MicroRNAs are major post-transcriptional regulators of gene expression. Here we show in the ancient protozoan Giardia lamblia a snoRNA-derived 26-nucleotide microRNA, miR3, which represses the translation of histone H2A mRNA containing an imperfect target but enhances translation when the target is made fully complementary. A stepwise mutational analysis of the fully complementary target showed that the activating effect of miR3 was significantly reduced when a single nucleotide at the 5′-end of the target was altered. The effect of miR3 became repressive when 12 of the nucleotides lost their complementation to miR3 with maximum repression reached when only 8 base-pairs remained between the miR3 seed sequence and the target. A synthetic 8-nucleotide RNA oligomer of the miR3 seed sequence was found capable of exerting a similar Argonaute-dependent translational repression. This is the first report showing a correlation between the extent of base-pairing with the target and a change in miRNA function.
Centrins are calcium binding proteins involved in cell division in eukaryotes. Previously, we have shown that depletion of centrin1 in Trypanosoma brucei (T. brucei) displayed arrested organelle segregation resulting in loss of cytokinesis. In this study we analyzed the role of T. brucei centrin2 (TbCen2) and T. brucei 3 (TbCen3) in the early events of T. brucei procyclic cell cycle. Both the immunofluorescence assay and electron microscopy showed that TbCen2 and 3-deficient cells were enlarged in size with duplicated basal bodies, multinuclei and new flagella that are detached along the length of the cell body. In both TbCen2 and TbCen3 depleted cells segregation of the organelles i.e. basal bodies, kinetoplast and nucleus was disrupted. Further analysis of the cells with defective organelle segregation identified three different sub configurations of organelle mis-segregations (Type 1–3). In addition, in majority of the TbCen2 depleted cells and in nearly half of the TbCen3 depleted cells, the kinetoplasts were enlarged and undivided. The abnormal segregations ultimately led to aborted cytokinesis and hence affected growth in these cells. Therefore, both centrin2 and 3 are involved in organelle segregation similar to centrin1 as was previously observed. In addition, we identified their role in kinetoplast division which may be also linked to overall mis-segregation.
The polo-like kinase in the deep branching eukaryote Trypanosoma brucei (TbPlk) has many unique features. Unlike all the other polo-like kinases known to associate with the nucleus and controlling both mitosis and cytokinesis, TbPlk localizes to the flagellum attachment zone (FAZ) and regulates only cytokinesis in T. brucei. TbPlk was, however, previously found capable of complementing all the multiple Plk (Cdc5) functions in Saccharomyces cerevisiae, indicating that it has acquired all the functions of Cdc5. In the present study, Cdc5 tagged with an enhanced yellow fluorescence protein (EYFP) localized exclusively in the FAZ of T. brucei, suggesting that the unusual localization and limited function of TbPlk are probably attributed to the particular environment in T. brucei cells. Structural basis for the FAZ localization of TbPlk was further investigated with TbPlk and TbPlk mutants tagged with EYFP and expressed in T. brucei. The results indicated that a kinase-inactive mutant N169A and a TbPlk mutant with the entire kinase domain (KD) deleted both localized to the FAZ. Substantial association with FAZ was also maintained when one of the two polo-boxes (PB1 or 2) or the linker region between them was deleted from TbPlk. But a deletion of both polo-boxes led to a complete exclusion of the protein from FAZ. All the deletion mutants retained the kinase activity, further indicating that the TbPlk kinase function does not play a role for FAZ localization. The two polo boxes in TbPlk are most likely instrumental in localizing the protein to FAZ through potential interactions with certain FAZ structural component(s). A putative cryptic bipartite nuclear targeting signal was identified in TbPlk, which was capable of directing TbPlk into the nucleus when either the kinase activity was lost or the PB1 was deleted from the protein.
Two core microRNA (miRNA) pathway proteins, Dicer and Argonaute, are found in Giardia lamblia, a deeply branching parasitic protozoan. There are, however, no apparent homologues of Drosha or Exportin5 in the genome. Here, we report a 26 nucleotide (nt) RNA derived from a 106 nt Box C/D snoRNA, GlsR2. This small RNA, designated miR5, localizes to the 3′ end of GlsR2 and has a 75 nt hairpin precursor. GlsR2 is processed by the Dicer from Giardia (GlDcr) and generated miR5. Immunoprecipitation of the Argonaute from Giardia (GlAgo) brought down miR5. When a Renilla Luciferase transcript with a 26 nt miR5 antisense sequence at the 3′-untranslated region (3′ UTR) was introduced into Giardia trophozoites, Luciferase expression was reduced ∼25% when synthetic miR5 was also introduced. The Luciferase mRNA level remained, however, unchanged, suggesting translation repression by miR5. This inhibition was fully reversed by introducing also a 2′-O-methylated antisense inhibitor of miR5, suggesting that miR5 acts by interacting specifically with the antisense sequence in the mRNA. A partial antisense knock down of GlDcr or GlAgo in Giardia indicated that the former is needed for miR5 biogenesis whereas the latter is required for miR5-mediated translational repression. Potential targets for miR5 with canonical seed sequences were predicted bioinformatically near the stop codon of Giardia mRNAs. Four out of the 21 most likely targets were tested in the Luciferase reporter assay. miR5 was found to inhibit Luciferase expression (∼20%) of transcripts carrying these potential target sites, indicating that snoRNA-derived miRNA can regulate the expression of multiple genes in Giardia.
