Andrographolide induced cytotoxicity and cell cycle arrest in Giardia trophozoites.

Giardiasis is a prevalent parasitic diarrheal disease caused by Giardia lamblia, affecting people worldwide. Recently, the availability of several drugs for its treatment has highlighted issues such as multidrug resistance, limited effectiveness and undesirable side effects. Therefore, it is necessary to develop alternative new drugs and treatment strategies that can enhance therapeutic outcomes and effectively treat giardiasis. Natural compounds show promise in the search for more potent anti-giardial agents. Our investigation focused on the effect of Andrographolide (ADG), an active compound of the Andrographis paniculata plant, on Giardia lamblia, assessing trophozoite growth, morphological changes, cell cycle arrest, DNA damage and inhibition of gene expression associated with pathogenic factors. ADG demonstrated anti-Giardia activity almost equivalent to the reference drug metronidazole, with an IC50 value of 4.99 µM after 24 h of incubation. In cytotoxicity assessments and morphological examinations, it showed significant alterations in trophozoite shape and size and effectively hindered the adhesion of trophozoites. It also caused excessive ROS generation, DNA damage, cell cycle arrest and inhibited the gene expression related to pathogenesis. Our findings have revealed the anti-giardial efficacy of ADG, suggesting its potential as an agent against Giardia infections. This could offer a natural and low-risk treatment option for giardiasis, reducing the risk of side effects and drug resistance.

Effect of magnetic field on the growth of the cultured Entamoeba histolytica isolated from patients in Palestine.

Static magnetic field (SMF) is generated in vicinity of moving charge or current passing through conductor. In this study, we aimed to investigate the effect of SMF on the growth of the cultured Entamoeba histolytica (E. histolytica) trophozoites. Different SMF strengths with maximum value equals 30 mT (mT) was applied on the E.histolytica for different periods of times: 0 h, 24 h, 48 h, and 72 h. A modified diphasic liver infusion agar medium was used for culturing E. histolytica in vitro. The results showed the successful stabilization of culture of E. histolytica trophozoites. If we kept the sample for longer time, e. g. 14 days, the growth rate decreases to zero. When applying 10 mT and 15 mT SMF on the sample, it is found that the cultivated E. histolytica trophozoites dies after 4 and 2 days respectively. The experiments suggested that the SMF inhibited the growth and the propagation of E. histolytica cells. In addition, it completely killed all the cells in a short time interval which depend on the SMF strength. It is concluded that the SMFs inhibits the growth of E. histolytica and change the morphology of these cells. Thus, we recommend to use SMF as treatment to mitigate the growth of E. histolytica.

In vitro anti-Acanthamoeba activity of flavonoid glycosides isolated from Delphinium gracile, D. staphisagria, Consolida oliveriana and Aconitum napellus.

Acanthamoeba spp. are widely distributed in the environment and cause serious infections in humans. Treatment of Acanthamoeba infections is very challenging and not always effective which requires the development of more efficient drugs against Acanthamoeba spp. The purpose of the present study was to test medicinal plants that may be useful in the treatment of Acanthamoeba spp. Here we evaluated the trophozoital and cysticidal activity of 13 flavonoid glycosides isolated from Delphinium gracile, D. staphisagria, Consolida oliveriana and from Aconitum napellus subsp. Lusitanicum against the amoeba Acanthamoeba castellanii. AlamarBlue Assay Reagent® was used to determine the activity against trophozoites of A. castellanii, and cytotoxic using Vero cells. Cysticidal activity was assessed on treated cysts by light microscopy using a Neubauer chamber to quantify cysts and trophozoites. Flavonoids 1, 2, 3 and 4 showed higher trophozoital activity and selectivity indexes than the reference drug chlorhexidine digluconate. In addition, flavonoid 2 showed 100% cysticidal activity at a concentration of 50 µm, lower than those of the reference drug and flavonoid 3 (100 µm). These results suggest that flavonoids 2 and 3 might be used for the development of novel therapeutic approaches against Acanthamoeba infections after satisfactory in vivo evaluations.

Protein Sumoylation Is Crucial for Phagocytosis in Entamoeba histolytica Trophozoites.

