Alpha-1 Giardin is an Annexin with Highly Unusual Calcium-Regulated Mechanisms

Alpha-giardins constitute the annexin proteome (group E annexins) in the intestinal protozoan parasite Giardia and, as such, represent the evolutionary oldest eukaryotic annexins. The dominance of alpha-giardins in the cytoskeleton of Giardia with its greatly reduced actin content emphasises the importance of the alpha-giardins for the structural integrity of the parasite, which is particularly critical in the transformation stage between cyst and trophozoite. In this study, we report the crystal structures of the apo- and calcium-bound forms of α1-giardin, a protein localised to the plasma membrane of Giardia trophozoites that has recently been identified as a vaccine target. The calcium-bound crystal structure of α1-giardin revealed the presence of a type III site in the first repeat as known from other annexin structures, as well as a novel calcium binding site situated between repeats I and IV. By means of comparison, the crystal structures of three different alpha-giardins known to date indicate that these proteins engage different calcium coordination schemes, among each other, as well as compared to annexins of groups A-D. Evaluation of the calcium-dependent binding to acidic phosphoplipid membranes revealed that this process is not only mediated but also regulated by the environmental calcium concentration. Uniquely within the large family of annexins, α1-giardin disengages from the phospholipid membrane at high calcium concentrations possibly due to formation of a dimeric species. The observed behaviour is in line with changing calcium levels experienced by the parasite during excystation and may thus provide first insights into the molecular mechanisms underpinning the transformation and survival of the parasite in the host.     

Giardia spp.: Distribution of contractile proteins in the attachment organelle

Giardia spp. trophozoites isolated from rat small intestine were examined by light microscopy, electron microscopy, SDS-gel electrophoresis, and immunocytochemistry. In SDS-gels of protein extracts of isolated Giardia spp. trophozoites protein bands corresponding to myosin, α-actinin, and actin were identified by comigration with avian myofibril proteins and molecular weight standards. Actin was specifically identified in SDS-gels by immunoautoradiography. Immunostaining for actin, α-actinin, myosin, and tropomyosin in trophozoites was demonstrated in the periphery of the ventral disc in an area corresponding to the lateral crest. Electron-dense fibrillar was observed in the lateral crest of the ventral disc by electron microscopy. Immunostaining for actin and α-actinin was also observed in the area of the median body, a microtubular organelle, and in electron-dense fibrillar material associated with the intracellular axonemes of the posterior-lateral flagella. The localization of these contractile proteins in the ventral disc suggests that they may play an important role in the mechanism of trophozoite attachment.     

Dual Acylation Accounts for the Localization of α19-Giardin in the Ventral Flagellum Pair of Giardia lamblia