Giardia lambia is a deeply branched parasitic protozoan and the pathogen causing the diarrhetic disorder, giardiasis. The mechanism of gene regulation in this organism is largely unknown. Here, we identified a 26 nucleotide (nt) small RNA from the 3′-end of a 106 nt small nucleolar RNA (GlsR2) in Giardia. GlsR2 is processed through the action of a Dicer protein in Giardia to generate the 26 nt RNA. The latter becomes associated with the Argonaute protein. The protein-RNA complex can repress the translation of messenger RNAs carrying the antisense sequence of the 26 nt RNA at the 3′-untranslated region. This small RNA, designated microRNA5 (miR5), has several potential targets identified in Giardia, among which four were further tested in Giardia and found their translation repressed by miR5. This is the second functioning microRNA we have indentified in Giardia. The microRNAs could be thus important regulators of gene expression in this ancient single cellular organism.
Translation of Giardiavirus (GLV) mRNA is initiated at an internal ribosome entry site (IRES) in the viral transcript. The IRES localizes to a downstream portion of 5′ untranslated region (UTR) and a part of the early downstream coding region of the transcript. Recent studies indicated that the IRES does not require a pre-initiation complex to initiate translation but may directly recruit the small ribosome subunit with the help of a number of trans-activating protein factors. A La autoantigen homologue in the viral host Giardia lamblia, GlLa, was proposed as one of the potential trans-activating factors based on its specific binding to GLV-IRES in vitro. In this study, we further elucidated the functional role of GlLa in GLV-IRES mediated translation in Giardia by knocking down GlLa with antisense morpholino oligo, which resulted in a reduction of GLV-IRES activity by 40%. An over-expression of GlLa in Giardia moderately stimulated GLV-IRES activity by 20%. A yeast inhibitory RNA (IRNA), known to bind mammalian and yeast La autoantigen and inhibit Poliovirus and Hepatitis C virus IRES activities in vitro and in vivo, was also found to bind to GlLa protein in vitro and inhibited GLV-IRES function in vivo. The C-terminal domain of La autoantigen interferes with the dimerization of La and inhibits its function. An over-expression of the C-terminal domain (200–348aa) of GlLa in Giardia showed a dominant-negative effect on GLV-IRES activity, suggesting a potential inhibition of GlLa dimerization. HA tagged GlLa protein was detected mainly in the cytoplasm of Giardia, thus supporting a primary role of GlLa in translation initiation in Giardiavirus.
Polo-like kinase in Trypanosoma brucei (TbPLK) is confined to the flagellum attachment zone (FAZ) and regulates only cytokinetic initiation. However, it apparently diffuses into the cytoplasm before the trans-localization of chromosomal passenger complex (CPC) from the midzone of central spindle to FAZ, which is known to be required for initiating cytokinesis. Synchronized T. brucei procyclic cells treated with a TbPLK inhibitor, GW843682X (GW), in late S phase were found to go through a full cell cycle at a normal pace before being arrested at cytokinetic initiation in the second cycle. However, synchronized cells treated with GW in G1 phase were arrested at cytokinetic initiation within the first cell cycle, suggesting that inhibition of TbPLK at its emergence blocks cytokinesis within the same cell cycle. To rule out potential off-target effects from GW, TbPLK RNA interference (RNAi) was induced to deplete TbPLK, and the progression of synchronized cells from late S phase was also found to be arrested at cytokinetic initiation within the first cell cycle. Apparently, TbPLK has accomplished its role in guiding cytokinesis before the late S phase, presumably by phosphorylating a certain substrate(s) during S phase, which may play a critical role in initiating the subsequent cytokinesis.