Posttranslational modifications provide Entamoeba histolytica proteins the timing and signaling to intervene during different processes, such as phagocytosis. However, SUMOylation has not been studied in E. histolytica yet. Here, we characterized the E. histolytica SUMO gene, its product (EhSUMO), and the relevance of SUMOylation in phagocytosis. Our results indicated that EhSUMO has an extended N-terminus that differentiates SUMO from ubiquitin. It also presents the GG residues at the C-terminus and the ΨKXE/D binding motif, both involved in target protein contact. Additionally, the E. histolytica genome possesses the enzymes belonging to the SUMOylation-deSUMOylation machinery. Confocal microscopy assays disclosed a remarkable EhSUMO membrane activity with convoluted and changing structures in trophozoites during erythrophagocytosis. SUMOylated proteins appeared in pseudopodia, phagocytic channels, and around the adhered and ingested erythrocytes. Docking analysis predicted interaction of EhSUMO with EhADH (an ALIX family protein), and immunoprecipitation and immunofluorescence assays revealed that the association increased during phagocytosis; whereas the EhVps32 (a protein of the ESCRT-III complex)-EhSUMO interaction appeared stronger since basal conditions. In EhSUMO knocked-down trophozoites, the bizarre membranous structures disappeared, and EhSUMO interaction with EhADH and EhVps32 diminished. Our results evidenced the presence of a SUMO gene in E. histolytica and the SUMOylation relevance during phagocytosis. This is supported by bioinformatics screening of many other proteins of E. histolytica involved in phagocytosis, which present putative SUMOylation sites and the ΨKXE/D binding motif.

A polo-like kinase modulates cytokinesis and flagella biogenesis in Giardia lamblia.

BACKGROUND: Polo-like kinases (PLKs) are conserved serine/threonine kinases that regulate the cell cycle. To date, the role of Giardia lamblia PLK (GlPLK) in cells has not been studied. Here, we report our investigation on the function of GlPLK to provide insight into the role of this PKL in Giardia cell division, especially during cytokinesis and flagella formation. METHODS: To assess the function of GIPLK, Giardia trophozoites were treated with the PLK-specific inhibitor GW843286X (GW). Using a putative open reading frame for the PLK identified in the Giardia genomic database, we generated a transgenic Giardia expressing hemagglutinin (HA)-tagged GlPLK and used this transgenic for immunofluorescence assays (IFAs). GlPLK expression was knocked down using an anti-glplk morpholino to observe its effect on the number of nuclei number and length of flagella. Giardia cells ectopically expressing truncated GlPLKs, kinase domain + linker (GlPLK-KDL) or polo-box domains (GlPLK-PBD) were constructed for IFAs. Mutant GlPLKs at Lys51, Thr179 and Thr183 were generated by site-directed mutagenesis and then used for the kinase assay. To elucidate the role of phosphorylated GlPLK, the phosphorylation residues were mutated and expressed in Giardia trophozoites RESULTS: After incubating trophozoites with 5 µM GW, the percentage of cells with > 4 nuclei and longer caudal and anterior flagella increased. IFAs indicated that GlPLK was localized to basal bodies and flagella and was present at mitotic spindles in dividing cells. Morpholino-mediated GlPLK knockdown resulted in the same phenotypes as those observed in GW-treated cells. In contrast to Giardia expressing GlPLK-PBD, Giardia expressing GlPLK-KDL was defective in terms of GIPLK localization to mitotic spindles and had altered localization of the basal bodies in dividing cells. Kinase assays using mutant recombinant GlPLKs indicated that mutation at Lys51 or at both Thr179 and Thr183 resulted in loss of kinase activity. Giardia expressing these mutant GlPLKs also demonstrated defects in cell growth, cytokinesis and flagella formation. CONCLUSIONS: These data indicate that GlPLK plays a role in Giardia cell division, especially during cytokinesis, and that it is also involved in flagella formation.

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.

Development of a simple host-free medium for efficient prezoosporulation of Perkinsus olseni trophozoites cultured in vitro.

