Killing the Dead: Chemotherapeutic Strategies Against Free‐Living Cyst‐Forming Protists (Acanthamoeba sp. and Balamuthia mandrillaris)

The opportunist free‐living protists such as Acanthamoeba spp. and Balamuthia mandrillaris have become a serious threat to human life. As most available drugs target functional aspects of pathogens, the ability of free‐living protists to transform into metabolically inactive cyst forms presents a challenge in treatment. It is hoped, that the development of broad spectrum antiprotist agents acting against multiple cyst‐forming protists to provide target‐directed inhibition will offer a viable drug strategy in the treatment of these rare infections. Here, we present a comprehensive report on upcoming drug targets, with emphasis on cyst wall biosynthesis along with the related biochemistry of encystment pathways, as we strive to bring ourselves a step closer to being able to combat these deadly diseases.     

Morphological Study of the Encystment and Excystment of Vermamoeba vermiformis Revealed Original Traits

Free‐living amoebae are ubiquitous protozoa commonly found in water. Among them, Acanthamoeba and Vermamoeba (formerly Hartmannella) are the most represented genera. In case of stress, such as nutrient deprivation or osmotic stress, these amoebae initiate a differentiation process, named encystment. It leads to the cyst form, which is a resistant form enabling amoebae to survive in harsh conditions and resist disinfection treatments. Encystment has been thoroughly described in Acanthamoeba but poorly in Vermamoeba. Our study was aimed to follow the encystment/excystment processes by microscopic observations. We show that encystment is quite rapid, as mature cysts were obtained in 9 h, and that cyst wall is composed of two layers. A video shows that a locomotive form is likely involved in clustering cysts together during encystment. As for Acanthamoeba, autophagy is likely active during this process. Specific vesicles, possibly involved in ribophagy, were observed within the cytoplasm. Remarkably, mitochondria rearranged around the nucleus within the cyst, suggesting high needs in energy. Unlike Acanthamoeba and Naegleria, no ostioles were observed in the cyst wall suggesting that excystment is original. During excystment, large vesicles, likely filled with hydrolases, were found in close proximity to cyst wall and digest it. Trophozoite moves inside its cyst wall before exiting during excystment. In conclusion, Vermamoeba encystment/excystment displays original trends as compare to Acanthamoeba.     

Scanning electron microscopy of pathogenic and non-pathogenic Naegleria cysts

Scanning electron microscopy of pathogenic and non-pathogenic Naegleria cysts. International journal for Parasitology4: 139–142. Cysts of 4 strains of non-pathogenic Naegleria gruberi and 5 strains of pathogenic Naegleria fowleri were examined in the scanning electron microscope. Excystment of the Naegleria gruberi amoebae occurred via preformed exit pores in the cyst wall. Similar structures were not found in the cysts of Naegleria fowleri, and excystment occurred by rupture of the cyst wall. The sequence of cyst wall rupture is illustrated for one of the pathogenic strains.     

Cyst‐motile stage relationship,morphology, ultrastructure,and molecular phylogeny of the gymnodinioid dinoflagellate Barrufeta resplendens comb. nov., formerly known as Gyrodinium resplendens,isolated from the Gulf of Mexico

In the present study, we redescribed Gyrodinium resplendens through incubation of process bearing cysts extracted from sediment collected in the northern Gulf of Mexico. The morphology and ultrastructure of the motile stage and cyst stage were examined using light microscopy, scanning electron microscopy, and transmission electron microscopy and this revealed that the species should be transferred to the genus Barrufeta. This genus differs from other gymnodinioid genera in possessing a Smurf‐cap apical structure complex (ASC) and currently encompasses only one species, Barrufeta bravensis. B. resplendens shows a Smurf‐cap ASC that consists of three rows of elongated vesicles with small knobs in the middle one. B. resplendens is very similar to B. bravensis in cell morphology, but can be separated using the ultrastructure such as the shape and location of nucleus and pyrenoids, which highlights the importance of ultrastructure at inter‐specific level in the genus Barrufeta. The unique cysts of B. resplendens are brown and process bearing, and have a tremic archeopyle with a zigzag margin on the dorsal side of the epicyst, and not polar as in cysts of Polykrikos. The cysts do not survive the palynological treatment used here and probably have a wide distribution. Maximum‐likelihood and Bayesian inference were carried out based on partial large subunit ribosomal DNA (LSU rDNA) sequences. Molecular phylogeny supports that the genus Barrufeta is monophyletic, and that the genus Gymnodinium is polyphyletic. Our results suggest that details of the ASC together with ultrastructure are potential features to subdivide the genus Gymnodinium.     