Giardiavirus (GLV) utilizes an internal ribosome entry site (IRES) for translation initiation in the early branching eukaryote Giardia lamblia. Unlike most of the viral IRESs among higher eukaryotes, which localize primarily within the 5′-untranslated region (UTR), the GLV IRES comprises 253 nts of 5′UTR and the initial 264 nts in the open-reading-frame (ORF). To test if GLV IRES also functions in higher eukaryotic systems, we examined it in rabbit reticulocyte lysate (RRL) and found that it functions much less efficiently than the IRES from the Encephalomyocarditis virus (EMCV) or Cricket paralysis virus (CrPV). In contrast, both EMCV-IRES and CrPV-IRESs were inactive in transfected Giardia cells. Structure-function analysis indicated that only the stem-loop U5 from the 5′UTR and the stem-loop I plus the downstream box (Dbox) from the ORF of GLV IRES are required for limited IRES function in RRL. Edeine, a translation initiation inhibitor, did not significantly affect the function of GLV IRES in either RRL or Giardia, indicating that a pre-initiation complex is not required for GLV IRES–mediated translation initiation. However, the small ribosomal subunit purified from Giardia did not bind to GLV IRES, indicating that additional protein factors may be necessary. A member of the helicase family IBP1 and two known viral IRES binding proteins La autoantigen and SRp20 have been identified in Giardia that bind to GLV IRES in vitro. These three proteins could be involved in facilitating small ribosome recruitment for initiating translation.
The Trypanosoma brucei cell cycle is regulated by combinations of cyclin/CRKs (cdc2 related kinases). Recently, two additional cyclins (CYC10, CYC11) and six new CRK (CRK7-12) homologues were identified in the T. brucei genome database [1,2].
Individual RNAi knockdowns of these new proteins in the procyclic form of T. brucei showed no apparent phenotype except for the CRK9 depletion, which enriched the cells in G2/M phase. But a similar CRK9 knockdown in the bloodstream form caused no apparent phenotype. CRK9 lacks the typical PSTAIRE motif for cyclin binding and the phenylalanine "gatekeeper" but binds to cyclin B2 in vitro and localizes to the nucleus in both forms of T. brucei. CRK9-depleted procyclic-form generated no detectable anucleate cells, suggesting an inhibition of cytokinesis by CRK9 depletion as well. The knockdown enriched cells with one nucleus, one kinetoplast and two closely associated basal bodies with an average distance of 1.08 mm in between, which was shorter than the control value of 1.36 μm, and the cells became morphologically deformed and rounded with time.
CRK9 may play a role in mediating the segregation between the two kinetoplast/basal body pairs prior to cytokinetic initiation. Since such a segregation over a relatively significant distance is essential for cytokinetic initiation only in the procyclic but may not be in the bloodstream form, CRK9 could be specifically involved in regulating cytokinetic initiation in the procyclic form of T. brucei.
Regulation of eukaryotic cell cycle progression requires sequential activation and inactivation of cyclin-dependent kinases. Previous RNA interference (RNAi) experiments in Trypanosoma brucei indicated that cyclin E1, cdc2-related kinase (CRK)1 and CRK2 are involved in regulating G1/S transition, whereas cyclin B2 and CRK3 play a pivotal role in controlling the G2/M checkpoint. To search for potential interactions between the other cyclins and CRKs that may not have been revealed by the RNAi assays, we used the yeast two-hybrid system and an in vitro GST pulldown assay and observed interactions between cyclin E1 and CRK1, CRK2 and CRK3. Cyclins E1-E4 are homologues of yeast Pho80 cyclin. But yeast complementation assays indicated that none of them possesses a Pho80-like function. Analysis of cyclin E1+CRK1 and cyclin E1+CRK2 double knockdowns in the procyclic form of T. brucei indicated that the cells were arrested more extensively in the G1 phase beyond the cumulative effect of individual knockdowns. But BrdU incorporation was significantly impaired only in cyclin E1+CRK1 depleted cells, whereas a higher percentage of cyclin E1+CRK2 knockdown cells assumed a grossly elongated posterior end morphology. A double knockdown of cyclin E1 and CRK3 arrested cells in G2/M much more efficiently than if CRK3 was depleted alone. Taken together, these data suggest multiple functions of cyclin E1. It forms a complex with CRK1 in promoting G1/S phase transition, with CRK2 in controlling the posterior morphogenesis during G1/S transition and with CRK3 in promoting passage across the G2/M checkpoint in the trypanosome.
Trypanosoma brucei; cell cycle; RNAi; morphology; cyclin-dependent kinases
Kinetoplastid membrane protein 11 (KMP-11) has been identified as a flagellar protein and is conserved among kinetoplastid parasites, but its potential function remains unknown. In a recent study, we identified KMP-11 as a microtubule-bound protein localizing to the flagellum as well as the basal body in both procyclic and bloodstream forms of Trypanosoma brucei (Z. Li, J. H. Lee, F. Chu, A. L. Burlingame, A. Gunzl, and C. C. Wang, PLoS One 3:e2354, 2008). Silencing of KMP-11 by RNA interference inhibited basal body segregation and cytokinesis in both forms and resulted in multiple nuclei of various sizes, indicating a continuous, albeit somewhat defective, nuclear division while cell division was blocked. KMP-11 knockdown in the procyclic form led to severely compromised formation of the new flagellum attachment zone (FAZ) and detachment of the newly synthesized flagellum. However, a similar phenotype was not observed in the bloodstream form depleted of KMP-11. Thus, KMP-11 is a flagellar protein playing critical roles in regulating cytokinesis in both forms of the trypanosomes. Its distinct roles in regulating FAZ formation in the two forms may provide a clue to the different mechanisms of cytokinetic initiation in procyclic and bloodstream trypanosomes.