A Giardia-specific protein family denominated as α-giardins, represents the major protein component, besides tubulin, of the cytoskeleton of the human pathogenic parasite Giardia lamblia. One of its members, α19-giardin, carries an N-terminal sequence extension of MGCXXS, which in many proteins serves as a target for dual lipid conjugation: myristoylation at the glycine residue after removal of the methionine and palmitoylation at the cysteine residue. As the first experimental evidence of a lipid modification, we found α19-giardin to be associated with the membrane fraction of disrupted trophozoites. After heterologous coexpression of α19-giardin with giardial N-myristoyltransferase (NMT) in Escherichia coli, we found the protein in a myristoylated form. Additionally, after heterologous expression together with the palmitoyl transferase Pfa3 in Saccharomyces cerevisiae, α19-giardin associates with the membrane of the main vacuole. Immunocytochemical colocalization studies on wild-type Giardia trophozoites with tubulin provide evidence that α19-giardin exclusively localizes to the ventral pair of the giardial flagella. A mutant in which the putatively myristoylated N-terminal glycine residue was replaced by alanine lost this specific localization. Our findings suggest that the dual lipidation of α19-giardin is responsible for its specific flagellar localization.The human pathogenic parasite Giardia lamblia (syn. Giardia intestinalis), a phylogenetically basal eukaryote (41), is the causative agent of giardiasis, an intestinal disease most prevalent in developing countries (39). The protist has a simple life cycle consisting of two stages, a vegetative trophozoite dwelling in the host intestine and an infective cyst form. Proliferating trophozoites are distinguished by a complex cytoskeleton whose most striking feature are eight flagella and a ventral disk, by which the parasite attaches to the intestinal epithelium of the host (11). As a diplomonad protist, the parasite possesses four different pairs of flagella, of which only the ventral and posterolateral ones are replicated in the first round of the cell cycle; the other two pairs require two further cell divisions for their complete renewal (32).Besides tubulin, the Giardia-specific giardins account for the major protein components of the giardial cytoskeleton (7, 36). From these 30- to 45-kDa proteins, the α-giardins have been recognized, based on sequence similarities, as annexin homologues (28). They represent a multiple set of all-helical proteins distinguished by four annexin domains each. We have previously confirmed that some α-giardins, indeed, satisfy a criterion of annexins, i.e., Ca²⁺-dependent association with phospholipids (1, 13, 43). Hence, these giardins have been nominated as protozoan annexins E1 to E3 (9). However, in contrast to annexins of multicellular organisms, some α-giardins contain specific sequence motifs in the fourth annexin domain, and these motifs are located at the cytoplasmic face and may make contact between the plasma membrane and the cytoskeleton within the cell (35, 43).The genome of G. lamblia possesses a total of 21 α-giardin-encoding genes (27, 30, 47). The particular importance of their gene products for the parasite may be indicated by the following: the human genome contains only 12 annexin genes, and the genome of Saccharomyces cerevisiae has none at all. Phylogenetic analyses of the α-giardin genes revealed that 19 members of this family evolved from two widely ramified branches, whereby the genes coding for α14-giardin (annexin E1, according to the annexin nomenclature) and α19-giardin jointly form a single arm (47). α19-Giardin, the subject of the present study, carries a predicted N-terminal sequence extension with an MGCXXS motif known as a target for dual fatty acylation, i.e., myristoylation at the N-terminal glycine and palmitoylation at the cysteine residue (8). However, no experimental data for a lipid conjugation to this protein and any other giardin are currently available.In the present study, we provide the first evidence that α19-giardin indeed can be both myristoylated and palmitoylated. In contrast to α14-giardin, which we found to be located in all giardial flagella as well as in the median body of the trophozoites (43, 46), α19-giardin appears exclusively localized to the ventral flagella of the trophozoites and is restricted to those portions protruding outside the cell body.     

Comparative study of sulfur utilization and speciation transformation of two elemental sulfur species by thermoacidophilic Archaea Acidianus manzaensis YN-25

The sulfur utilization and speciation transformation of two elemental sulfur species (orthorhombic α-S₈ and amorphous μ-S) by thermoacidophilic Archaea strain Acidianus manzaensis YN-25 was comparatively studied. The results of cell growth and sulfur oxidation behavior showed that A. manzaensis cultured on μ-S grew faster (about 24 h earlier to reach stationary phase than that on α-S₈) and produced more H⁺ and SO₄^2?. Results of scanning electron microscopy indicated that the surfaces of μ-S and α-S₈ were differently bio-corroded by A. manzaensis into loose porosities and pits, respectively. Fourier transform-infrared spectroscopy analysis indicated both μ-S and α-S₈ were adsorbed by the cells. X-ray diffraction and Raman spectra analysis indicated that μ-S was mostly converted into α-S₈ after A. manzaensis cell growth while α-S₈ was not transformed into a different allotrope. The fitted result of sulfur K edge XANES spectra further showed that the μ-S after growth of A. manzaensis were composed of 92.1% α-S₈ and 7.9% μ-S, while no change in composition for α-S₈ was found.     

Regulation of the α-expansin gene OsEXPA8 expression affects root system architecture in transgenic rice plants

Expansins are cell wall proteins implicated in the control of plant growth via loosening of the extracellular matrix, and are encoded by a large gene family. However, data linked to loss of function of single genes which support the role of expansins in root growth remain limited. In this study, we used RNA interference to examine the biological functions of the rice α-expansin gene OsEXPA8. Repression of OsEXPA8 expression in rice impaired the root system architecture and plant growth significantly, leading to shorter primary roots and fewer lateral roots. Accordingly, the cell size of the root vascular bundle reduced drastically. Notably, OsEXPA8 silencing impaired root hair elongation; moreover, plant height was clearly reduced. Transient expression of OsEXPA8-GFP in onion epidermal cells verified that OsEXPA8 is located on the cell wall. OsEXPA8 was expressed predominantly in the root and shoot of one-week-old rice seedlings, and highly induced by NaCl but suppressed by nitrate and phosphate starvation. In addition, atomic force microscopy was used to explore alterations in cell wall nanomechanics caused by OsEXPA8 protein reduction, which showed that the wall stiffness (Young’s modulus) of OsEXPA8-silenced suspension cells was increased significantly. Taken together, our results suggest that OsEXPA8 is critical for root system architecture, which supports the hypothesis that expansins are involved in enhancing plant growth by mediating cell wall loosening.     