The In Vitro Efficacy of Antimicrobial Agents Against the Pathogenic Free‐Living Amoeba Balamuthia mandrillaris

The free‐living amoeba Balamuthia mandrillaris causes usually fatal encephalitis in humans and animals. Only limited studies have investigated the efficacy of antimicrobial agents against the organism. Assay methods were developed to assess antimicrobial efficacy against both the trophozoite and cyst stage of B. mandrillaris (ATCC 50209). Amphotericin B, ciclopirox olamine, miltefosine, natamycin, paromomycin, pentamidine isethionate, protriptyline, spiramycin, sulconazole and telithromycin had limited activity with amoebacidal levels of > 135–500 μM. However, diminazene aceturate (Berenil^®) was amoebacidal at 7.8 μM and 31.3–61.5 μM for trophozoites and cysts, respectively. Assays for antimicrobial testing may improve the prognosis for infection and aid in the development of primary selective culture isolation media.     

Balamuthia mandrillaris: In Vitro Interactions with Selected Protozoa and Algae

Although Balamuthia mandrillaris was identified more than two decades ago as an agent of fatal granulomatous encephalitis in humans and other animals, little is known about its ecological niche, biological behavior in the environment, food preferences and predators, if any. When infecting humans or other animals, Balamuthia feeds on tissues; and in vitro culture, it feeds on mammalian cells (monkey kidney cells, human lung fibroblasts, and human microvascular endothelial cells). According to recent reports, it is believed that Balamuthia feeds on small amebae, for example, Acanthamoeba that are present in its ecological niche. To test this hypothesis, we associated Balamuthia on a one‐on‐one basis with selected protozoa and algae. We videotaped the behavior of Balamuthia in the presence of a potential prey, its ability to hunt and attack its food, and the time required to eat and cause damage to the target cell by direct contact. We found that B. mandrillaris ingested trophozoites of Naegleria fowleri, Naegleria gruberi, Acanthamoeba spp., Trypanosoma cruzi epimastigotes, Toxoplasma gondii tachyzoites, and Giardia. However, it did not feed on Acanthamoeba cysts or algae. Balamuthia caused cytolysis of T. cruzi epimastigotes and T. gondii tachyzoites by direct contact. Balamuthia trophozoites and cysts were, however, eaten by Paramecium sp.     

Characterization of the extracellular matrix of Phaeodactylum tricornutum (Bacillariophyceae): structure,composition, and adhesive characteristics

The extracellular matrix of the ovoid and fusiform morphotypes of Phaeodactylum tricornutum (Bohlin) was characterized in detail. The structural and nanophysical properties were analyzed by microscopy. Of the two morphotypes, only the ovoid form secretes adhesive mucilage; light microscopy and scanning electron microscopy images showed that the mucilage was secreted from the girdle band region of the cell as cell‐substratum tethers, accumulating on the surface forming a biofilm. After 7 d, the secreted mucilage became entangled, forming adhesive strands that crisscrossed the substratum surface. In the initial secreted mucilage atomic force microscopy identified a high proportion of adhesive molecules without regular retraction curves and some modular‐like adhesive molecules, in the 7 d old biofilm, the adhesive molecules were longer with fewer adhesive events but greater adhesive strength. Chemical characterization was carried out on extracted proteins and polysaccharides. Differences in protein composition, monosaccharide composition, and linkage analysis are discussed in relation to the composition of the frustule and secreted adhesive mucilage. Polysaccharide analysis showed a broad range of monosaccharides and linkages across all fractions with idiosyncratic enrichment of particular monosaccharides and linkages in each fraction. 3‐linked Mannan was highly enriched in the cell frustule fractions indicating a major structural role, while Rhamnose and Fucose derivatives were enriched in the secreted fractions of the ovoid morphotype suggesting involvement in cell adhesion. Comparison of SDS‐PAGE of extracellular proteins showed two major bands for the ovoid morphotype and four for the fusiform morphotype of which only one appeared to be common to both morphotypes.     

Development and evaluation of an off‐the‐slide genotyping technique for identifying Giardia cysts and Cryptosporidium oocysts directly from US EPA Method 1623 slides

Aims

This study developed and systematically evaluated performance and limit of detection of an off‐the‐slide genotyping procedure for both Cryptosporidium oocysts and Giardia cysts.