Trypanosoma brucei, the etiologic agent of African sleeping sickness, divides into insect (procyclic) and bloodstream forms. These two forms are subject to distinct cell cycle regulations, with cytokinesis controlled primarily by basal body/kinetoplast segregation in the procyclic form but by mitosis in the bloodstream form. Polo-like kinases (PLKs), known to play essential roles in regulating both mitosis and cytokinesis among eukaryotes, have a homologue in T. brucei, TbPLK, which regulates only cytokinesis. In our previous study, overexpressed triply hemagglutinin-tagged TbPLK (TbPLK-3HA) in the procyclic form localized to a mid-dorsal point and the anterior tip of the cell along the flagellum attachment zone (FAZ). In our current study, TbPLK-3HA expressed at the endogenous level was identified at the same dorsal location of both procyclic and bloodstream forms, albeit it was no longer detectable at the anterior tip of the cell. Endogenously expressed TbPLK fused with an enhanced yellow fluorescent protein (EYFP) localized to the same dorsal location along the FAZs in living procyclic and bloodstream cells. Fluorescence-activated cell sorter analysis of hydroxyurea-synchronized procyclic cells revealed that TbPLK-EYFP emerges during S phase, persists through G2/M phase, and vanishes in G1 phase. An indicated TbPLK-EYFP association with the FAZs of G2/M cells may thus represent a timely localization to a potential initiation site of cytokinesis, which agrees with the recognized role of TbPLK in cytokinetic initiation.
An Argonaute homolog and a functional Dicer have been identified in the ancient eukaryote Giardia lamblia, which apparently lacks the ability to perform RNA interference (RNAi). The Giardia Argonaute plays an essential role in growth and is capable of binding specifically to the m7G-cap, suggesting a potential involvement in microRNA (miRNA)-mediated translational repression. To test such a possibility, small RNAs were isolated from Giardia trophozoites, cloned, and sequenced. A 26-nucleotide (nt) small RNA (miR2) was identified as a product of Dicer-processed snoRNA GlsR17 and localized to the cytoplasm by fluorescence in situ hybridization, whereas GlsR17 was found primarily in the nucleolus of only one of the two nuclei in Giardia. Three other small RNAs were also identified as products of snoRNAs, suggesting that the latter could be novel precursors of miRNAs in Giardia. Putative miR2 target sites were identified at the 3′-untranslated regions (UTR) of 22 variant surface protein mRNAs using the miRanda program. In vivo expression of Renilla luciferase mRNA containing six identical miR2 target sites in the 3′-UTR was reduced by 40% when co-transfected with synthetic miR2, while the level of luciferase mRNA remained unaffected. Thus, miR2 likely affects translation but not mRNA stability. This repression, however, was not observed when Argonaute was knocked down in Giardia using a ribozyme-antisense RNA. Instead, an enhancement of luciferase expression was observed, suggesting a loss of endogenous miR2-mediated repression when this protein is depleted. Additionally, the level of miR2 was significantly reduced when Dicer was knocked down. In all, the evidence indicates the presence of a snoRNA-derived miRNA-mediated translational repression in Giardia.
Gene regulation in Giardia lamblia, a primitive parasitic protozoan responsible for the diarrheal disease giardiasis, is poorly understood. There is no consensus promoter sequence. A simple eight–base pair AT-rich region is sufficient to initiate gene transcription in this organism. Thus, the main control of gene expression may occur after the stage of transcription. The presence of Dicer and Argonaute homologs in Giardia suggested that microRNA (miRNA)-mediated translational repression could be one mechanism of gene regulation. In this work, we characterized the presence of the miRNA pathway in Giardia as well as identified the novel use of small nucleolar RNA (snoRNA) as miRNA precursors. Potential target sites for one small RNA (miR2) were identified with the miRanda program. In vivo reporter assays confirmed the specific interaction between the target sites and miR2. A ribozyme-mediated reduction of Dicer and Argonaute in Giardia showed that the former is required for miR2 production whereas the latter functions in mediating the inhibition of reporter expression, which agrees with the roles of these two proteins. This is the first evidence of miRNA-mediated gene regulation in Giardia and the first demonstration of the use of snoRNAs as miRNA precursors.
