Activation of artemisinin and heme degradation in Leishmania tarentolae promastigotes: A possible link.

Endoperoxides (EPs) appear to be promising drug candidates against protozoal diseases, including malaria and leishmaniasis. Previous studies have shown that these drugs need an intracellular activation to exert their pharmacological potential. The efficiency of these drugs is linked to the extensive iron demand of these intracellular protozoal parasites. An essential step of the activation mechanism of these drugs is the formation of radicals in Leishmania. Iron is a known trigger for intracellular radical formation. However, the activation of EPs by low molecular iron or by heme iron may strongly depend on the structure of the EPs themselves. In this study, we focused on the activation of artemisinin (Art) in Leishmania tarentolae promastigotes (LtP) in comparison to reference compounds. Viability assays in different media in the presence of different iron sources (hemin/fetal calf serum) showed that IC50 values of Art in LtP were modulated by assay conditions, but overall were within the low micromolar range. Low temperature electron paramagnetic resonance (EPR) spectroscopy of LtP showed that Art shifted the redox state of the labile iron pool less than the EP ascaridole questioning its role as a major activator of Art in LtP. Based on the high reactivity of Art with hemin in previous biomimetic experiments, we focused on putative heme-metabolizing enzymes in Leishmania, which were so far not well described. Inhibitors of mammalian heme oxygenase (HO; tin and chromium mesoporphyrin) acted antagonistically to Art in LtP and boosted its IC50 value for several magnitudes. By inductively coupled plasma methods (ICP-OES, ICP-MS) we showed that these inhibitors do not block iron (heme) accumulation, but are taken up and act within LtP. These inhibitors blocked the conversion of hemin to bilirubin in LtP homogenates, suggesting that an HO-like enzyme activity in LtP exists. NADPH-dependent degradation of Art and hemin was highest in the small granule and microsomal fractions of LtP. Photometric measurements in the model Art/hemin demonstrated that hemin requires reduction to heme and that subsequently an Art/heme complex (λmax 474 nm) is formed. EPR spin-trapping in the system Art/hemin revealed that NADPH, ascorbate and cysteine are suitable reductants and finally activate Art to acyl-carbon centered radicals. These findings suggest that heme is a major activator of Art in LtP either via HO-like enzyme activities and/or chemical interaction of heme with Art.

Ultrastructure of vegetative cells and resting cysts, and live observations of the encystation and excystation processes in Diophrys oligothrix Borror, 1965 (Protista, Ciliophora).

Ciliated protists can form cysts to resist unfavorable environmental conditions and then excyst when environmental conditions become favorable. This study used electron and light microscopy to investigate the structure of vegetative cells and resting cysts, as well as the encysting and excysting processes of Diophrys oligothrix. For the first time, ampules were revealed beneath the pellicle in the genus Diophrys, and their extrusome types differed between Diophrys species. Membrane-packed discs of diverse shapes were found in the cytoplasm just beneath the pellicle around the cytopharynx and were separated by rows of microtubule units. Beneath the discs, some double-layer microtubule structures were detected as well. During encystment, the ventral ciliature was folded in a ventral cavity of the cell, and the caudal cirri were retracted directly into the cyst in a separate cavity on the dorsal side. In the resting cysts, high autophagic activity occurred, possibly including digestion of membrane-packed discs and ampules. Two macronuclear nodules kept their basic shape, although the chromatin aggregation and fusion region were observed in ultrathin sections. The cyst wall contained two layers, namely, the ectocyst, and endocyst. In mature cysts, basal bodies and ciliary shafts were observed, demonstrating that D. oligothrix forms non-kinetosome-resorbing cysts. The process of excystment occurred in two modes, either with or without participation of a contractile vacuole.

Islandinium minutum subsp. barbatum subsp. nov. (Dinoflagellata), a New Organic-Walled Dinoflagellate Cyst from the Western Arctic: Morphology, Phylogenetic Position Based on SSU rDNA and LSU rDNA, and Distribution.