Methods and Results

Slide standards containing flow‐sorted (oo)cysts were used to evaluate the off‐the‐slide genotyping procedure by microscopy and PCR. Results show approximately 20% of cysts and oocysts are lost during staining. Although transfer efficiency from the slide to the PCR tube could not be determined by microscopy, it was observed that the transfer process aided in the physical lysis of the (oo)cysts likely releasing DNA. PCR detection rates for a single event on a slide were 44% for Giardia and 27% for Cryptosporidium, and a minimum of five cysts and 20 oocysts are required to achieve a 90% PCR detection rate. A Poisson distribution analysis estimated the relative PCR target densities and limits of detection, it showed that 18 Cryptosporidium and five Giardia replicates are required for a 95% probability of detecting a single (oo)cyst on a slide.

Conclusions

This study successfully developed and evaluated recovery rates and limits of detection of an off‐the‐slide genotyping procedure for both Cryptosporidium and Giardia (oo)cysts from the same slide.

Significance and Impact of the Study

This off‐the‐slide genotyping technique is a simple and low cost tool that expands the applications of US EPA Method 1623 results by identifying the genotypes and assemblages of the enumerated Cryptosporidium and Giardia. This additional information will be useful for microbial risk assessment models and watershed management decisions.     

Subdiffraction‐resolution live‐cell imaging for visualizing thylakoid membranes

The chloroplast is the chlorophyll‐containing organelle that produces energy through photosynthesis. Within the chloroplast is an intricate network of thylakoid membranes containing photosynthetic membrane proteins that mediate electron transport and generate chemical energy. Historically, electron microscopy (EM) has been a powerful tool for visualizing the macromolecular structure and organization of thylakoid membranes. However, an understanding of thylakoid membrane dynamics remains elusive because EM requires fixation and sectioning. To improve our knowledge of thylakoid membrane dynamics we need to consider at least two issues: (i) the live‐cell imaging conditions needed to visualize active processes in vivo; and (ii) the spatial resolution required to differentiate the characteristics of thylakoid membranes. Here, we utilize three‐dimensional structured illumination microscopy (3D‐SIM) to explore the optimal imaging conditions for investigating the dynamics of thylakoid membranes in living plant and algal cells. We show that 3D‐SIM is capable of examining broad characteristics of thylakoid structures in chloroplasts of the vascular plant Arabidopsis thaliana and distinguishing the structural differences between wild‐type and mutant strains. Using 3D‐SIM, we also visualize thylakoid organization in whole cells of the green alga Chlamydomonas reinhardtii. These data reveal that high light intensity changes thylakoid membrane structure in C. reinhardtii. Moreover, we observed the green alga Chromochloris zofingiensis and the moss Physcomitrella patens to show the applicability of 3D‐SIM. This study demonstrates that 3D‐SIM is a promising approach for studying the dynamics of thylakoid membranes in photoautotrophic organisms during photoacclimation processes.     

Formation of the Giardia Cyst Wall: Studies on Extracellular Assembly Using Immunogold Labeling and High Resolution Field Emission SEM

Encystment of the intestinal protozoan, Giardia, is a key step in the life cycle that enables this parasite to be transmitted from host to host via either fecal oral, waterborne, or foodborne transmission. The process of encystment was studied by localizing cyst wall specific antigens with immunofluorescence for light microscopy and immunogold staining for field emission scanning electron microscopy. Chronological sampling of Giardia cultures stimulated with endogenous bile permitted identification of an intracellular and extracellular phase in cyst wall formation, a process which required a total of 14-16 h. The intracellular phase lasted for 8-10 h, while the extracellular phase, involved the appearance of cyst wall antigen on the trophozoite membrane, and the assembly of the filamentous layer, a process requiring an additional 4-6 h for completion of mature cysts. The extracellular phase was initiated with the appearance of cyst wall antigen on small protrusions of the trophozoite membrane (-15 nm), which became enlarged with time to caplike structures ranging up to 100 nm in diameter. Caplike structures involved with filament growth were detected over the entire surface of the trophozoite including the adhesive disc and flagella. Encysting cells rounded up, lost attachment to the substratum, and became enclosed in a layer of filaments. Late stages in encystment included a “tailed” cyst in which flagella were not fully retracted into the cyst. Clusters of cysts were seen in which filaments at the surface of one cyst were connected with the surface of adjacent cysts or the “tailed” processes of adjacent cysts, suggesting that the growth of cyst wall filaments may be at the terminal end. In conclusion, the process of encystment has been shown to consist of two morphologically different stages (intracellular and extracellular) which requires 16 h for completion. Further investigation of the extracellular stage with regard to assembly of the filamentous layer of the cyst wall may lead to innovative methods for interfering with production of an intact functional cyst wall, and thereby, regulation of viable Giardia cyst release from the host.     

