (PAM) system with DC10B E. coli cells (dcm mutants) to bypass the barriers of both type I and IV R-M of S. aureus, thus allowing E. coli plasmid DNA to be transformed directly into clinical MRSA strains MW2, N315 and LAC, representing three of the most common clonal complexes. Successful transformation of clinical S. aureus isolates with E. coli-derived plasmids should greatly increase the ability to genetically modify relevant S. aureus strains and advance our understanding of S. aureus pathogenesis.