A study of modern sediment from the Western Arctic has revealed the presence of a distinctive brown-colored cyst with a spherical central body bearing unbranched processes that are usually solid with a small basal pericoel. Distinctive barbs project from some processes, and process tips are usually minutely expanded into conjoined barbs. The archeopyle is apical and saphopylic. This cyst corresponds to Islandinium? cezare morphotype 2 of Head et al. (2001, J. Quat. Sci., 16:621). Phylogenetic analyses based on the small and large subunit rRNA genes infer close relationship with Islandinium minutum, the type of which is that of the genus. Re-examination of specimens of I. minutum reveals the presence of minute barbs on its processes, but differences with Islandinium? cezare morphotype 2 remain based on size, process distribution, and barb development. Furthermore, the internal transcribed spacer shows I. minutum to be distinct from this morphotype. On the basis of these small but discrete differences, we propose the new subspecies Islandinium minutum subsp. barbatum subsp. nov. Molecular sequencing of other cysts encountered, namely Echinidinium karaense, an unidentified flattened cyst, and "Polykrikos quadratus", places them in the Monovela clade, the latter showing greater morphological variability than previously thought.

A New Heterolobosean Amoeboflagellate, Tetramitus dokdoensis n. sp., Isolated from a Freshwater Pond on Dokdo Island in the East Sea, Korea.

The genus Tetramitus is a representative amoeboflagellate group within the Heterolobosea, and currently contains over a dozen species. Here, a new heterolobosean amoeboflagellate was isolated from a freshwater pond on Dokdo Island, Korea. The amoebae have eruptive pseudopodia, no uroidal filament, and a nucleus with a central nucleolus. The length and width of the amoebae are 15.5-28.0 µm and 5.4-12.6 µm, respectively. The flagellates are conical, with 4 flagella of equal length (~10 µm). There is a discrete rostrum in the subapical region of the flagellate form. The cyst has thin endo- and ectocyst layers and no cyst pores. The amoeba shows slow movement at 37 °C, but does not move at 42 °C under a light microscope. Phylogenies of the 18S rRNA gene and the ITS1-5.8S rRNA gene-ITS2 sequence show that the strain belongs to a subclade of Tetramitus that includes Tetramitus rostratus, Tetramitus waccamawensis and Tetramitus entericus, amongst others. Nonetheless, the strain is distinct from other species in both molecular phylogenetic trees. Thus the strain isolated from the Dokdo Island is proposed as a novel species, Tetramitus dokdoensis n. sp.

Morphological re-description and molecular characterization of Kudoa pagrusi (Myxosporea: Multivalvulida) infecting the heart muscles of the common sea bream fish Pagrus pagrus (Perciformes: Sparidae) from the Red Sea, Egypt.

In the present study, 100 samples of different sizes of the common sea bream fish Pagrus pagrus were collected from the Egyptian water along the Gulf of Suez, Red Sea and examined for the prevalence of myxosporidian parasites in general and Kudoa spp. in particular. Fish samples were thoroughly externally examined. After dissection, all the internal organs were removed and examined. A total of 60 out of 100 fish specimens were found to be infected with Kudoa stages. Parasitic infection was restricted to the heart muscles of the examined fish. None of the other organs was found to be infected. Macroscopic cysts (plasmodia) heavily infested the different parts of the heart muscles. Each plasmodium measured 1.2-2.5 (1.53 ± 0.2) mm × 0.63-0.80 (0.65 ± 0.2) mm. Mature spores are quadratic in shape in the apical view showing four equal valves and four symmetrical polar capsules. Fresh spores were 5.0-7.1 (5.7 ± 0.2) µm long × 5.4-8.5 (6.1 ± 0.3) µm wide. On the basis of spore morphology, the present species was identified as Kudoa pagrusi. Morphometric characterization revealed that the relatively small size of this Kudoa species was the distinctive feature that separates it from all previously described species. Molecular analysis based on small subunit ribosomal DNA (SSU rDNA) sequences revealed that the highest percentage of identity was observed with K. scomberomori and followed by K. shiomitsui, K. hypoepicarclialis, K. amamiensis, and K. kenti. The kudoid spores showed morphometric variations to some extents but had essentially identical nucleotide sequences of the SSU rDNA gene sequences closest to those of K. scomberomori and K. shiomitsui recorded from elasmobranchs in the Indo-Pacific Ocean. The present findings support the identification of an ancestral marine origin of the present Kudoa species.

Stress management in cyst-forming free-living protists: programmed cell death and/or encystment.