Carbohydrate analysis of Acanthamoeba castellanii

We analyzed biochemically Acanthamoeba castellanii trophozoites, intact cysts and cyst walls belonging to the T4 genotype using gas chromatography combined with mass spectrometry. Cyst walls were prepared by removing intracellular material from cysts by pre-treating them with sodium dodecyl sulphate (SDS) containing dithiothreitol, and then subjecting these to a series of sequential enzymatic digestions using amyloglucosidase, papain, DNase, RNase and proteinase K. The resulting “cyst wall” material was subsequently lyophilized and subjected to glycosyl composition analysis. Transmission electron microscopy confirmed the removal of intracystic material following enzymatic treatment. Our results showed that treated A. castellanii trophozoites, intact cysts and cyst walls contained various sugar moieties, of which a high percentage was galactose and glucose, in addition to small amounts of mannose, and xylose. Linkage analysis revealed several types of glycosidic linkages including the 1,4-linked glucosyl conformation, indicative of cellulose. Inhibitor studies suggested that, beside sugar synthesis, cytoskeletal re-arrangement and mitogen-activated protein kinase-mediated pathways are involved in A. castellanii encystment.     

Pseudoparamoeba garorimi n. sp., with Notes on Species Distinctions within the Genus

A new marine species of naked lobose amoebae Pseudoparamoeba garorimi n. sp. (Amoebozoa, Dactylopodida) isolated from intertidal marine sediments of Garorim Bay, Korea was studied with light and transmission electron microscopy. This species has a typical set of morphological characters for a genus including the shape of the locomotive form, type of subpseudopodia and the tendency to form the single long waving pseudopodium in locomotion. Furthermore, it has the same cell surface structures as were described for the type species, Pseudoparamoeba pagei: blister‐like glycostyles with hexagonal base and dome‐shaped apex; besides, cell surface bears hair‐like outgrowths. The new species described here lacks clear morphological distinctions from the two other Pseudoparamoeba species, but has considerable differences in the 18S rDNA and COX1 gene sequences. Phylogenetic analysis based on 18S rDNA placed P. garorimi n. sp. at the base of the Pseudoparamoeba clade with high PP/BS support. The level of COX1 sequence divergence was 22% between P. garorimi n. sp. and P. pagei and 25% between P. garorimi n. sp. and P. microlepis. Pseudoparamoeba species are hardly distinguishable by morphology alone, but display clear differences in 18S rDNA and COX1 gene sequences.     

Evaluation of prokaryotic and eukaryotic cells as food source for Balamuthia mandrillaris

Balamuthia mandrillaris is a recently identified free-living protozoan pathogen that can cause fatal granulomatous encephalitis in humans. Recent studies have shown that B. mandrillaris consumes eukaryotic cells such as mammalian cell cultures as food source. Here, we studied B. mandrillaris interactions with various eukaryotic cells including, monkey kidney fibroblast-like cells (COS-7), human brain microvascular endothelial cells (HBMEC) and Acanthamoeba (an opportunistic protozoan pathogen) as well as prokaryotes, Escherichia coli. B. mandrillaris exhibited optimal growth on HBMEC compared with Cos-7 cells. In contrast, B. mandrillaris did not grow on bacteria but remained in the trophozoite stage. When incubated with Acanthamoeba trophozoites, B. mandrillaris produced partial Acanthamoeba damage and the remaining Acanthamoeba trophozoites underwent encystment. However, B. mandrillaris were unable to consume Acanthamoeba cysts. Next, we observed that B. mandrillaris-mediated Acanthamoeba encystment is a contact-dependent process that requires viable B. mandrillaris. In support, conditioned medium of B. mandrillaris did not stimulate Acanthamoeba encystment nor did lysates of B. mandrillaris. Overall, these studies suggest that B. mandrillaris target Acanthamoeba in the trophozoite stage; however, Acanthamoeba possess the ability to defend themselves by forming cysts, which are resistant to B. mandrillaris. Further studies will examine the mechanisms associated with food selectivity in B. mandrillaris.     