Aurora B kinase plays essential roles in mitosis and cytokinesis in eukaryotes. In the procyclic form of Trypanosoma brucei, the Aurora B homolog TbAUK1 regulates mitosis and cytokinesis, phosphorylates the Tousled-like kinase TbTLK1, interacts with two mitotic kinesins TbKIN-A and TbKIN-B and forms a novel chromosomal passenger complex (CPC) with two novel proteins TbCPC1 and TbCPC2. Here we show with time-lapse video microscopy the time course of CPC trans-localization from the spindle midzone in late anaphase to the dorsal side of the cell where the anterior end of daughter cell is tethered, and followed by a glide toward the posterior end to divide the cell, representing a novel mode of cytokinesis in eukaryotes. The three subunits of CPC, TbKIN-B and TbTLK1 interact with one another suggesting a close association among the five proteins. An ablation of TbTLK1 inhibited the subsequent trans-localization of CPC and TbKIN-B, whereas a knockdown of CPC or TbKIN-B disrupted the spindle pole localization of TbTLK1 during mitosis. In the bloodstream form of T. brucei, the five proteins also play essential roles in chromosome segregation and cytokinesis and display subcellular localization patterns similar to that in the procyclic form. The CPC in bloodstream form also undergoes a trans-localization during cytokinesis similar to that in the procyclic form. All together, our results indicate that the five-protein complex CPC-TbTLK1-TbKIN-B plays key roles in regulating chromosome segregation in the early phase of mitosis and that the highly unusual mode of cytokinesis mediated by CPC occurs in both forms of trypanosomes.
Trichomonas vaginalis is a parasitic protozoan purine auxotroph possessing a unique purine salvage pathway consisting of a bacterial type purine nucleoside phosphorylase (PNP) and a purine nucleoside kinase. Thus, T. vaginalis PNP (TvPNP) functions in the reverse direction relative to PNPs in other organisms. Immucillin-A (ImmA) and DADMe-Immucillin-A (DADMe-ImmA) are transition state mimics of adenosine with geometric and electrostatic features that resemble early and late transition states of adenosine at the transition state stabilized by TvPNP. ImmA demonstrates slow-onset tight-binding inhibition with TvPNP, to give an equilibrium dissociation constant of 87 pM, an inhibitor release half-time of 17.2 min and a Km/Kd ratio of 70,100. DADMe-ImmA resembles a late ribooxacarbenium ion transition state for TvPNP to give a dissociation constant of 30 pM, an inhibitor release half-time of 64 min and a Km/Kd ratio of 203,300. Tight binding of DADMe-ImmA supports a late SN1 transition state. Despite their tight binding to TvPNP, ImmA and DADMe-ImmA are weak inhibitors of human and P. falciparum PNPs. The crystal structures of the TvPNP•ImmA•PO4 and TvPNP•DADMe-ImmA•PO4 ternary complexes differ from previous structures with substrate analogues. The tight binding with DADMe-ImmA is in part due to a 2.7 Å ionic interaction between a PO4 oxygen and the N1’ cation of the hydroxypyrrolidine and is weaker in the TvPNP•ImmA•PO4 structure at 3.5 Å. However, the TvPNP•ImmA•PO4 structure includes hydrogen bonds between the 2’-hydroxyl and the protein that are not present in TvPNP•DADMe-ImmA•PO4. These structures explain why DADMe-ImmA binds tighter than ImmA. Immucillin-H is a 12 nM inhibitor of TvPNP but a 56 pM inhibitor of human PNP. And this difference is explained by isotope-edited difference infrared spectroscopy with [6-18O]ImmH to establish that O6 is the keto tautomer in TvPNP•ImmH•PO4, causing an unfavorable leaving-group interaction.
Aurora B kinase is a key component of the chromosomal passenger complex (CPC), which regulates chromosome segregation and cytokinesis. An ortholog of Aurora B was characterized in Trypanosoma brucei (TbAUK1), but other conserved components of the complex have not been found. Here we identified four novel TbAUK1 associated proteins by tandem affinity purification and mass spectrometry. Among these four proteins, TbKIN-A and TbKIN-B are novel kinesin homologs, whereas TbCPC1 and TbCPC2 are hypothetical proteins without any sequence similarity to those known CPC components from yeasts and metazoans. RNAi-mediated silencing of each of the four genes led to loss of spindle assembly, chromosome segregation and cytokinesis. TbKIN-A localizes to the mitotic spindle and TbKIN-B to the spindle midzone during mitosis, whereas TbCPC1, TbCPC2 and TbAUK1 display the dynamic localization pattern of a CPC. After mitosis, the CPC disappears from the central spindle and re-localizes at a dorsal mid-point of the mother cell, where the anterior tip of the daughter cell is tethered, to start cell division toward the posterior end, indicating a most unusual CPC-initiated cytokinesis in a eukaryote.