In the face of harsh conditions and given a choice, a cell may (i) undergo programmed cell death, (ii) transform into a cancer cell, or (iii) enclose itself into a cyst form. In metazoans, the available evidence suggests that cellular machinery exists only to execute or avoid programmed cell death, while the ability to form a cyst was either lost or never developed. For cyst-forming free-living protists, here we pose the question whether the ability to encyst was gained at the expense of the programmed cell death or both functions coexist to counter unfavorable environmental conditions with mutually exclusive phenotypes.

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.

Description of Dientamoeba fragilis cyst and precystic forms from human samples.

Dientamoeba fragilis is a common enteropathogen of humans. Recently a cyst stage of the parasite was described in an animal model; however, no cyst stage has been described in detail from clinical samples. We describe both cyst and precystic forms from human clinical samples.

Commitment to cyst formation in Giardia.

Giardia trophozoites differentiate into infectious cysts (encystment) in response to physiological stimuli; encystment is crucial for Giardia's transmission, survival and pathogenesis. In vitro, Giardia encysts when bile sequesters lipids necessary for this lipid auxotroph, and in vivo they encyst to infect new hosts. In this study, we investigated, for the first time, commitment to encystment in Giardia using both molecular and cellular techniques. We show that after 3-6 h in inducing conditions, encysting trophozoites continue to encyst regardless of whether the inducing stimulus remains. We propose that a trophozoite's inability to revert to a growing or dividing trophozoite represents a commitment to encystment. The onset of commitment correlated with the appearance of encystment specific vesicles (ESVs) and encystment specific protein synthesis. These observations suggest the involvement of regulatory pathways with the ability to 'remember' a transient signal long after its removal; a property that enables encysting trophozoites to complete the encystment process should the unfavourable triggering condition(s) change. The ability to form cysts in response to transient signals or, as we have highlighted in this paper, the ability of a small percentage of the population to form cysts without an inducer is vital for the maintenance of infection within populations.

ABC transporters in Dictyostelium discoideum development.

ATP-binding cassette (ABC) transporters can translocate a broad spectrum of molecules across the cell membrane including physiological cargo and toxins. ABC transporters are known for the role they play in resistance towards anticancer agents in chemotherapy of cancer patients. There are 68 ABC transporters annotated in the genome of the social amoeba Dictyostelium discoideum. We have characterized more than half of these ABC transporters through a systematic study of mutations in their genes. We have analyzed morphological and transcriptional phenotypes for these mutants during growth and development and found that most of the mutants exhibited rather subtle phenotypes. A few of the genes may share physiological functions, as reflected in their transcriptional phenotypes. Since most of the abc-transporter mutants showed subtle morphological phenotypes, we utilized these transcriptional phenotypes to identify genes that are important for development by looking for transcripts whose abundance was unperturbed in most of the mutants. We found a set of 668 genes that includes many validated D. discoideum developmental genes. We have also found that abcG6 and abcG18 may have potential roles in intercellular signaling during terminal differentiation of spores and stalks.

Morphological and phylogenetic description of a new xenoma-inducing microsporidian, Microsporidium aurata nov. sp., parasite of the gilthead seabream Sparus aurata from the Red Sea.

A new species of Microsporidia found in the marine teleost Sparus aurata collected from Hurghada coasts along the Red Sea, Egypt was described based on light and ultrastructural studies. Twenty three (30.6%) out of 75 of the examined fish were parasitized with a microsporidian parasite. Numerous macroscopic whitish cysts embedded in the peritoneal cavity were observed to infect many organs of the body including muscles, connective tissues, and the intestinal epithelium. The infection was developed as tumor-like masses of often up to 5 mm in diameter inducing an enormous hypertrophy to the infected organs. Fresh spores appeared mostly ovoid to pyriform in shape reaching a size of 1.7 ± 0.5 (1.5-2.5) µm × 1.3 ± 0.4 (1-2) µm; they possessed a large vacuole at the posterior end. These spores were located within a sporophorous vesicle which was bound by a thick amorphous wall. The ultrastructural features support the placement of the present species within the genus Microsporidium. The developmental stages were enclosed within a xenoma structure that was bounded by a double-layered cyst wall. The life cycle of the microsporidian pathogen described herein included four stages: proliferation (merogony), sporogony, sporoblast, spores, and liberation. Mature spores appeared electron dense, uninucleate, and were ellipsoidal in shape. At the anterior end of the spore, the anchoring disk was found in a central position. There was a definite number (5-11) of turns of the polar tube. A 538-bp region of the SSU rDNA gene of the studied species was sequenced (GenBank accession number: KF0220444). Multiple sequence alignment calculated a high degree of similarity (>92%) with six microsporidian species. The most closely related sequence was provided by the GenBank entry AF151529 for Microsporidium prosopium isolated from Hyperoplus lanceolatus differing in 67 nucleotide positions in its SSU rDNA with the highest percentage of identity (97.2%) and the lowest divergence value (0.20). Variations in the morphology of the spores and developmental stages between the two species revealed that the two species are different. The site of infection in the host and description of the onset of parasite development are strong criteria for the placement of the microsporidian parasite of the fish S. aurata within the genus Microsporidium as a new species, and we propose to name it Microsporidium aurata nov. sp.

