Acanthamoeba Castellani Research Articles

Isolates of Acanthamoeba species in the marine environment in the Philippines.

Acanthamoebae spp. are considered the most commonly occurring free-living amoebae (FLA) in the environment. Their high resilience enables them to thrive in different types of environments. Using purposive sampling, 80 surface water samples were collected from identified coastal sites in Mariveles, Bataan, and Lingayen Gulf (40 water samples for each). Nineteen (23.75%) of the 80 water samples yielded positive amoebic growth during the 14-day culture and microscopic examination. The polymerase chain reaction confirmed Acanthamoeba spp. DNA in isolates MB1, A3, A4, A7, C5, and D3 using JDP1 and JDP2 primer sets. Further sequencing revealed that the isolates belonged to Acanthamoeba sp., Acanthamoeba culbertsoni, Acanthamoeba castellani, and Acanthamoeba genotype T4. The sequences were deposited in GenBank and registered under accession numbers PP741651, PP767364, PP741728, PP741729, PP767365, and PP767366, respectively. Potential risk factors such as waste disposal, expansion of human settlements to coastal locations, and soil runoffs in these environments should be controlled to mitigate the proliferation of potentially pathogenic strains of FLAs.

Structural basis for the toxicity of Legionella pneumophila effector SidH

Legionella pneumophila (LP) secretes more than 300 effectors into the host cytosol to facilitate intracellular replication. One of these effectors, SidH, 253 kDa in size with no sequence similarity to proteins of known function is toxic when overexpressed in host cells. SidH is regulated by the LP metaeffector LubX which targets SidH for degradation in a temporal manner during LP infection. The mechanism underlying the toxicity of SidH and its role in LP infection are unknown. Here, we determined the cryo-EM structure of SidH at 2.7 Å revealing a unique alpha helical arrangement with no overall similarity to known protein structures. Surprisingly, purified SidH came bound to a E. coli EF-Tu/t-RNA/GTP ternary complex which could be modeled into the cryo-EM density. Mutation of residues disrupting the SidH-tRNA interface and SidH-EF-Tu interface abolish the toxicity of overexpressed SidH in human cells, a phenotype confirmed in infection of Acanthamoeba castellani. We also present the cryo-EM structure of SidH in complex with a U-box domain containing ubiquitin ligase LubX delineating the mechanism of regulation of SidH. Our data provide the basis for the toxicity of SidH and into its regulation by the metaeffector LubX.

Activity of Apo-Lactoferrin on Pathogenic Protozoa.

Parasites and other eventually pathogenic organisms require the ability to adapt to different environmental conditions inside the host to assure survival. Some host proteins have evolved as defense constituents, such as lactoferrin (Lf), which is part of the innate immune system. Lf in its iron-free form (apo-Lf) and its peptides obtained by cleavage with pepsin are microbicides. Parasites confront Lf in mucosae and blood. In this work, the activity of Lf against pathogenic and opportunistic parasites such as Cryptosporidium spp., Eimeria spp., Entamoeba histolytica, Giardia duodenalis, Leishmania spp., Trypanosoma spp., Plasmodium spp., Babesia spp., Toxoplasma gondii, Trichomonas spp., and the free-living but opportunistic pathogens Naegleria fowleri and Acanthamoeba castellani were reviewed. The major effects of Lf could be the inhibition produced by sequestering the iron needed for their survival and the production of oxygen-free radicals to more complicated mechanisms, such as the activation of macrophages to phagocytes with the posterior death of those parasites. Due to the great interest in Lf in the fight against pathogens, it is necessary to understand the exact mechanisms used by this protein to affect their virulence factors and to kill them.