The parasitizing stage (trophozoite) of the protozoan parasite Perkinsus olseni progresses to the dormant stage (prezoosporangium) immediately after the death of the host through physiologically and morphologically drastic changes. This development is reproducible in Ray's fluid thioglycollate medium (RFTM). In this study, supplementation with tissue extract from a host, the Manila clam, significantly improved the efficiency of development, as determined by the numbers and sizes of developed prezoosporangia. Similar results were seen following supplementation with boiled host tissue extract, which indicates that a thermally stable component of the host is required for the parasite's development. Subsequently, we found that a commercially available lipid concentrate significantly increased prezoosporulation without host tissue, suggesting that the lipids in host tissue enhance prezoosporangia development. Moreover, we determined that yeast extract, sodium thioglycollate, and sodium chloride were the only components of RFTM required for prezoosporulation. Based on these findings, we prepared a simple, host-free medium for P. olseni prezoosporulation-Lipid concentrate Yeast extract Medium (LpcYM)-consisting of yeast extract, lipid concentrate, sodium thioglycollate, and sodium chloride. We confirmed that the prezoosporangia developed in LpcYM produce zoospores that are infectious to Manila clams and that trophozoites of other Perkinsus species (P. marinus, P. honshuensis, and P. chesapeaki) also develop to prezoosporangia in this host-free medium. As LpcYM has the simplest composition of prezoosporulation media available thus far, it enables us to conduct molecular and biochemical studies examining the drastic transformation process of this parasite.

In vitro Cultivation of Plasmodium falciparum.

OBJECTIVE: Plasmodium falciparum is a protozoan parasite that causes many deaths worldwide. It's cultivation in an in vitro culture setting contributes significantly to scientific studies. However, there are no laboratories in Turkey that cultivate P. falciparum in vitro. Hence, the purpose of this study was to cultivate P. falciparum in vitro. METHODS: Five P. falciparum strains were used in our study and were kept frozen in liquid nitrogen tanks. These parasite strains were then thawed in a 37 °C water bath and transferred to the Albumax-complete medium that was previously prepared. After that, the petri dishes were placed in the chamber. For 30 seconds, a special gas mixture containing 5% CO2, 5% O2 and 90% N2 was added into the chamber which was placed in a 37 °C oven and left for incubation for 2 days. At the end of the incubation period, thin smear preparations were prepared from the medium, stained with Giemsa and examined using an immersion lens. RESULTS: Examination of the smears revealed that trophozoite and schizont forms of all P. falciparum isolates were present at a rate of 2% in in vitro culture medium. CONCLUSION: As a result of our study, the in vitro culture of P. falciparum was successfully developed. With this, several projects such as biological and chemical characteristics, pathogenicity, phenotypic and molecular-level drug sensitivities and parasite vaccination studies can be carried out more easily in our country.

Four-Dimensional Characterization of the Babesia divergens Asexual Life Cycle, from the Trophozoite to the Multiparasite Stage.

Babesia is an apicomplexan parasite of significance that causes the disease known as babesiosis in domestic and wild animals and in humans worldwide. Babesia infects vertebrate hosts and reproduces asexually by a form of binary fission within erythrocytes/red blood cells (RBCs), yielding a complex pleomorphic population of intraerythrocytic parasites. Seven of them, clearly visible in human RBCs infected with Babesia divergens, are considered the main forms and named single, double, and quadruple trophozoites, paired and double paired pyriforms, tetrad or Maltese Cross, and multiparasite stage. However, these main intraerythrocytic forms coexist with RBCs infected with transient parasite combinations of unclear origin and development. In fact, little is understood about how Babesia builds this complex population during its asexual life cycle. By combining cryo-soft X-ray tomography and video microscopy, main and transitory parasites were characterized in a native whole cellular context and at nanometric resolution. The architecture and kinetics of the parasite population was observed in detail and provide additional data to the previous B. divergens asexual life cycle model that was built on light microscopy. Importantly, the process of multiplication by binary fission, involving budding, was visualized in live parasites for the first time, revealing that fundamental changes in cell shape and continuous rounds of multiplication occur as the parasites go through their asexual multiplication cycle. A four-dimensional asexual life cycle model was built highlighting the origin of several transient morphological forms that, surprisingly, intersperse in a chronological order between one main stage and the next in the cycle.IMPORTANCE Babesiosis is a disease caused by intraerythrocytic Babesia parasites, which possess many clinical features that are similar to those of malaria. This worldwide disease is increasing in frequency and geographical range and has a significant impact on human and animal health. Babesia divergens is one of the species responsible for human and cattle babesiosis causing death unless treated promptly. When B. divergens infects its vertebrate hosts, it reproduces asexually within red blood cells. During its asexual life cycle, B. divergens builds a population of numerous intraerythrocytic (IE) parasites of difficult interpretation. This complex population is largely unexplored, and we have therefore combined three- and four-dimensional imaging techniques to elucidate the origin, architecture, and kinetics of IE parasites. Unveiling the nature of these parasites has provided a vision of the B. divergens asexual cycle in unprecedented detail and is a key step to develop control strategies against babesiosis.