Starvation and Encystment of a Soil Amoeba Hartmannella castellanii*

SYNOPSIS. The behavior of the amoeba H. castellanii was investigated in various carbon and nitrogen deficient media with a view to developing a satisfactory replacement medium for the study of encystment and excystment. Media which had been devised for other soil amoebae did not cause H. castellanii to encyst. In these media there was an efflux of material from the cells which was independent of osmolarity but which was minimized by the addition of magnesium. Maximal encystment occurred in a medium containing magnesium chloride alone. The cysts produced in the magnesium chloride replacement medium are viable and readily excyst when resuspended in the growth medium. The cysts contain cellulose, which is not present in the vegetative amoebae, and differ from the amoebae in their greater resistance to induced lysis and mechanical injury.     

Relationship between the dinoflagellate cyst Spiniferites pachydermus and Gonyaulax ellegaardiae sp. nov. from Izmir Bay,Turkey, and molecular characterization

Here, we established the cyst‐motile stage relation‐ship for Spiniferites pachydermus through incubation of cysts with a characteristically microreticulate/perforate surface isolated from Izmir Bay in the eastern Aegean Sea of the eastern Mediterranean. The morphology of the motile stage was similar to Gonyaulax spinifera but had a different size, overhang, displacement and reticulations. Based on the distinct morphology of the cyst and morphological differences in motile cells, we assigned S. pachydermus from Izmir Bay to the new species Gonyaulax ellegaardiae. We elucidate the phylogenetic relationship of G. ellegaardiae through large and small subunit ribosomal DNA and show that it forms a clade with other species that belong to the G. spinifera complex.     

<Emphasis Type="Italic">Acanthamoeba castellanii</Emphasis> an environmental host for <Emphasis Type="Italic">Shigella dysenteriae</Emphasis> and <Emphasis Type="Italic">Shigella sonnei</Emphasis>

The interaction between Shigella dysenteriae or Shigella sonnei and Acanthamoeba castellanii was studied by viable counts, gentamicin assay and electron microscopy. The result showed that Shigella dysenteriae or Shigella sonnei grew and survived in the presence of amoebae for more than 3 weeks. Gentamicin assay showed that the Shigella were viable inside the Acanthamoeba castellanii which was confirmed by electron microscopy that showed the Shigella localized in the cytoplasm of the Acanthamoeba castellanii. In conclusion, the relationship between Shigella dysenteriae and Shigella sonnei with Acanthamoeba castellanii is symbiotic, and accordingly free-living amoebae may serve as a transmission reservoir for Shigella in water.     

Catecholamines inEntamoebae: Recent (re)discoveries

Free-living and enteric amoebae have similar two-stage life cycles, and both organisms depend on being able to monitor environmental conditions to determine whether to continue multiplying as trophozoites, or to differentiate into dormant or transmissible cysts. Conditions that support high trophozoite densities might also be expected to select for mechanisms of information exchange between these cells. We recently determined that trophozoites of at least one species ofEntamoeba release and respond to catecholamine compounds during differentiation from the trophozoite stage into the cyst stage. It turns out that this is not an entirely novel finding, as a number of previous studies have demonstrated parts of this story in free-living or enteric amoebae. We briefly review here major points of the previous studies and describe some of our recent results that have extended them.     

Of mudsnails,terrapins, and flukes: Use of trematodes as a field‐based project in parasitology research

We present an exercise for counting trematode cysts on mudsnails that can be implemented as a field‐based project in course work by students or by volunteers. The exercise involves the digenetic trematode Pleurogonius malaclemys, which infects diamondback terrapins (Malaclemys terrapin) as its definitive host, and eastern mudsnails (Tritia obsoleta) as its intermediate host. The trematode forms macroscopic metacercarial cysts on the shells and opercula of the mudsnails, and the life cycle is completed when terrapins ingest the intermediate hosts with cysts. Previous research has suggested that quantifying cysts on mudsnails can be a relatively simple method to estimate terrapin population sizes where terrapins co‐occur with mudsnails. Three non‐professional scientist groups (including students and volunteers) were tested in their estimates of prevalence and intensity data compared to verified cyst counts. Prevalence data were relatively accurate (~76–78%) and reliable (68–89%) across the groups. The intensity of cysts (on shells and opercula of mudsnails) were mostly within 12% of the verified counts (total mean intensity of 1.81–2.97 cysts/parasitized mudsnail) but varied significantly in two cases due to false positives (likely due to sand grains or debris scored as cysts) or negatives (likely due to missed cysts behind the operculum). The exercise can be completed as a lab or series of labs in courses, and data from such projects could be used in terrapin conservation efforts, particularly in helping scientists determine where terrapins occur. We discuss modification of the exercise for other trematode species in regions around the world and hope this expands recognition by the public of the important role of parasites in ecosystems.