Investigation of the anti-parasitic effect of the water extract of Thymbra spicata on Acanthamoeba castellani (L.) trophozoites and cysts

This study aimed to determine the anti-parasitic activity of the water extract obtained from Thymbra spicata (L.). The plant material was extracted with methanol in a Soxhlet apparatus. The extract was then fractionated with water and chloroform. The water phase was frozen and freeze-dried. Afterward, this extract was applied on A. castellani trophozoites and cysts at various concentrations, and the viability rates were determined by counting under the microscope. At the end of the experimental process, it was determined that there was a strong correlation between the increasing extract concentration and the anti-parasitic effect. T. spicata extract was not effective enough to neutralize all cysts at any of the concentrations examined. T. spicata extract, at concentrations of 16.0 and 32.0 mg/ml, removed all trophozoites in the medium from the 24th h of the experiment. Based on this result, it was determined that the plant species evaluated here could be used to treat A. castellani infections. It will be possible to achieve a good effect on the cyst forms of the parasite species by testing higher extract concentrations, which cannot be evaluated during the experimental process. In addition, with advanced analyzes, it will be possible to reveal the chemical substance responsible for the activity in the plant species in question and to perform further analyzes on this substance.

Two Ecdysteroids Isolated from Micropropagated Lychnis flos-cuculi and the Biological Activity of Plant Material.

Genetically uniform plant material, derived from Lychnis flos-cuculi propagated in vitro, was used for the isolation of 20-hydroxyecdysone and polypodine B and subjected to an evaluation of the antifungal and antiamoebic activity. The activity of 80% aqueous methanolic extracts, their fractions, and isolated ecdysteroids were studied against pathogenic Acanthamoeba castellani. Additionally, a Microtox® acute toxicity assay was performed. It was found that an 80% methanolic fraction of root extract exerts the most potent amoebicidal activity at IC50 of 0.06 mg/mL at the 3rd day of treatment. Both ecdysteroids show comparable activity at IC50 of 0.07 mg/mL. The acute toxicity of 80% fractions at similar concentrations is significantly higher than that of 40% fractions. Crude extracts exhibited moderate antifungal activity, with a minimum inhibitory concentration (MIC) within the range of 1.25–2.5 mg/mL. To the best of our knowledge, the present report is the first to show the biological activity of L. flos-cuculi in terms of the antifungal and antiamoebic activities and acute toxicity. It is also the first isolation of the main ecdysteroids from L. flos-cuculi micropropagated, ecdysteroid-rich plant material.

Domestic and Peri-Domestic Study Reveals the Presence of Probably <i>Acanthamoeba castellanii</i>

Acanthamoeba is free living amoeba consisting of many species that are naturally pathogenic and have been isolated from different environmental sources. The purpose of this study was to determine the prevalence and relative abundance of Acanthamoeba species in soil and water samples within the University of Jos environment and to create public health awareness on the dangers of this parasitic protozoan. The study was conducted in the eleven (11) Faculties of the University of Jos. Soil and water samples were collected from each of the faculties, cultured and morphologically identified for positive samples. In addition, quantitative data on occurrence were examined to help better understand the potential risk to the university community. The prevalence of Acanthamoeba based on the chi-square analysis indicates that there is a significant difference between the number of Acanthamoeba species in the soil and water samples collected (P Acanthamoeba based on the Man-Whitney test indicates that there was no significant difference in the distribution of Acanthamoeba species in the water and soil samples between different faculties (P > 0.05). Morphological identification indicates the presence of probably Acanthamoeba castellani. The demonstration of the presence of Acanthamoeba species in soil and water sources calls for awareness among the clinical community, as cases of keratitis and granulomatous amoebic encephalitis might have never been recorded due to lack of expertise or unawareness amongst the clinical community. An improved treatment of water supply and strict adherence to water act needs to be strictly encouraged.

Construction of <i>Yersinia pestis</i> Strain Producing Fluorescent Protein GFP and Prospects of Its Usage

Objective of the study was to construct the recombinant Y. pestis strain producing fluorescent protein GFP and analysis of the prospects of its usage for investigation of interaction of the agent with the protozoa, and also macroorganism of mammals (rodents). Materials and methods . To create the strain producing fluorescent protein GFP, we used the natural Y. pestis strain isolated in 2016 in Gorno-Altai high-mountain plague focus. Fluorescent protein gen was inserted into commercial vector pTurboGFP-B via electroporation. Properties of the obtained recombinant Y. pestis strain 367 pTurboGFP-B were studied using microbiological, biological, and molecular-genetic methods. Results and conclusions . The plague agent strain, containing vector plasmid pTurboGFP-B which encodes synthesis of green fluorescent protein GFP, and being resistant to ampicillin was constructed applying electroporation. The designed Y. pestis strain, producing green fluorescent protein, does not differ from the stock strain by its cultural-morphological, biochemical properties, virulence and survivability in mixed culture with Acanthamoeba castellani . On solid nutrient media, recombinant strain and its subcultures, obtained from infected animals, formed colonies of greenish-yellow color, becoming fluorescent under UV-light. In preparations from animals and from co-cultures with amoeba, fluorescent cells of plague agent were observed. The constructed strain can be utilized as a model one for investigation of interactions between plague agent and cells of protozoa (soil amoeba, nematodes) and mammals under laboratory conditions.