Identification of α-11 giardin as a flagellar and surface component of Giardia lamblia

Giardia lamblia is a protozoan pathogen with distinct cytoskeletal structures, including median bodies and eight flagella. In this study, we examined components comprising G. lamblia flagella. Crude flagellar extracts were prepared from G. lamblia trophozoites, and analyzed by two-dimensional (2-D) gel electrophoresis. The 19 protein spots were analyzed by MALDI–TOF mass spectrometry, identifying ten metabolic enzymes, six distinct giardins, Giardia trophozoite antigen 1, translational initiation factor eIF-4A, and an extracellular signal-regulated kinase 2. Among the identified proteins, we studied α-11 giardin which belongs to a group of cytoskeletal proteins specific to Giardia. Western blot analysis and real-time PCR indicated that expression of α-11 giardin is not significantly increased during encystation of G. lamblia. Immunofluorescence assays using anti-α-11 giardin antibodies revealed that α-11 giardin protein mainly localized to the plasma membranes and basal bodies of the anterior flagella of G. lamblia trophozoites, suggesting that α-11 giardin is a genuine component of the G. lamblia cytoskeleton.     

Application of Genotyping during an Extensive Outbreak of Waterborne Giardiasis in Bergen, Norway, during Autumn and Winter 2004

During the autumn and winter of 2004 and 2005, an extensive outbreak of waterborne giardiasis occurred in Bergen, Norway. Over 1,500 patients were diagnosed with giardiasis. Analysis of water from the implicated source revealed low numbers of Giardia cysts, but the initial contamination event probably occurred up to 10 weeks previously. While sewage leakage from a residential area is now considered to be the probable source of contamination, during the episode waste from one particular septic tank was thought to be a possible source. Genotyping of cysts from the septic tank demonstrated that they were assemblage A cysts, although the sequences were not identical to any previously published sequences. For the β-giardin gene, the closest published subgenotype was subgenotype A3; for the gdh gene, the closest published subgenotype was subgenotype A2. Genotyping of cysts from 21 patient samples revealed that they were assemblage B cysts; thus, the septic tank was unlikely to be the contamination source. Sequencing of the β-giardin and gdh genes from patient samples and a comparison of the sequences gave complex results. For the β-giardin gene, three isolates had sequences identical to subgenotype B3 sequences. However, other isolates had between one and four single-nucleotide polymorphisms (SNPs). For the gdh gene, none of the sequences were identical to the sequence published for subgenotype B3, and the sequences had between one and three SNPs. One isolate, which was identical to subgenotype B3 at the β-giardin gene, was more similar to subgenotype B2 at the gdh gene. Grouping the isolates on the basis of SNPs resulted in different groups for the two genes. The results are discussed in relation to giardiasis in Norway and to other Giardia genotyping studies.     

Giardia lamblia behavior during encystment: How morphological changes in shape occur

Giardia is an intestinal parasite that undergoes adaptation for survival outside the host. Different stages in the Giardia cyst formation include distinctive changes in the trophozoite shape and polarization, from the characteristic flattened dorsal–ventral axis found in motile trophozoites to a rounded appearance and also the appearance of a “tail-like” appendage in later stages of cyst formation. In addition, the flagella disappear and the cyst is oval or rounded and immotile. Since we found no clear information describing how the cells change shape and how the flagella disappear, we applied videomicroscopy, scanning and transmission electron microscopy to follow the gradual modifications that occur in the trophozoite, including alterations in the cell shape, the manner of flagella internalization and changes in disc behavior. Based on the data presented here, it was possible to construct a temporal sequence of changes during Giardia encystation. In this article we show how the membrane growth of the flange contributes to cell shape changes during encystment. In addition, an operculum and flagella internalization is shown. There is a video as a supplement showing these modifications. In other procedure, the plasma membrane was removed and the disc was seen by high resolution scanning electron microscopy where the modifications of the disc spiral can be followed.     