ATP-dependent protease complexes are present in all living organisms, including the 26S proteasome in eukaryotes, Archaea, and Actinomycetales, and the HslVU protease in eubacteria. The structure of HslVU protease resembles that of the 26S proteasome, and the simultaneous presence of both proteases in one organism was deemed unlikely. However, HslVU homologs have been identified recently in some primordial eukaryotes, though their potential function remains elusive. We characterized the HslVU homolog from Trypanosoma brucei, a eukaryotic protozoan parasite and the causative agent of human sleeping sickness. TbHslVU has ATP-dependent peptidase activity and, like its bacterial counterpart, has essential lysine and N-terminal threonines in the catalytic subunit. By epitope tagging, TbHslVU localizes to mitochondria and is associated with the mitochondrial genome, kinetoplast DNA (kDNA). RNAi of TbHslVU dramatically affects the kDNA by causing over-replication of the minicircle DNA. This leads to defects in kDNA segregation and, subsequently, to continuous network growth to an enormous size. Multiple discrete foci of nicked/gapped minicircles are formed on the periphery of kDNA disc, suggesting a failure in repairing the gaps in the minicircles for kDNA segregation. TbHslVU is a eubacterial protease identified in the mitochondria of a eukaryote. It has a novel function in regulating mitochondrial DNA replication that has never been observed in other organisms.
ATP-dependent protein-hydrolyzing enzyme complexes are present in all living organisms, including the 26S proteasome in eukaryotes and the HslVU complex in bacteria. A simultaneous presence of both complexes in an organism was originally deemed unlikely until some HslVU homologs were found in certain ancient eukaryotes, though their potential function in these organisms remains unclear. We characterized an HslVU complex in Trypanosoma brucei, a protozoan parasite that causes human sleeping sickness in Africa. The complex is an active enzyme localized to the mitochondria of the parasite and closely associated with the mitochondrial DNA complex, which consists of several thousand small circular DNAs and a few dozen mitochondrial genomic DNAs. Depletion of this HslVU from the parasite resulted in a continuous synthesis of the small circular DNA, which led to aberrant segregation and incessant growth of the mitochondrial DNA complex to an enormous size that eventually blocks cell division. This novel HslVU function, which has not been observed in other organisms previously, could be a potential target for anti-sleeping sickness chemotherapy.
Centrin is a calcium-binding centrosome/basal body–associated protein involved in duplication and segregation of these organelles in eukaryotes. We had shown that disruption of one of the centrin genes (centrin1) in Leishmania amastigotes resulted in failure of both basal body duplication and cytokinesis. Here, we undertook to define the role of centrin1 (TbCen1) in the duplication and segregation of basal body and its associated organelles kinetoplast and Golgi, as well as its role in cytokinesis of the procyclic form of Trypanosoma brucei by depleting its protein using RNA inhibition methodology. TbCen1-depleted cells showed significant reduction in growth compared with control cells. Morphological analysis of these cells showed they were large and pleomorphic with multiple detached flagella. Both immunofluorescence assays using organelle-specific antibodies and electron microscopic analysis showed that TbCen1-deficient cells contained multiple basal bodies, kinetoplasts, Golgi, and nuclei. These multiple organelles were, however, closely clustered together, indicating duplication without segregation in the absence of centrin. This failure in organelle segregation may be the likely cause of inhibition of cytokinesis, suggesting for the first time a new and unique role for centrin in the segregation of organelles without affecting their multiplication in the procyclic form of T. brucei.
Eukaryotic translation initiation factor 4E (eIF4E) binds to the m7GTP of capped mRNAs and is an essential component of the translational machinery that recruits the 40S small ribosomal subunit. We describe here the identification and characterization of two eIF4E homologues in an ancient protist, Giardia lamblia. Using m7GTP-Sepharose affinity column chromatography, a specific binding protein was isolated and identified as Giardia eIF4E2. The other homologue, Giardia eIF4E1, bound only to the m2,2,7GpppN structure. Although neither homologue can rescue the function of yeast eIF4E, a knockdown of eIF4E2 mRNA in Giardia by a virus-based antisense ribozyme decreased translation, which was shown to use m7GpppN-capped mRNA as a template. Thus, eIF4E2 is likely the cap-binding protein in a translation initiation complex. The same knockdown approach indicated that eIF4E1 is not required for translation in Giardia. Immunofluorescence assays showed wide distribution of both homologues in the cytoplasm. But eIF4E1 was also found concentrated and colocalized with the m2,2,7GpppN cap, 16S-like rRNA, and fibrillarin in the nucleolus-like structure in the nucleus. eIF4E1 depletion from Giardia did not affect mRNA splicing, but the protein was bound to Giardia small nuclear RNAs D and H known to have an m2,2,7GpppN cap, thus suggesting a novel function not yet observed among other eIF4Es in eukaryotes.