Investigation of the Incidence of Legionella and Free-Living Amoebae in Swimming Pool Waters and Biofilm Specimens in Istanbul by Different Methods

Legionella bacteria living in free form or in biofilm and free-living amoebae (FLA) can infect humans through swimming pools and can cause various diseases. FLA may also threaten the health of swimmers because they are capable of being hosts for Legionella and some other bacteria. The aim of this study was to investigate the presence of total aerobic heterotrophic bacteria (TAHB), FLA and Legionella bacteria in swimming pool waters and biofilm samples in Istanbul by using culture and FISH methods. Water plate count agar (wPCA), buffered charcoal yeast extract (BCYE) agar supplemented with glycinevancomycin-polymyxin-cycloheximide (GVPC) and Escherichia coli cultivated non-nutrient agar (NNA) were used for the culture of TAHB, Legionella and FLA. For the FISH method analysis , Leg 705 and Leg PNE1 probes labeled with fluorescent dye for Legionella and ACANTHA probe for Acanthamoeba genus FLA were used. Legionella pneumophila serogroup 1 ATCC 33152, L.pneumophila serogroup 3 ATCC 33155 and Acanthamoeba castellani ATCC 50373 were used as positive controls. TAHB were grown in 92% and 84% of water and biofilm samples. Although Legionella bacteria could not be grown in any of the water samples, it was detected in 6 (24%) water samples by FISH method. Although these bacteria could be grown in 1 (4%) of biofilm samples, 7 (28%) were detected by FISH method. FLA were found to be 16% by culture in water samples and 28% by FISH analysis. These amoebae were detected 8% and 20% in biofilm samples by culture and FISH method, respectively. It was determined that one of the isolates of FLA had thermotolerant activity (potentially pathogenic). L.pneumophila serogroup 1 was detected in one water sample and in four biofilm samples. According to the culture method, TAHB and FLA were found to be more common in water samples than in biofilm samples and Legionella bacteria were more common in biofilm samples than in water samples (p≤ 0.05). In the detection of Legionella bacteria, the superiority of FISH method compared to culture method was found to be statistically significant (p≤ 0.05). In this study, it was found that the number of TAHB in the controlled swimming pools was within the limits determined by the Ministry of Health (≤ 200 cfu/ml). It will be appropriate to examine both water and biofilm samples for the investigation of TAHB, FLA and Legionella. It may be appropriate to use both culture and FISH methods to detect the presence of FLA in water and biofilm samples. This study is the first study to investigate the presence of Legionella and FLA in swimming pools in Istanbul, and further studies are needed to examine more pool water and biofilm samples. With the data obtained, the health principles and controls of swimming pools will be re-considered and will be contributed to public health.

The effect of antiamoebic agents and Ce6‐PDT on acanthamoeba castellani trophozoites and cysts, in vitro