Giardia lamblia: Evaluation of roller bottle cultivation

A modified “outside-in” roller bottle with a high ratio of surface area to volume was used to cultivate Giardia lamblia. Yields were high, more so when bottles were rotated at 6 rph (9.3 ± 4.0 × 10⁸ trophozoites/bottle) than at 12 rph (4.2 ± 1.9 × 10⁸ trophozoites/bottle). The method was more efficient than stationary tube culture with respect to utilization of culture medium; trophozoite concentration after roller bottle culture (1.7 ± 0.8 × 10⁶ trophozoites/ml) was significantly higher (by a factor of 2.8) than concentrations obtained from stationary tube culture (0.6 ± 0.4 × 10⁶ trophozoites/ml, P < 0.002). Increased yields from roller bottle culture were not accounted for by a reduction in mean trophozoite generation time (roller culture, 10.7 ± 1.2 hr; stationary tube culture, 10.3 ± 0.6 hr) but may be related to prolongation of the period of log phase growth or increased trophozoite survival. Trophozoite yields expressed per unit surface area were significantly higher from roller bottle culture (7.2 ± 3.1 × 10⁵ trophozoites/cm²) than from stationary tubes (1.9 ± 1.0 × 10⁵ trophozoites/cm², P < 0.002). Attempts to cultivate G. lamblia in spin culture using polystyrene beads (Biosilon) as a microcarrier were unsuccessful, trophozoite growth being inhibited rather than promoted. Roller bottle culture of G. lamblia, however, is efficient, economical, and less laborious than stationary tube culture, particularly when more than 10⁸ trophozoites are required.     

Interaction of Pasteurella multocida with Free-Living Amoebae

Pasteurella multocida is a highly infectious, facultative intracellular bacterium which causes fowl cholera in birds. This study reports, for the first time, the observed interaction between P. multocida and free-living amoebae. Amoebal trophozoites were coinfected with fowl-cholera-causing P. multocida strain X-73 that expressed the green fluorescent protein (GFP). Using confocal fluorescence microscopy, GFP expressing X-73 was located within the trophozoite. Transmission electron microscopy of coinfection preparations revealed clusters of intact X-73 cells in membrane-bound vacuoles within the trophozoite cytoplasm. A coinfection assay employing gentamicin to kill extracellular bacteria was used to assess the survival and replication of P. multocida within amoebae. In the presence of amoebae, the number of recoverable intracellular X-73 cells increased over a 24-h period; in contrast, X-73 cultured alone in assay medium showed a consistent decline in growth. Cytotoxicity assays and microscopy showed that X-73 was able to lyse and exit the amoebal cells approximately 18 h after coinfection. The observed interaction between P. multocida and amoebae can be considered as an infective process as the bacterium was able to invade, survive, replicate, and lyse the amoebal host. This raises the possibility that similar interactions occur in vivo between P. multocida and host cells. Free-living amoebae are ubiquitous within water and soil environments, and P. multocida has been observed to survive within these same ecosystems. Thus, our findings suggest that the interaction between P. multocida and amoebae may occur within the natural environment.     

Members of the Entamoeba histolytica transmembrane kinase family play non-redundant roles in growth and phagocytosis

Entamoeba histolytica contains a large and novel family of transmembrane kinases (TMKs). The expression patterns of the E. histolytica TMKs in individual trophozoites and the roles of the TMKs for sensing and responding to extracellular cues were incompletely characterised. Here we provide evidence that single cells express multiple TMKs and that TMK39 and TMK54 likely serve non-redundant cellular functions. Laser-capture microdissection was used in conjunction with microarray analysis to demonstrate that single trophozoites express more than one TMK gene. Anti-peptide antibodies were raised against unique regions in the extracellular domains of TMK39, TMK54 and PaTMK, and TMK expression was analysed at the protein level. Flow cytometric assays revealed that populations of trophozoites homogeneously expressed TMK39, TMK54 and PaTMK, while confocal microscopy identified different patterns of cell surface expression for TMK39 and TMK54. The functions of TMK39 and TMK54 were probed by the inducible expression of dominant-negative mutants. While TMK39 co-localised with ingested beads and expression of truncated TMK39 interfered with trophozoite phagocytosis of apoptotic lymphocytes, expression of a truncated TMK54 inhibited growth of amoebae and altered the surface expression of the heavy subunit of the E. histolytica Gal/GalNAc lectin. Overall, our data indicates that multiple members of the novel E. histolytica TMK family are utilised for non-redundant functions by the parasite.     