Eukaryote-Conserved Methylarginine Is Absent in Diplomonads and Functionally Compensated in Giardia.

Methylation is a common posttranslational modification of arginine and lysine in eukaryotic proteins. Methylproteomes are best characterized for higher eukaryotes, where they are functionally expanded and evolved complex regulation. However, this is not the case for protist species evolved from the earliest eukaryotic lineages. Here, we integrated bioinformatic, proteomic, and drug-screening data sets to comprehensively explore the methylproteome of Giardia duodenalis-a deeply branching parasitic protist. We demonstrate that Giardia and related diplomonads lack arginine-methyltransferases and have remodeled conserved RGG/RG motifs targeted by these enzymes. We also provide experimental evidence for methylarginine absence in proteomes of Giardia but readily detect methyllysine. We bioinformatically infer 11 lysine-methyltransferases in Giardia, including highly diverged Su(var)3-9, Enhancer-of-zeste and Trithorax proteins with reduced domain architectures, and novel annotations demonstrating conserved methyllysine regulation of eukaryotic elongation factor 1 alpha. Using mass spectrometry, we identify more than 200 methyllysine sites in Giardia, including in species-specific gene families involved in cytoskeletal regulation, enriched in coiled-coil features. Finally, we use known methylation inhibitors to show that methylation plays key roles in replication and cyst formation in this parasite. This study highlights reduced methylation enzymes, sites, and functions early in eukaryote evolution, including absent methylarginine networks in the Diplomonadida. These results challenge the view that arginine methylation is eukaryote conserved and demonstrate that functional compensation of methylarginine was possible preceding expansion and diversification of these key networks in higher eukaryotes.

The role of the Acanthamoeba castellanii Sir2-like protein in the growth and encystation of Acanthamoeba.

BACKGROUND: The encystation of Acanthamoeba leads to the development of resilient cysts from vegetative trophozoites. This process is essential for the survival of parasites under unfavorable conditions. Previous studies have reported that, during the encystation of A. castellanii, the expression levels of encystation-related factors are upregulated. However, the regulatory mechanisms for their expression during the encystation process remains unknown. Proteins in the sirtuin family, which consists of nicotinamide adenine dinucleotide-dependent deacetylases, are known to play an important role in various cellular functions. In the present study, we identified the Acanthamoeba silent-information regulator 2-like protein (AcSir2) and examined its role in the growth and encystation of Acanthamoeba. METHODS: We obtained the full-length sequence for AcSir2 using reverse-transcription polymerase chain reaction. In Acanthamoeba transfectants that constitutively overexpress AcSir2 protein, SIRT deacetylase activity was measured, and the intracellular localization of AcSir2 and the effects on the growth and encystation of trophozoites were examined. In addition, the sirtuin inhibitor salermide was used to determine whether these effects were caused by AcSir2 overexpression RESULTS: AcSir2 was classified as a class-IV sirtuin. AcSir2 exhibited functional SIRT deacetylase activity, localized mainly in the nucleus, and its transcription was upregulated during encystation. In trophozoites, AcSir2 overexpression led to greater cell growth, and this growth was inhibited by treatment with salermide, a sirtuin inhibitor. When AcSir2 was overexpressed in the cysts, the encystation rate was significantly higher; this was also reversed with salermide treatment. In AcSir2-overexpressing encysting cells, the transcription of cellulose synthase was highly upregulated compared with that of control cells, and this upregulation was abolished with salermide treatment. Transmission electron microscope-based ultrastructural analysis of salermide-treated encysting cells showed that the structure of the exocyst wall and intercyst space was impaired and that the endocyst wall had not formed. CONCLUSIONS: These results indicate that AcSir2 is a SIRT deacetylase that plays an essential role as a regulator of a variety of cellular processes and that the regulation of AcSir2 expression is important for the growth and encystation of A. castellanii.