AbstractPurposeTo analyze the concentration dependent effect of diamidines (hexamidine‐diisethionat (HD), propamidin‐isethionate (PD), dibromopropamidine‐diisethionat (DD)); biguanides (polyhexamethylen biguanid (PHMB), chlorhexidine (CH)); natamycin (NM); miltefosine (MF); povidone iodine (PVPI) and chlorin e6 photodynamic therapy (PDT) on Acanthamoeba castellani trophozoites and cysts, in vitro.MethodsAcanthamoeba castellani strain was cultured in 712 peptone‐yeast extract‐glucose (PYG) medium. Thereafter, trophozoites or cysts were cultured in 0.25–0.1% HD, PD or DD, or 0.005–0.02% PHMB, CH or NM or 0.001625–0.0065 MF or 0.25–1% PVPI containing PYG medium for 2 h or underwent Chlorin e6‐PDT. Then, the percentage of dead trophozoites was determined by CytoTox 96® Non‐Radioactive Cytotoxicity assay and trypan blue staining and those of cysts using trypan blue staining. Treated trophozoites and cysts were also inoculated to non‐nutrient agar Eshericia coli plates and were observed for 5 weeks.ResultsAll concentrations of different antiamoebic agents had a significant cytotoxic effect on AK trophozoites and cysts (p < 0.05), except 0.02% PHMB and CH, using trypan blue assay for trophozoites, and Ce6‐PDT for cysts. Observing the agar plates, HD, PD, PHMB, CH, NM and PVPI led to morphological changes of acanthamoeba trophozoites, which could not form cysts within 5 weeks. DD and MF treated cysts could excyst and later encyst again.ConclusionsConservative treatment using different antiamoebic agents or Ce6‐PDT can not eradicate all Acanthamoeba castellani trophozoites and cysts. In vitro analyzis of treatment efficacy of different antiamoebic agents may alow a more specific treatment in acanthamoeba keratitis.

Ex vivo Cytopathic Effect Study of Human Acanthamoeba keratitis and Use of E. coli in Parasitic Culture

Acanthamoeba is free living amoeba and can cause Acanthamoeba keratitis, a serious disease which affects the cornea. It is widely existing in fresh water and soil. Acanthamoeba has three phases during the life cycle; trophozoite and a resistant double walled cyst and protocysts. The pathogenesis starts when the trophozoites bind to the surface of cornea by expressing mannose-binding protein which can bind to mannose glycoproteins on the corneal surface. The contaminated contact lenses can increase the infection by increasing the binding ability of the trophozoites. This project aimed to study the cytopathic effect of different stages Acanthamoeba keratitis. Study strains of Acanthamoeba castellani (ATCC 50370) was cultured and seeded in Hep2 cell lines. E. coli (strain M12) was used to isolate the trophozoites. The third stage, protocysts was made using Neff's medium. The cytopathic effect was checked trophozoites invasion to monolayer cells cultured in a small flask and in 24 well tissue culture plate. The time of destroying Hep2 cells by the trophozoites is less than that of other Acanthamoeba stages. The double ring-shaped cell wall of the cysts may increase the time-kill of Hep2 due to their resistant which was very slow to hatch. Reactivating the trophozoites from cysts then seeding in Hep2 destroyed the cells at less time compared fresh trophozoites as the virulence of the reactivated trophozoites being higher. The result could suggest that the infection of corneas with reactivated trophozoites may be more complex. Finally, the study found that the number of the trophozoites can limit the cytopathic effect of Acanthamoeba.

Microbiocidal characterization of a novel povidone-iodine based rigid contact lens disinfecting solution.

This study investigated the efficacy of a novel povidone-iodine based disinfection solution for rigid gas permeable (RGP) lenses compared to three other currently available multipurpose and hydrogen peroxide solutions. Bactericidal and fungicidal activities were assessed using ISO 14729 reference methods, whilst amoebicidal properties were evaluated using an automated viability counter. All solutions were also assessed for long term storage stability over a 3-month period. The FDA guidelines were adequately achieved by all solutions tested and 3-month storage did not affect their activity against bacterial and fungal agents. Activity against Acanthamoeba castellani trophozoites reached 1-log reduction for the povidone-iodine solution and approached this level for the remaining solutions. Activity against cysts was somewhat reduced and ranged from 78 to 86% reduction in viability. A povidone-iodine based solution, which may offer advantages as resistance to this agent has not been demonstrated and it lacks toxicity, provided equivalent antimicrobial activity to other RGP solutions.

Amoebae, Giant Viruses, and Virophages Make Up a Complex, Multilayered Threesome.