Expression of the cdc2-related kinase 3 (CRK3) together with expression of CRK1, -2, -4, or -6, were knocked down in pairs in the procyclic and bloodstream forms of Trypanosoma brucei, using the RNA interference technique. Double knockdowns of CRK3 and CRK2, CRK4, or CRK6 exerted significant growth inhibition and enriched the cells in G2/M phase, whereas a CRK3 plus CRK1 (CRK3 + CRK1) knockdown arrested cells in both G1/S and G2/M transitions. Thus, CRK1 and CRK3 are apparently the kinases regulating the G1/S and G2/M checkpoint passages, respectively, whereas the other CRKs are probably playing only minor roles in cell cycle regulation. A CRK1 + CRK2 knockdown in the procyclic form was found to cause aberrant posterior cytoskeletal morphogenesis (X. M. Tu and C. C. Wang, Mol. Biol. Cell 16:97-105, 2005). A CRK3 + CRK2 knockdown, however, did not lead to such a change, suggesting that CRK2 depletion can lead to the abnormal morphogenesis only when procyclic-form cells are arrested in the G1 phase. The G2/M-arrested procyclic form produces up to 20% stumpy anucleated cells (zoids) in the population, suggesting that cytokinesis and cell division are not blocked by mitotic arrest but are apparently driven to completion by the kinetoplast cycle. In the bloodstream form, however, G2/M arrest resulted in little zoid formation but, instead, enriched a population of cells each containing multiple kinetoplasts, basal bodies, and flagella and an aggregate of multiple nuclei, indicating failure in entering cytokinesis. The two different cytokinetic regulations between two distinct stage-specific forms of the same organism may provide an interesting and useful model for further understanding the evolution of cytokinetic control among eukaryotes.
Translation of uncapped giardiavirus (GLV) mRNA in Giardia lamblia requires the presence of a 5′-untranslated region (5′-UTR) and a viral capsid coding region. We used dicistronic viral constructs to show that the downstream 253 nucleotides (nt) of the 5′-UTR plus the initial 264-nt capsid coding region constitute an internal ribosome entry site (IRES). Predicted secondary structures in the 253-nt 5′-UTR include stem-loops U3, U4a, U4b, U4c, and U5. Chemical and enzymatic probing analysis confirmed the presence of all predicted stem-loops except U4a. Disruption of stem-loop structures U3 and U5 by site-directed mutagenesis resulted in a drastic reduction in translation of a monocistronic viral transcript, which could be restored by compensatory sequence changes. Mutations disrupting stem-loops U4b and U4c do not exert an appreciable effect on translation, but certain sequences in the U4a region and in U4b do appear to play important roles in the IRES. Structural analysis also suggests that an 8-nt U3 loop sequence (nt 147 to 154) pairs with an 8-nt downstream sequence (nt 168 to 175) to form a pseudoknot. Disruption of this pseudoknot by mutagenesis resulted in a drastic reduction in translation, which could be restored by compensatory sequence changes. This study has defined the secondary structure in the 5′-UTR of the IRES. Together with the previous results, we have now completed analysis of the entire structure of GLV IRES and fully defined the functionally essential structural elements in it.
The expression levels of four Cdc2-related kinases (CRK1, 2, 4, and 6) in the procyclic form of Trypanosoma brucei were knocked down in pairs using the RNA interference (RNAi) technique. A double knockdown of CRK1 and CRK2 resulted in arrested cell growth in the G1 phase accompanied by an apparent cessation of nuclear DNA synthesis. The arrested cells became elongated at the posterior end like the G1-phase cells generated by knockdown of CycE1/CYC2 in a previous study. However, ∼5% of the G1 cells in the current study also possessed multiply branched posterior ends, which have not previously been observed in T. brucei. DAPI and immunofluorescence staining showed a single nucleus, kinetoplast, basal body, and flagellum in the anterior portion of each G1 cell. The split and grossly extended posterior ends were heavily stained with antibodies to tyrosinated α-tubulin, suggesting an accumulation of newly synthesized microtubules. A significant population of anucleate cells (zoids), apparently derived from kinetoplast-dictated cytokinesis and cell division of the G1 cells, also had extended and branched posterior ends filled with newly synthesized microtubules. This continued posterior extension of microtubules in the G1 cells and zoids suggests that CRK1 and CRK2 exert a coordinated control on G1/S passage and the limited growth of the microtubule corset toward the posterior end. This connection may provide a new insight into the mechanism of morphological maintenance of an ancient protist during its cell cycle progression.