Occurrence and Molecular Identification of Giardia duodenalis from Stray Cats in Guangzhou,Southern China

The objective of this study was to genetically characterize isolates of Giardia duodenalis and to determine if zoonotic potential of G. duodenalis could be found in stray cats from urban and suburban environments in Guangzhou, China. Among 102 fresh fecal samples of stray cats, 30 samples were collected in Baiyun district (urban) and 72 in Conghua district (suburban). G. duodenalis specimens were examined using light microscopy, then the positive specimens were subjected to PCR amplification and subsequent sequencing at 4 loci such as glutamate dehydrogenase (gdh), triose phosphate isomerase (tpi), β-giardin (bg), and small subunit ribosomal RNA (18S rRNA) genes. The phylogenetic trees were constructed using obtained sequences by MEGA5.2 software. Results show that 9.8% (10/102) feline fecal samples were found to be positive by microscopy, 10% (3/30) in Baiyun district and 9.7% (7/72) in Conghua district. Among the 10 positive samples, 9 were single infection (8 isolates, assemblage A; 1 isolate, assemblage F) and 1 sample was mixed infection with assemblages A and C. Based on tpi, gdh, and bg genes, all sequences of assemblage A showed complete homology with AI except for 1 isolate (CHC83). These findings not only confirmed the occurrence of G. duodenalis in stray cats, but also showed that zoonotic assemblage A was found for the first time in stray cats living in urban and suburban environments in China.     

Induction of cell death on Plasmodium falciparum asexual blood stages by Solanum nudum steroids

Solanum nudum Dunal (Solanaceae) is a plant used in traditional medicine in Colombian Pacific Coast, from which five steroids denominated SNs have been isolated. The SNs compounds have antiplasmodial activity against asexual blood stages of Plasmodium falciparum strain 7G8 with an IC₅₀ between 20–87 µM. However, their mode of action is unknown. Steroids regulate important cellular functions including cell growth, differentiation and death. Thus, the aim of this work was to determine the effects of S. nudum compounds on P. falciparum asexual blood stages and their association with cell death. We found that trophozoite and schizont stages were the most sensitive to SNs. By Giemsa-stained smears, induction of crisis forms was observed. Transmission electron microscopy of treated parasites showed morphological abnormalities such as a cytoplasm rich in vesicles and myelinic figures. The Mitochondria presented no morphological alterations and the nuclei showed no abnormal chromatin condensation. By the use of S. nudum compounds, cell death in P. falciparum was evident by a decrease in mitochondrial membrane potential, DNA fragmentation and cytoplasmic acidification. The asexual blood stages of P. falciparum showed some apoptotic-like and autophagic-like cell death characteristics induced by SNs treatment.     

Acanthamoeba castellanii Neff: In vitro activity against the trophozoite stage of a natural sesquiterpene and a synthetic cobalt(II)-lapachol complex

In this study, the in vitro activities of a natural sesquiterpene, α-cyperotundone, isolated from the root bark of Maytenus retusa and a cobalt(II)-complex of a natural occurring prenyl hydroxynaphthoquinone (lapachol) were evaluated against the trophozoite stage of Acanthamoeba castellanii Neff using a previously developed colorimetric 96-well microtiter plate assay, based on the oxido-reduction of Alamar Blue®. The obtained activities showed that these two compounds were able to inhibit the in vitro growth of the amoebae at relatively low concentrations. Further identification of the molecular targets of these products and their effects on acanthamoebae should be determined to evaluate their possible therapeutic use.     