Viral infection had not been observed for amoebae, until the Acanthamoeba polyphaga mimivirus (APMV) was discovered in 2003. APMV belongs to the nucleocytoplasmatic large DNA virus (NCLDV) family and infects not only A. polyphaga, but also other professional phagocytes. Here, we review the Megavirales to give an overview of the current members of the Mimi- and Marseilleviridae families and their structural features during amoebal infection. We summarize the different steps of their infection cycle in A. polyphaga and Acanthamoeba castellani. Furthermore, we dive into the emerging field of virophages, which parasitize upon viral factories of the Megavirales family. The discovery of virophages in 2008 and research in recent years revealed an increasingly complex network of interactions between cell, giant virus, and virophage. Virophages seem to be highly abundant in the environment and occupy the same niches as the Mimiviridae and their hosts. Establishment of metagenomic and co-culture approaches rapidly increased the number of detected virophages over the recent years. Genetic interaction of cell and virophage might constitute a potent defense machinery against giant viruses and seems to be important for survival of the infected cell during mimivirus infections. Nonetheless, the molecular events during co-infection and the interactions of cell, giant virus, and virophage have not been elucidated, yet. However, the genetic interactions of these three, suggest an intricate, multilayered network during amoebal (co-)infections. Understanding these interactions could elucidate molecular events essential for proper viral factory activity and could implicate new ways of treating viruses that form viral factories.

Amoebicidal, antimicrobial and in vitro ROS scavenging activities of Tunisian Rubus ulmifolius Schott, methanolic extract

The present study aimed to evaluate the activity of methanolic extract of Rubus ulmifolius Schott against the Acanthamoeba castellani Neff Strain as well as its antioxidant and antimicrobial effects. The tested extract has a good amoebicidal activity with low IC50 (61.785 ± 1.322 μg/ml) and also has significant activity against both Gram-positive (S. aureus, S. agalactiae) and Gram-negative bacteria (E. coli, S. typhimurium) and against C. albicans. The inhibition zones diameters (IZD) and minimal inhibitory concentration (MIC) values were in the range of 22.5–50 mm and 02.29–4.76 mg ml−1, respectively. In the other hand, the in vitro ROS scavenging activity was evaluated, the tested extract exhibited a good effect on the ·OH radical (89.99% at a concentration of 100 μg/ml) when compared to the ascorbic acid (68.81%). Moreover, the inhibition percentage of superoxide generation by R. ulmifolius extract at 100 μg/ml was greater than ascorbic acid (79.55; 64.79%, respectively). Also, the tested extract showed a high percentage of H2O2 scavenging activity (99.95% at 100 μg/ml).Our findings suggest that R. ulmifolius could be a potential source of natural antioxidant in preventing many diseases associated with oxidative stress, amoebic and bacterial infections.

Amoebicidal activity of α-bisabolol, the main sesquiterpene in chamomile (Matricaria recutita L.) essential oil against the trophozoite stage of Acanthamoeba castellani Neff.

Acanthamoeba genus includes opportunistic pathogens which are distributed worldwide and are causative agents of a fatal encephalitis and severe keratitis in humans and other animals. Until present there are not fully effective therapeutic agents against this pathogen and thus the need to search for novel anti-amoebic compounds is urgent. Recently, essential oils of aromatic and medicinal plants have shown activity against Acanthamoeba strains. Therefore, this study was aimed to evaluate the activity of main component of chamomile essential oil (a sesquiterpene) namely α-bisabolol against the Acanthamoeba castellani Neff strain. After evaluation of the activity and toxicity of this molecule, IC50 values of 20.839 ± 2.015 for treated amoebae as well as low citotoxicty levels in a murine macrophage cell line was observed. Moreover, in order to elucidate mechanism of action of this molecule, changes in chromatin condensation levels, permeability of the plasmatic membrane, the mitochondrial membrane potential and the ATP levels in the treated amoebic strains were checked. The obtained results revealed that α-bisabolol was able to induce apoptosis, increase the permeability of the plasmatic membrane and decrease both mitochondrial and ATP levels in the treated amoebae. Therefore, and given the obtained results, α-bisabolol could be used a future therapeutic agent against Acanthamoeba infections.

Effects of Lead Phytochemicals of Radix Scutellariae on Acanthamoeba.