Anti-Acanthamoeba effect of potassium isostearate for use as a multipurpose solution.

Acanthamoeba is one of the organisms that cause corneal infection. In this study, attention was focused on potassium isostearate (iso-C18K, a branched chain fatty acid salt) for use in a multipurpose solution (MPS) against Acanthamoeba. An anti-amoebic test against Acanthamoeba castellanii ATCC 30010 (trophozoites type) was conducted. As a result, a growth reduction effect of 4 log units (99.99% suppression) was observed after incubation with 150 mM (5.0 w/v%) iso-C18K for 10 minutes. Furthermore, after the amoeba suspension was mixed with iso-C18K, disruption of cell membranes were observed, and the minimum amoebacidal concentration (MAC) at that time was 9.6 mM (0.31 w/v%). To evaluate the effectiveness as an MPS, assessment by verification tests was conducted using contact lenses. Reducing the concentration of iso-C18K caused a decrease in the number of viable cells, which was confirmed at a MAC of 1.2 mM (0.039 w/v%).

Trichomonas vaginalis NTPDase inhibited by lycorine modulates the parasite-neutrophil interaction.

Lycorine is an Amaryllidaceae alkaloid that presents anti-Trichomonas vaginalis activity. T. vaginalis causes trichomoniasis, the most common non-viral sexually transmitted infection. The modulation of T. vaginalis purinergic signaling through the ectonucleotidases, nucleoside triphosphate diphosphohydrolase (NTPDase), and ecto-5'-nucleotidase represents new targets for combating the parasite. With this knowledge, the aim of this study was to investigate whether NTPDase and ecto-5'-nucleotidase inhibition by lycorine could lead to extracellular ATP accumulation. Moreover, the lycorine effect on the reactive oxygen species (ROS) production by neutrophils and parasites was evaluated as well as the alkaloid toxicity. The metabolism of purines was assessed by HPLC. ROS production was measured by flow cytometry. Cytotoxicity against epithelial vaginal cells and fibroblasts was tested, as well as the hemolytic effect of lycorine and its in vivo toxicity in Galleria mellonella larvae. Our findings showed that lycorine caused ATP accumulation due to NTPDase inhibition. The alkaloid did not affect the ROS production by T. vaginalis; however, it increased ROS levels in neutrophils incubated with lycorine-treated trophozoites. Lycorine was cytotoxic against vaginal epithelial cells and fibroblasts; conversely, it was not hemolytic neither exhibited toxicity against the in vivo model of G. mellonella larvae. Overall, besides having anti-T. vaginalis activity, lycorine modulates ectonucleotidases and stimulates neutrophils to secrete ROS. This mechanism of action exerted by the alkaloid could enhance the susceptibility of T. vaginalis to host immune cell, contributing to protozoan clearance.

Giardia lamblia: Laboratory Maintenance, Lifecycle Induction, and Infection of Murine Models.

Giardia lamblia is a protozoan parasite that is found ubiquitously throughout the world and is a major contributor to diarrheal disease. Giardia exhibits a biphasic lifestyle existing as either a dormant cyst or a vegetative trophozoite. Infections are typically initiated through the consumption of cyst-contaminated water or food. Giardia was first axenized in the 1970s and can be readily maintained in a laboratory setting. Additionally, Giardia is one of the few protozoans that can be induced to complete its complete lifecycle using laboratory methods. In this article, we outline protocols to maintain Giardia and induce passage through its lifecycle. We also provide protocols for infecting and quantifying parasites in an animal infection model. © 2020 Wiley Periodicals LLC. Basic Protocol 1: In vitro maintenance and growth of Giardia trophozoites Basic Protocol 2: In vitro encystation of Giardia cysts Basic Protocol 3: In vivo infections using Giardia trophozoites.