A new heterotrophic dinoflagellate from the North-eastern Pacific, Protoperidinium fukuyoi: cyst-theca relationship, phylogeny, distribution and ecology.

The cyst-theca relationship of Protoperidinium fukuyoi n. sp. (Dinoflagellata, Protoperidiniaceae) is established by incubating resting cysts from estuarine sediments off southern Vancouver Island, British Columbia, Canada, and San Pedro Harbor, California, USA. The cysts have a brown-coloured wall, and are characterized by a saphopylic archeopyle comprising three apical plates, the apical pore plate and canal plate; and acuminate processes typically arranged in linear clusters. We elucidate the phylogenetic relationship of P. fukuyoi through large and small subunit (LSU and SSU) rDNA sequences, and also report the SSU of the cyst-defined species Islandinium minutum (Harland & Reid) Head et al. 2001. Molecular phylogenetic analysis by SSU rDNA shows that both species are closely related to Protoperidinium americanum (Gran & Braarud 1935) Balech 1974. Large subunit rDNA phylogeny also supports a close relationship between P. fukuyoi and P. americanum. Three subgroups in total are further characterized within the Monovela group. The cyst of P. fukuyoi shows a wide geographical range along the coastal tropical to temperate areas of the North-east Pacific, its distribution reflecting optimal summer sea-surface temperatures of ~14-18 °C and salinities of 22-34 psu.

Redescription of Eimeria zarudnyi Alyousif & Al-Shawa, 2003 as Choleoeimeria zarudnyi n. comb. (Apicomplexa: Eimeriidae).

Coprological examination of the worm lizard Diplometopon zarudnyi Nikolskii revealed the presence of oöcysts of Choleoeimeria zarudnyi (Alyousif & Al-Shawa, 2003) n. comb. in five (17%) of the 30 lizards examined. Sporulated oöcysts were found in the faeces and the gallbladder contents. These are tetrasporocystic, ellipsoidal, 25-32 × 18-25 (mean 27 × 22) µm, with a smooth bi-layered wall. The dizoic sporocysts are ovoidal, 10-13 × 6-9 (mean 11 × 7) µm, with a granulated sporocyst residuum. Sporozoites are banana-shaped with an average size of 13 × 3 µm. Endogenous stages (meronts, gamonts and gametes) are confined to the gallbladder epithelium and the infected cells were hypertrophied. Based on the morphological features of the exogenous stages and the endogenous development of the present parasite, its generic affiliation is revised and Eimeria zarudnyi Alyousif & Al-Shawa, 2003 is transferred to the genus Choleoeimeria.

Two-gene phylogeny of bright-spored Myxomycetes (slime moulds, superorder Lucisporidia).

Myxomycetes, or plasmodial slime-moulds, are one of the largest groups in phylum Amoebozoa. Nonetheless, only ∼10% are in the database for the small subunit (SSU) ribosomal RNA gene, the most widely used gene for phylogenetics and barcoding. Most sequences belong to dark-spored Myxomycetes (order Fuscisporida); the 318 species of superorder Lucisporidia (bright-spored) are represented by only eleven genuine sequences. To compensate for this, we provide 66 new sequences, 37 SSU rRNA and 29 elongation factor 1-alpha (EF-1α), for 82% of the genera of Lucisporidia. Phylogenetic analyses of single- and two-gene alignments produce congruent topologies and reveal both morphological characters that have been overemphasised and those that have been overlooked in past classifications. Both classical orders, Liceida and Trichiida, and several families and genera are para/polyphyletic; some previously unrecognised clades emerge. We discuss possible evolutionary pathways. Our study fills a gap in the phylogeny of Amoebozoa and provides an extensive SSU rRNA sequence reference database for environmental sampling and barcoding. We report a new group I intron insertion site for Myxomycetes in one Licea.