Crystal structure of α-galactosidase from Lactobacillus acidophilus NCFM: insight into tetramer formation and substrate binding

Lactobacillus acidophilus NCFM is a probiotic bacterium known for its beneficial effects on human health. The importance of α-galactosidases (α-Gals) for growth of probiotic organisms on oligosaccharides of the raffinose family present in many foods is increasingly recognized. Here, the crystal structure of α-Gal from L. acidophilus NCFM (LaMel36A) of glycoside hydrolase (GH) family 36 (GH36) is determined by single-wavelength anomalous dispersion. In addition, a 1.58-Å-resolution crystallographic complex with α-d-galactose at substrate binding subsite − 1 was determined. LaMel36A has a large N-terminal twisted β-sandwich domain, connected by a long α-helix to the catalytic (β/α)₈-barrel domain, and a C-terminal β-sheet domain. Four identical monomers form a tightly packed tetramer where three monomers contribute to the structural integrity of the active site in each monomer. Structural comparison of LaMel36A with the monomeric Thermotoga maritima α-Gal (TmGal36A) reveals that O2 of α-d-galactose in LaMel36A interacts with a backbone nitrogen in a glycine-rich loop of the catalytic domain, whereas the corresponding atom in TmGal36A is from a tryptophan side chain belonging to the N-terminal domain. Thus, two distinctly different structural motifs participate in substrate recognition. The tetrameric LaMel36A furthermore has a much deeper active site than the monomeric TmGal36A, which possibly modulates substrate specificity. Sequence analysis of GH36, inspired by the observed structural differences, results in four distinct subgroups having clearly different active-site sequence motifs. This novel subdivision incorporates functional and architectural features and may aid further biochemical and structural analyses within GH36.     

Raised calcium and oxidative stress cooperatively promote alpha-synuclein aggregate formation

Cell loss in Parkinson’s and Parkinson’s-plus diseases is linked to abnormal, aggregated forms of the cytoplasmic protein, α-synuclein (α-syn). The factors causing α-syn aggregation may include oxidative stress, changes in protein turnover and dysregulation of calcium homeostasis, resulting in cytotoxic aggregated α-syn species. Recently, we showed that raised calcium can promote α-syn aggregation. We have now investigated the effects of raised calcium combined with oxidation/oxidative stress on α-syn aggregation both in vitro and in vivo. We treated monomeric α-syn with calcium, hydrogen peroxide or calcium plus hydrogen peroxide in vitro and used size exclusion chromatography, fluorescence correlation spectroscopy, atomic force microscopy and scanning electron microscopy to investigate protein aggregation. Our in vitro data is consistent with a cooperative interaction between calcium and oxidation resulting in α-syn oligomers. In cell culture experiments, we used thapsigargin or ionophore A23187 to induce transient increases of intracellular free calcium in human 1321N1 cells expressing an α-syn-GFP construct both with and without co-treatment with hydrogen peroxide and observed α-syn aggregation by fluorescence microscopy. Our in vivo cell culture data shows that either transient increase in intracellular free calcium or hydrogen peroxide treatment individually were able to induce significantly (P = 0.01) increased 1–4 μm cytoplasmic α-syn aggregates after 12 h in cells transiently transfected with α-syn-GFP. There was a greater proportion of cells positive for aggregates when both raised calcium and oxidative stress were combined, with a significantly increased proportion (P = 0.001) of cells with multiple (3 or more) discrete α-syn focal accumulations per cell in the combined treatment compared to raised calcium only. Our data indicates that calcium and oxidation/oxidative stress can cooperatively promote α-syn aggregation both in vitro and in vivo and suggests that oxidative stress may play an important role in the calcium-dependent aggregation mechanism.     

Morphology of Giardia agilis: Observation by Scanning Electron Microscopy and Interference Reflexion Microscopy1

The flagellated protozoan, Giardia agilis, was isolated from tadpole small intestine and examined by scanning electron microscopy and interference reflexion microscopy. The general morphology of the G. agilis trophozoite is similar to G. muris and G. duodenalis, but with modifications that reflect its elongated form. Interference reflexion microscopic analysis of attachment of G. agilis reveals a pattern of focal contacts by the lateral crest of the ventral disc, the ventrolateral flange, the lateral shield, and by numerous microvillus-like appendages found along the lateral border of the trophozoite. The pattern of focal contacts was observed to be dynamic; trophozoites were observed to make and break the focal contacts in a relatively short time and to glide along the surface of the substratum without breaking focal contacts.     