Anti-PfGARP activates programmed cell death of parasites and reduces severe malaria.

Malaria caused by Plasmodium falciparum remains the leading single-agent cause of mortality in children1, yet the promise of an effective vaccine has not been fulfilled. Here, using our previously described differential screening method to analyse the proteome of blood-stage P. falciparum parasites2, we identify P. falciparum glutamic-acid-rich protein (PfGARP) as a parasite antigen that is recognized by antibodies in the plasma of children who are relatively resistant-but not those who are susceptible-to malaria caused by P. falciparum. PfGARP is a parasite antigen of 80 kDa that is expressed on the exofacial surface of erythrocytes infected by early-to-late-trophozoite-stage parasites. We demonstrate that antibodies against PfGARP kill trophozoite-infected erythrocytes in culture by inducing programmed cell death in the parasites, and that vaccinating non-human primates with PfGARP partially protects against a challenge with P. falciparum. Furthermore, our longitudinal cohort studies showed that, compared to individuals who had naturally occurring anti-PfGARP antibodies, Tanzanian children without anti-PfGARP antibodies had a 2.5-fold-higher risk of severe malaria and Kenyan adolescents and adults without these antibodies had a twofold-higher parasite density. By killing trophozoite-infected erythrocytes, PfGARP could synergize with other vaccines that target parasite invasion of hepatocytes or the invasion of and egress from erythrocytes.

Ceramide synthase 2 knockdown suppresses trophozoite growth, migration, in vitro encystment and excystment of Entamoeba invadens.

Entamoeba invadens is the protozoan which causes multiple damages in reptiles and is considered a prototype for the study of the Entamoeba encystment/excystment in vitro. Here we report that EinCerS2 knockdown promoted decrease in sphingomyelin (SM) subspecies with long-chain fatty acids (24:0) down to 50% but increase sphingolipids with short-chain fatty acids (16:0) up to three times in both trophozoites and cysts of E. invadens. EinCerS2 silencing also resulted in decreased trophozoites' movement, proliferation, cysts formation, and trophozoites hatched after excystment. By immunofluorescence assays, a polyclonal antibody against EinCerS2 detected the enzyme in the cytoplasm of E. invadens trophozoites, colocalizing with Endoplasmic Reticulum-resident cognate EiSERCA. Interestingly, EinCerS2 was redistributed close to the plasma membrane during encystation, suggesting that the generation of diacylglycerol (DAG) via synthesis of sphingolipids and the activation protein kinase C might participate in the encystment process of E. invadens.

Vitamin supplementation increases the virulence of Entamoeba histolytica grown axenically.

As a consequence of axenic growth and the elimination of accompanying bacterial flora, Entamoeba histolytica virulence decreases rapidly, and pathogenicity is lost. This paper evaluated the impact of vitamin supplementation on the pathogenicity of E. histolytica. Growth of E. histolytica trophozoites, cultured axenically in PEHPS (a Spanish acronym for the main ingredients - casein peptone, liver, pancreas extract and bovine serum) medium, with or without vitamins, exhibited a similar growth rate. However, the vitamin-enriched PEHPS preparations expressed 2.65 times more haemolytic activity (at 60 min: 98 vs 48%, P < 0.05), 2.5 times more phospholipase A2 activity at 150 min of incubation and generated more hepatic abscesses (88 vs 60%, P = 0.05) than the preparations without vitamins. The haemolytic and phospholipase A2 activity for the PEHPS - V preparations were restored following vitamin supplementation with A and D. These data highlight, for the first time, that vitamins and specifically vitamin A and D were essential for the recovery of amoebic virulence, lost through axenic growth.

Plasma membrane damage repair is mediated by an acid sphingomyelinase in Entamoeba histolytica.