The Amoebozoa.

The model organism Dictyostelium discoideum is a member of the Amoebozoa, one of the six major -divisions of eukaryotes. Amoebozoa comprise a wide variety of amoeboid and flagellate organisms with single cells measuring from 5 µm to several meters across. They have adopted many different life styles and sexual behaviors and can live in all but the most extreme environments. This chapter provides an overview of Amoebozoan diversity and compares roads towards multicellularity within the Amoebozoa with inventions of multicellularity in other protist divisions. The chapter closes with a scenario for the evolution of Dictyostelid multicellularity from an Amoebozoan stress response.

Thelohanellus niloticus sp. nov. (Myxozoa: Myxosporea), a parasite of the Nile carp Labeo niloticus from the River Nile, Egypt.

In the present study, the morphology and morphometric characterization of Thelohanellus niloticus sp. nov., a new myxozoan belonging to genus Thelohanellus Kudo, 1933 (Myxosporea, Bivalvulida) infecting the gills of Labeo niloticus (Osteichthyes, Cyprinidae), were described for the first time from the River Nile at El-Minia Governorate, Egypt. Forty-one out of 78 (52.6 %) of the examined fish were infected. The infection was observed as irregular, milky whitish, cyst-like plasmodia (up to 0.8 mm in diameter) attached to the gill filaments of the host fish. These plasmodia contained tear-shaped myxospores with slightly tapering anterior and rounded posterior ends. Each spore has a single pyriform polar capsule. Spores measured about 23.3 ± 0.3 (20.4-27.1) µm long and 13.4 ± 0.4 (11.5-14.2) µm wide. The polar capsule was 11.7 ± 0.3 (9.2-12.5) µm long and 4.7 ± 0.3 (3.5-6.2) µm wide, containing a polar filament coiled perpendicular to the longitudinal axis of the spore body making eight turns. Occasionally, an oblong, irregular-shaped mass of protoplasm with a slightly oval nucleus (1.4 µm in diameter) and a small iodinophilous vacuole measured 0.85 ± 0.2 µm (0.73-1.2 µm) were observed in the spore. Due to the lack of the second polar capsule characterizing Myxobolus sp., the present parasite is placed within the genus Thelohanellus. Based on morphological differences (compared with other members of Thelohanellus Kudo, 1933) and the host specificity, this species is described as a new one of the genus Thelohanellus recorded for the first time in Egypt.

Amoeba stages in the deepest branching heteroloboseans, including Pharyngomonas: evolutionary and systematic implications.

The taxon Heterolobosea (Excavata) is a major group of protists well known for its diversity of life stages. Most are amoebae capable of transforming into flagellates (amoeboflagellates), while others are known solely as flagellates or solely as amoebae. The deepest-branching heterolobosean taxon confirmed previously, Pharyngomonas, was generally assumed to be a pure flagellate, suggesting that the amoeba form arose later in the evolution of Heterolobosea sensu lato. Here we report that multiple isolates of Pharyngomonas are actually amoeboflagellates that also have cyst stages, with only amoebae transforming into cysts. The amoeba form of Pharyngomonas showed heterolobosean characteristics (e. g. eruptive movement), but also possessed unusual morphological features like slow-flowing crenulated hyaline crescents with conical subpseudopodia, finger-like projections and branching posterior extensions. Furthermore, phylogenetic analyses of 18S ribosomal RNA gene sequences that included two undescribed species of amoebae showed that Pharyngomonas is not the only deep-branching heterolobosean to possess an amoeba stage. These results suggest that possession of an amoeba stage was ancestral for Heterolobosea, unifying this taxon as a group of species with amoeba stages in their lifecycle or derived from organisms with such stages.

The Toxoplasma nuclear factor TgAP2XI-4 controls bradyzoite gene expression and cyst formation.