Organization and expression of a-tubulin genes in Drosophila melanogaster: One member of the a-tubulin multigene family is transcribed in both oogenesis and later embryonic development

Genomic clones containing α-tubulin sequences were isolated from a library of Drosophila melanogaster DNA and identified by a hybridization-selection and in vitro-translation procedure. The in vitro translation products were identical to the two electrophoretic variants of α-tubulin present in Drosophila embryos. They co-assembled with an embryonic tubulin fraction to form microtubules in vitro and generated the same partial proteolytic fragments as Drosophila α-tubulins. Hybridization in situ to polytene chromosomes revealed that the α-tubulin sequences constitute a multigene family localized on the right arm of chromosome 3 at sites 84 B3–6, 84 D4–8 and 85 E6–10. Clones hybridizing to these sites corresponded to the three major α-tubulin sequences in genomic DNA. The α-tubulin sequences at 84 B3–6 were present twice per haploid genome, embedded in a large duplicated DNA segment. The sequences of the three genomic α-tubulin genes showed considerable divergence, making it possible to conclude that both of the α-tubulin variants present in embryos are encoded by the genes at 84 B3–6. Furthermore, the abundance of this α-tubulin messenger RNA changes with the requirements for microtubule synthesis during embryo development. The genes at 84 B3–6 encoded both the stored maternal mRNA of the oocyte and the major mRNA transcribed during embryonic development.     

α-crystallin-type heat-shock protein from mycoplasma Acholeplasma laidlawii (Mollicutes)

An increased amount of major heat-shock proteins (HSPs) after heat treatment has been revealed in Acholeplasma laidlawii cells grown in liquid culture, with the quantity of small HSPs, known as P17, being enhanced by hundreds times. The P17 protein was isolated and identified as an α-crystallin-type HSP (α-HSP) by sequencing the N-terminal 15 amino acids of the P17 polypeptide chain followed by finding the corresponding open reading frame (ORF) in the completely sequenced genome of A. laidlawii PG 8A. A computer-based search for homologous ORFs in the genomes of all 14 species of the Mycoplasmataceae family (mycoplasmas themselves) that have been completely sequenced to date yields a negative result. However, among the representatives of the Mollicutes (mycoplasma) class, genes encoding α-HSPs were found in two phytoplasma species (Phytoplasmataceae family) and the acholeplasma examined (Acholeplasmataceae family). It is supposed that the presence or absence of α-HSPs in microorganisms might be related to their inhabitancy; representatives of Acholeplasmataceae and Phytoplasmataceae families mostly reside in plant tissues, which is in contrast to the majority of the Mycoplasmataceae family, which lives in animal and human tissues, i.e., use ecological niches with relatively constant temperature.     

Rab11 and Actin Cytoskeleton Participate in Giardia lamblia Encystation,Guiding the Specific Vesicles to the Cyst Wall

Background

Giardia passes through two stages during its life cycle, the trophozoite and the cyst. Cyst formation involves the synthesis of cyst wall proteins (CWPs) and the transport of CWPs into encystation-specific vesicles (ESVs). Active vesicular trafficking is essential for encystation, but the molecular machinery driving vesicular trafficking remains unknown. The Rab proteins are involved in the targeting of vesicles to several intracellular compartments through their association with cytoskeletal motor proteins.

Methodology and Principal Findings

In this study, we found a relationship between Rab11 and the actin cytoskeleton in CWP1 transport. Confocal microscopy showed Rab11 was distributed throughout the entire trophozoite, while in cysts it was translocated to the periphery of the cell, where it colocalized with ESVs and microfilaments. Encystation was also accompanied by changes in rab11 mRNA expression. To evaluate the role of microfilaments in encystation, the cells were treated with latrunculin A. Scanning electron microscopy showed this treatment resulted in morphological damages to encysted parasites. The intensity of fluorescence-labeled Rab11 and CWP1 in ESVs and cyst walls was reduced, and rab11 and cwp1 mRNA levels were down-regulated. Furthermore, knocking down Rab11 with a hammerhead ribozyme resulted in an up to 80% down-regulation of rab11 mRNA. Although this knockdown did not appear lethal for trophozoites and did not affect cwp1 expression during the encystation, confocal images showed CWP1 was redistributed throughout the cytosol.

Conclusions and Significance

Our results indicate that Rab11 participates in the early and late encystation stages by regulating CWP1 localization and the actin-mediated transport of ESVs towards the periphery. In addition, alterations in the dynamics of actin affected rab11 and cwp1 expression. Our results provide new information about the molecules involved in Giardia encystation and suggest that Rab11 and actin may be useful as novel pharmacological targets.