Acanthamoeba keratitis (AK), which is associated with noncompliant use of contact lenses, remains difficult to treat due to delayed diagnosis and paucity of therapeutic agents. Although improvements in activity against Acanthamoeba infection have been achieved in disinfecting solutions for soft contact lenses, such modifications have not been extended to those for special rigid gas permeable (RGP) contact lenses, which are increasingly used for myopia control in children. Phytochemicals present in herbs used for traditional Chinese medicine may be effective as therapeutic or preventive agents. The purpose of this study was to investigate amoebicidal properties of lead phytochemicals of Radix scutellariae alone and in combination with multipurpose (disinfecting) solutions (MPS) for RGP lenses. Viability of Acanthamoeba castellani and A. polyphaga trophozoites was determined following exposure to four phytochemicals: baicalin, baicalein, wogonoside, and oroxylin A and both alone and in combination with four RGP MPS, using a modified stand-alone technique. As individual agents, wogonoside and oroxylin A showed highest activity against A. castellani and A. polyphaga trophozoites, respectively. For both organisms, the combination of baicalein and oroxylin A was superior. Effectiveness of MPS alone did not exceed 0.27 log reduction, but addition of combined baicalein and oroxylin A resulted in 0.92 and 0.64 log reductions of A. castellani and A. polyphaga, respectively. The combination of baicalein and oroxylin A enhanced the activity of MPS for RGP contact lenses against trophozoites of two pathogens, A. castellani, and A. polyphaga, and offers a potential therapeutic and/or preventative agent for AK.

Efficacité anti-amibienne in vitro de l’association riboflavine/UV-A

Acanthamoeba keratitis is a rare but serious disease and is particularly difficult to treat when the diagnosis is delayed, partly because of the limitations of current therapies. The purpose of our study is to evaluate the anti-amoebic effectiveness of riboflavin and UV-A on Acanthamoeba castellani. We tested the effect of 0.02% chlorhexidine alone (C), the combination of riboflavin 1% and UV-A (UV-A+R), and the combination of the two treatments (R+C+UV-A) on cultures of vegetative and cystic forms of A.castellani. We conducted a parasite count under optical microscopy for each treated area at day 1, 4 and 8. There was a decrease in the number of cysts for all three treatments (C, UV-A+R, R+C+UV-A). This reduction was greater for the plates treated with R+UV-A (P <0.01 at D8) and those treated with C+R+UV-A (P<0.001 at D8) compared to those exposed to chlorhexidine alone (C). There was no decrease in the number of amoebic trophozoites for the three treatments (C, UV-A+R, R+C+UV-A), but encystment was observed. Given the in vitro efficacy of riboflavin combined with UV-A against cystic forms of A.castellani and excellent in vivo tolerance of the procedure, the treatment of acanthamoeba keratitis might be improved by this new therapeutic approach.

Stationary phase and mature infectious forms of Legionella pneumophila produce distinct viable but non‐culturable cells

Legionella pneumophila is an intracellular bacterial parasite of freshwater protozoa and an accidental waterborne human pathogen. L. pneumophila is highly pleomorphic showing several forms that differentiate within its developmental cycle. In water, L. pneumophila produces viable but non-culturable cells (VBNCCs), which remain largely uncharacterized. We produced VBNCCs from two developmental forms of L. pneumophila [stationary phase forms (SPFs) and mature infectious forms (MIFs)] in two water microcosms [double-deionized (dd) and tap water] at 45°C. In contrast with SPFs, MIFs upheld a robust ultrastructure and high viability in the two water microcosms. In dd-water, MIFs and SPFs lost their culturability faster than in tap water and did not consume their poly-β-hydroxybutyrate inclusions. Resuscitation in Acanthamoeba castellani was only possible for VBNCCs produced from SPFs in tap water. Addition of salts to dd-water prolonged L. pneumophila culturability to tap water levels, suggesting that L. pneumophila requires ions to maintain its readiness to resume growth. VBNCCs resisted detergent lysis and digestion in the ciliate Tetrahymena, except for VBNCCs produced from SPFs in dd-water. L. pneumophila VBNCCs thus show distinct traits according to its originating developmental form and the surrounding water microcosm.