Toxoplasma gondii undergoes many phenotypic changes during its life cycle. The recent identification of AP2 transcription factors in T. gondii has provided a platform for studying the mechanisms controlling gene expression. In the present study, we report that a recombinant protein encompassing the TgAP2XI-4 AP2 domain was able to specifically bind to a DNA motif using gel retardation assays. TgAP2XI-4 protein is localized in the parasite nucleus throughout the tachyzoite life cycle in vitro, with peak expression occurring after cytokinesis. We found that the TgAP2XI-4 transcript level was higher in bradyzoite cysts isolated from brains of chronically infected mice than in the rapidly replicating tachyzoites. A knockout of the TgAP2XI-4 gene in both T. gondii virulent type I and avirulent type II strains reveals its role in modulating expression and promoter activity of genes involved in stage conversion of the rapidly replicating tachyzoites to the dormant cyst forming bradyzoites. Furthermore, mice infected with the type II KO mutants show a drastically reduced brain cyst burden. Thus, our results validate TgAP2XI-4 as a novel nuclear factor that regulates bradyzoite gene expression during parasite differentiation and cyst formation.

Role of the Skp1 prolyl-hydroxylation/glycosylation pathway in oxygen dependent submerged development of Dictyostelium.

BACKGROUND: Oxygen sensing is a near universal signaling modality that, in eukaryotes ranging from protists such as Dictyostelium and Toxoplasma to humans, involves a cytoplasmic prolyl 4-hydroxylase that utilizes oxygen and α-ketoglutarate as potentially rate-limiting substrates. A divergence between the animal and protist mechanisms is the enzymatic target: the animal transcriptional factor subunit hypoxia inducible factor-α whose hydroxylation results in its poly-ubiquitination and proteasomal degradation, and the protist E3SCF ubiquitin ligase subunit Skp1 whose hydroxylation might control the stability of other proteins. In Dictyostelium, genetic studies show that hydroxylation of Skp1 by PhyA, and subsequent glycosylation of the hydroxyproline, is required for normal oxygen sensing during multicellular development at an air/water interface. Because it has been difficult to detect an effect of hypoxia on Skp1 hydroxylation itself, the role of Skp1 modification was investigated in a submerged model of Dictyostelium development dependent on atmospheric hyperoxia. RESULTS: In static isotropic conditions beneath 70-100% atmospheric oxygen, amoebae formed radially symmetrical cyst-like aggregates consisting of a core of spores and undifferentiated cells surrounded by a cortex of stalk cells. Analysis of mutants showed that cyst formation was inhibited by high Skp1 levels via a hydroxylation-dependent mechanism, and spore differentiation required core glycosylation of Skp1 by a mechanism that could be bypassed by excess Skp1. Failure of spores to differentiate at lower oxygen correlated qualitatively with reduced Skp1 hydroxylation. CONCLUSION: We propose that, in the physiological range, oxygen or downstream metabolic effectors control the timing of developmental progression via activation of newly synthesized Skp1.

Bestatin inhibits cell growth, cell division, and spore cell differentiation in Dictyostelium discoideum.

Bestatin methyl ester (BME) is an inhibitor of Zn(2+)-binding aminopeptidases that inhibits cell proliferation and induces apoptosis in normal and cancer cells. We have used Dictyostelium as a model organism to study the effects of BME. Only two Zn(2+)-binding aminopeptidases have been identified in Dictyostelium to date, puromycin-sensitive aminopeptidase A and B (PsaA and PsaB). PSA from other organisms is known to regulate cell division and differentiation. Here we show that PsaA is differentially expressed throughout growth and development of Dictyostelium, and its expression is regulated by developmental morphogens. We present evidence that BME specifically interacts with PsaA and inhibits its aminopeptidase activity. Treatment of cells with BME inhibited the rate of cell growth and the frequency of cell division in growing cells and inhibited spore cell differentiation during late development. Overexpression of PsaA-GFP (where GFP is green fluorescent protein) also inhibited spore cell differentiation but did not affect growth. Using chimeras, we have identified that nuclear versus cytoplasmic localization of PsaA affects the choice between stalk or spore cell differentiation pathway. Cells that overexpressed PsaA-GFP (primarily nuclear) differentiated into stalk cells, while cells that overexpressed PsaAΔNLS2-GFP (cytoplasmic) differentiated into spores. In conclusion, we have identified that BME inhibits cell growth, division, and differentiation in Dictyostelium likely through inhibition of PsaA.