Entamoeba histolytica is a pathogen that during its infective process confronts the host defenses, which damages the amoebic plasma membrane (PM), resulting in the loss of viability. However, it is unknown whether amoebic trophozoites are able to repair their PM when it is damaged. Acid sphingomyelinases (aSMases) have been reported in mammalian cells to promote endocytosis and removal of PM lesions. In this work, six predicted amoebic genes encoding for aSMases were found to be transcribed in the HM1:IMSS strain, finding that the EhaSM6 gene is the most transcribed in basal growth conditions and rendered a functional protein. The secreted aSMase activity detected was stimulated by Mg+2 and inhibited by Co+2. Trophozoites that overexpress the EhaSM6 gene (HM1-SM6HA) exhibit an increase of 2-fold in the secreted aSMase activity. This transfectant trophozoites exposed to pore-forming molecules (SLO, Magainin, ß-Defensin 2 and human complement) exhibited an increase from 6 to 25-fold in the secreted aSMase activity which correlated with higher amoebic viability in a Ca+2 dependent process. However, other agents that affect the PM such as hydrogen peroxide also induced an increase of secreted aSMase, but to a lesser extent. The aSMase6 enzyme is N- and C-terminal processed. Confocal and transmission electron microscopy showed that trophozoites treated with SLO presented a migration of lysosomes containing the aSMase towards the PM, inducing the formation of membrane patches and endosomes in the control strain. These cellular structures were increased in the overexpressing strain, indicating the involvement of the aSMase6 in the PM injury repair. The pore-forming molecules induced an increase in the expression of EhaSM1, 2, 5 and 6 genes, meanwhile, hydrogen peroxide induced an increase in all of them. In all the conditions evaluated, the EhaSM6 gene exhibited the highest levels of induction. Overall, these novel findings show that the aSMase6 enzyme from E. histolytica promotes the repair of the PM damaged with pore-forming molecules to prevent losing cell integrity. This novel system could act when encountered with the lytic defense systems of the host.

Motility and cytoskeletal organisation in the archigregarine Selenidium pygospionis (Apicomplexa): observations on native and experimentally affected parasites.

Representatives of Apicomplexa perform various kinds of movements that are linked to the different stages of their life cycle. Ancestral apicomplexan lineages, including gregarines, represent organisms suitable for research into the evolution and diversification of motility within the group. The vermiform trophozoites and gamonts of the archigregarine Selenidium pygospionis perform a very active type of bending motility. Experimental assays and subsequent light, electron, and confocal microscopic analyses demonstrated the fundamental role of the cytoskeletal proteins actin and tubulin in S. pygospionis motility and allowed us to compare the mechanism of its movement to the gliding machinery (the so-called glideosome concept) described in apicomplexan zoites. Actin-modifying drugs caused a reduction in the movement speed (cytochalasin D) or stopped the motility of archigregarines completely (jasplakinolide). Microtubule-disrupting drugs (oryzalin and colchicine) had an even more noticeable effect on archigregarine motility. The fading and disappearance of microtubules were documented in ultrathin sections, along with the formation of α-tubulin clusters visible after the immunofluorescent labelling of drug-treated archigregarines. The obtained data indicate that subpellicular microtubules most likely constitute the main motor structure involved in S. pygospionis bending motility, while actin has rather a supportive function.

The peripheral vesicles gather multivesicular bodies with different behavior during the Giardia intestinalis life cycle.

Giardia intestinalis presents an intriguing endomembrane system, which includes endoplasmic reticulum and peripheral vesicles (PVs). The PVs have previously been considered to be organelles that display early and late endosomal and lysosomal properties. Some of these vesicles accumulate macromolecules ingested by the protozoan and show acid phosphatase activity. It has been previously shown that the parasite releases microvesicles, which contribute to giardiasis pathogenesis; however, the vesicles' origin and the way in which they are released by the parasite still remain unclear. In this study, we induced the parasites to encyst in vitro and analyzed these events using advanced electron microscopy techniques, including focused ion beam and electron microscopy tomography followed by three-dimensional reconstruction, in order to better understand protozoal multivesicular body (MVB) biogenesis. In addition, we performed an ultrastructural analysis of phosphatase activity during differentiation. We demonstrated that some vegetative trophozoites' PVs exhibited morphological characteristics of MVBs with a mean diameter of 50 nm, containing intraluminal vesicles (ILVs).