Horizontal Transfer and Evolution of Prokaryote Transposable Elements in Eukaryotes

Horizontal transfer (HT) of transposable elements (TEs) plays a key role in prokaryotic evolution, and mounting evidence suggests that it has also had an important impact on eukaryotic evolution. Although many prokaryote-to-prokaryote and eukaryote-to-eukaryote HTs of TEs have been characterized, only few cases have been reported between prokaryotes and eukaryotes. Here, we carried out a comprehensive search for all major groups of prokaryotic insertion sequences (ISs) in 430 eukaryote genomes. We uncovered a total of 80 sequences, all deriving from the IS607 family, integrated in the genomes of 14 eukaryote species belonging to four distinct phyla (Amoebozoa, Ascomycetes, Basidiomycetes, and Stramenopiles). Given that eukaryote IS607-like sequences are most closely related to cyanobacterial IS607 and that their phylogeny is incongruent with that of their hosts, we conclude that the presence of IS607-like sequences in eukaryotic genomes is the result of several HT events. Selection analyses further suggest that our ability to detect these prokaryote TEs today in eukaryotes is because HT of these sequences occurred recently and/or some IS607 elements were domesticated after HT, giving rise to new eukaryote genes. Supporting the recent age of some of these HTs, we uncovered intact full-length, potentially active IS607 copies in the amoeba Acanthamoeba castellani. Overall, our study shows that prokaryote-to-eukaryote HT of TEs occurred at relatively low frequency during recent eukaryote evolution and it sets IS607 as the most widespread TE (being present in prokaryotes, eukaryotes, and viruses).

RpkA, a Highly Conserved GPCR with a Lipid Kinase Domain, Has a Role in Phagocytosis and Anti-Bacterial Defense

RpkA (Receptor phosphatidylinositol kinase A) is an unusual seven-helix transmembrane protein of Dictyostelium discoideum with a G protein coupled receptor (GPCR) signature and a C-terminal lipid kinase domain (GPCR-PIPK) predicted as a phosphatidylinositol-4-phosphate 5-kinase. RpkA-homologs are present in all so far sequenced Dictyostelidae as well as in several other lower eukaryotes like the oomycete Phytophthora, and in the Legionella host Acanthamoeba castellani. Here we show by immunofluorescence that RpkA localizes to endosomal membranes and is specifically recruited to phagosomes. RpkA interacts with the phagosomal protein complex V-ATPase as proteins of this complex co-precipitate with RpkA-GFP as well as with the GST-tagged PIPK domain of RpkA. Loss of RpkA leads to a defect in phagocytosis as measured by yeast particle uptake. The uptake of the pathogenic bacterium Legionella pneumophila was however unaltered whereas its intra-cellular replication was significantly enhanced in rpkA-. The difference between wild type and rpkA- was even more prominent when L. hackeliae was used. When we investigated the reason for the enhanced susceptibility for L. pneumophila of rpkA- we could not detect a difference in endosomal pH but rpkA- showed depletion of phosphoinositides (PIP and PIP2) when we compared metabolically labeled phosphoinositides from wild type and rpkA-. Furthermore rpkA- exhibited reduced nitrogen starvation tolerance, an indicator for a reduced autophagy rate. Our results indicate that RpkA is a component of the defense system of D. discoideum as well as other lower eukaryotes.

In vitro amoebicidal activities of Teucrium polium and T. chamaedrys on Acanthamoeba castellanii trophozoites and cysts

Amoebic keratitis is difficult to treat without total efficacy in some patients because of cysts, which are less susceptible than trophozoites to the usual treatments. The aim of this study is to evaluate the in vitro amoebicidal activity of the methanolic extracts of Teucrium polium and Teucrium chamaedrys. In the presence of methanolic extracts (ranging from 1.0 to 32.0 mg/ml), numbers of the viable Acanthamoeba castellani trophozoites and cysts were decreased during the experimental process. Both extracts showed time- and dose-dependent amoebicidal action on the trophozoites and cysts. Among the extracts tested, T. chamaedrys showed the strongest amoebicidal effect on the trophozoites. In the presence of 16 mg/ml or above extract concentrations, no viable trophozoites were observed within 48 h. In the case of T. polium, no viable trophozoites were observed within 48 h at 32 mg/ml concentration. As expected, cysts were found more resistant to the extracts than the trophozoites.