Schistosoma mansoni, an intravascular parasite, has evolved a number of immune evasion mechanisms to establish itself in the host, such as antioxidant enzymes. Our laboratory has demonstrated that the highest levels of certain antioxidant enzymes are found in adult worms, which are the least susceptible to immune killing. Vaccination of mice with naked DNA constructs containing the gene encoding Cu/Zn cytosolic superoxide dismutase (SmCT-SOD) showed significant levels of protection compared to a control group, and our data demonstrate that the adult worms are a target of the immune response that confers resistance in SmCT-SOD DNA-vaccinated mice. Because SmCT-SOD shows significant identity with the human homologue, we evaluated the reactivity of anti-SmCT-SOD antibodies derived from SmCT-SOD-immunized mice and rabbits and from S. mansoni-infected individuals to human superoxide dismutase (hSOD) and SmCT-SOD parasite-specific peptides to assess the potential for autoimmune responses from immunization with the recombinant molecule. In addition, we evaluated the ability of various SmCT-SOD adjuvant-delivered immunizations to induce cross-reactive antibodies. Both mouse and rabbit antibodies generated against SmCT-SOD recognized the denatured form of hSOD. The same antibodies did not recognize nondenatured hSOD. Sera from infected individuals with different clinical forms of schistosomiasis recognized SmCT-SOD but not hSOD. Antibodies from mice immunized with different SmCT-SOD-containing formulations of both DNA and protein were able to recognize SmCT-SOD-derived peptides but not soluble hSOD. All together, these findings serve as a basis for developing a subunit vaccine against schistosomiasis.
The RNA polymerase of giardiavirus (GLV) is synthesized as a fusion protein through a −1 ribosomal frameshift in a region where gag and pol open reading frames (ORFs) overlap. A heptamer, CCCUUUA, and a potential pseudoknot found in the overlap were predicted to be required for the frameshift. A 68-nucleotide (nt) cDNA fragment containing these elements was inserted between the GLV 5′ 631-nt cDNA and the out-of-frame luciferase gene that required a −1 frameshift within the 68-nt fragment for expression. Giardia lamblia trophozoites transfected with the transcript of this construct showed a frameshift frequency at 1.7%, coinciding with the polymerase-to-capsid protein ratio in GLV. The heptamer is required for the frameshift but can be replaced with other sequences of the same motif. Mutations placing stop codons in the 0 or −1 frame, located directly before or after the heptamer, implicated the latter as the site for the −1 frameshift. Shortening or destroying the putative stem decreased the frameshift efficiency threefold; the efficiency was fully recovered by mutations to restore the stem. Deleting 18 nt from the 3′ end of the 68-nt fragment, which formed the second stem in the putative pseudoknot, had no effect on the frequency of the frameshift. Chemical probing of the RNA secondary structure in the frameshift region showed that bases resistant to chemical modification were clustered in the putative stem structures, thus confirming the presence of the postulated stem-loop, while all the bases in the loop were chemically modified, thus ruling out their capability of forming a pseudoknot. These results confirmed the conclusion based on data from the mutation study that there is but a simple stem-loop downstream from the heptamer. Together, they constitute the structural elements for a −1 ribosomal frameshift in the GLV transcript.
Parasitic protozoa lack the ability to synthesize purine nucleotides de novo, relying instead on purine salvage enzymes for their survival. Guanine phosphoribosyltransferase (GPRT) from the protozoan parasite Giardia lamblia is a potential target for rational antiparasitic drug design, based on the experimental evidence, which indicates the lack of interconversion between adenine and guanine nucleotide pools. The present study is a continuation of our efforts to use three-dimensional structures of parasitic phosphoribosyltransferases (PRTs) to design novel antiparasitic agents. Two micromolar phthalimide-based GPRT inhibitors were identified by screening the in-house phthalimide library. A combination of structure-based scaffold selection using virtual library screening across the PRT gene family and solid phase library synthesis led to identification of smaller (molecular weight, <300) ligands with moderate to low specificity for GPRT; the best inhibitors, GP3 and GP5, had Ki values in the 23 to 25 μM range. These results represent significant progress toward the goal of designing potent inhibitors of purine salvage in Giardia parasites. As a second step in this process, altering the phthalimide moiety to optimize interactions in the guanine-binding pocket of GPRT is expected to lead to compounds with promising activity against G. lamblia PRT.
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.
The encystation process is crucial for survival and transmission of Giardia lamblia to new hosts. During this process, vesicular trafficking and the cytoskeleton play important roles. In eukaryotic cells, intracellular transport is regulated by proteins, including Rab-GTPases and SNAREs, which regulate vesicle formation along with recognition of and binding to the target membrane. Cytoskeletal structures are also involved in these processes. In this study, we demonstrate the participation of Rab11 in the transport of encystation-specific vesicles (ESVs). Additionally, we demonstrate that disruption of actin microfilaments affects ESVs transport. The modification of actin dynamics was also correlated with a reduction in rab11 and cwp1 expression. Furthermore, down-regulation of rab11 mRNA by a specific hammerhead ribozyme caused nonspecific localization of CWP1. We thus provide new information about the molecular machinery that regulates Giardia lamblia encystation. Given our findings, Rab11 and actin may be useful targets to block Giardia encystation.