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1.
BMC Evol Biol ; 20(1): 92, 2020 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-32727367

RESUMO

BACKGROUND: Hypotrichia are a group with the most complex morphology and morphogenesis within the ciliated protists. The classification of Gastrostyla-like species, a taxonomically difficult group of hypotrichs with a common ventral cirral pattern but various dorsal and ontogenetic patterns, is poorly understood. Hence, systematic relationships within this group and with other taxa in the subclass Hypotrichia remain unresolved. RESULTS: 18S rRNA gene sequence of a new Gastrostyla-like taxon was obtained. Phylogenetic analyses based on the 18S rRNA gene sequences indicate that this ciliate represents a new genus that is closely related to Heterourosomoida and Kleinstyla within the oxytrichid clade of the Hypotrichia. However, the position of this cluster remains unresolved. All three genera deviate from the typical oxytrichids by their incomplete (or lack of) dorsal kinety fragmentation during morphogenesis. Morphology and morphogenesis of this newly discovered form, Heterogastrostyla salina nov. gen., nov. spec., are described. Heterogastrostyla nov. gen., is characterised as follows: more than 18 fronto-ventral-transverse cirri, cirral anlagen V and VI develop pretransverse cirri, and dorsal ciliature in Urosomoida-like pattern. CONCLUSIONS: Similar to the CEUU-hypothesis about convergent evolution of urostylids and uroleptids, we speculate that the shared ventral cirral patterns of Gastrostyla-like taxa might have resulted from convergent evolution.


Assuntos
Cilióforos/classificação , Classificação , Salinidade , Solo , Animais , Sequência de Bases , Núcleo Celular/genética , DNA Ribossômico/genética , Hypotrichida/classificação , Hypotrichida/genética , Funções Verossimilhança , Morfogênese/genética , Filogenia , Subunidades Ribossômicas Menores/genética , Especificidade da Espécie
2.
Parasitol Res ; 119(8): 2733-2740, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32617726

RESUMO

Amebiasis is a worldwide parasitic zoonosis, with symptoms of abdominal discomfort, indigestion, diarrhea, and even death. However, limited information about the prevalence of Entamoeba spp. in experimental nonhuman primates (NHPs) in southwestern China is available. The objective of the current study was to investigate the frequency and species identity of Entamoeba to evaluate potential zoonotic risk factors for Entamoeba spp. infection in experimental NHPs. A total of 505 fecal samples were collected from NHPs (macaques) and analyzed by PCR analysis the small subunit rRNA (SSU rRNA) gene of Entamoeba spp. Forty-seven specimens were positive for Entamoeba spp., and the prevalence of Entamoeba spp. was 9.31% (47/505). Significant differences in the prevalence rates among the three breeds (P = 0.002 < 0.01, df = 2, χ2 = 12.33) and feed types (P = 0.001 < 0.01, df = 1, χ2 = 10.12) were observed. Altogether, four Entamoeba species, including E. dispar (57.44%), E. chattoni (29.78%), E. histolytica (6.38%), and E. coli (6.38%), were identified by DNA sequence analysis. The results suggested a low prevalence but high diversity of Entamoeba species in experimental NHPs in Yunnan Province, southwestern China. Results of this study contribute to the knowledge of the genetic characteristics of Entamoeba spp. in NHPs.


Assuntos
Entamoeba/genética , Entamebíase/veterinária , Macaca/parasitologia , Infecções Protozoárias em Animais/epidemiologia , Infecções Protozoárias em Animais/parasitologia , Animais , Animais de Laboratório , China/epidemiologia , DNA de Protozoário/genética , Entamoeba/classificação , Entamoeba/isolamento & purificação , Entamebíase/epidemiologia , Entamebíase/parasitologia , Entamebíase/transmissão , Fezes/parasitologia , Epidemiologia Molecular , Prevalência , Infecções Protozoárias em Animais/transmissão , RNA Ribossômico/genética , Subunidades Ribossômicas Menores/genética , Análise de Sequência de DNA
3.
Parasitol Res ; 119(8): 2741-2745, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32577820

RESUMO

Blastocystis is a zoonotic protozoan parasite frequently identified in the intestinal tract of humans and a vast variety of animals, worldwide. Here, we assessed the prevalence of Blastocystis and its subtypes in stool samples of raccoons. Stool samples from 30 raccoons were collected. Total DNA was extracted, and the barcoding region of the small subunit ribosomal rRNA (SSU rRNA) gene was amplified and sequenced. Specific fragment for Blastocystis was successfully amplified in five samples (16.66%). Sequencing analysis revealed ST1, ST2, and ST3 among 1, 2, and 2 Blastocystis-positive samples. Our results documented the presence of Blastocystis subtypes 1-3 in raccoons. Subtype 1 showed higher similarity to the human isolates of Blastocystis. However, it seems that raccoons may emerge as reservoirs for Blastocystis and may be linked to zoonotic transmission of the protist.


Assuntos
Infecções por Blastocystis/veterinária , Blastocystis/isolamento & purificação , Infecções Protozoárias em Animais/parasitologia , Guaxinins/parasitologia , Animais , Sequência de Bases , Blastocystis/classificação , Blastocystis/genética , Infecções por Blastocystis/epidemiologia , Infecções por Blastocystis/parasitologia , Infecções por Blastocystis/transmissão , DNA de Protozoário/genética , Fezes/parasitologia , Variação Genética , Genótipo , Irã (Geográfico)/epidemiologia , Prevalência , Infecções Protozoárias em Animais/epidemiologia , Infecções Protozoárias em Animais/transmissão , RNA Ribossômico/genética , Subunidades Ribossômicas Menores/genética
4.
Parasitol Res ; 119(8): 2431-2438, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32394002

RESUMO

The specimens of Trachinus draco collected from the Bay of Bizerte were found to be infected with a new Ceratomyxa species described as Ceratomyxa draconis n. sp. The sequence of small subunit ribosomal RNA gene obtained in this study differs from other Ceratomyxa sequences available in GenBank. Mature spores of this species were elongated and crescent-shaped in sutural view, measuring 7.4 ± 0.77 (6.4-8.0) µm in thickness and 30.8 ± 1.65 (28.8-32.8) µm in width. The polar capsules were spherical, equal in size, and measuring 3.3 ± 0.2 (3.6-4.0) µm in diameter. The Ceratomyxa draconis n. sp. showed a clearly seasonal variation of prevalence with highest prevalence noted during summer months.


Assuntos
Doenças dos Peixes/parasitologia , Myxozoa , Doenças Parasitárias em Animais/parasitologia , Perciformes/parasitologia , Animais , Baías , DNA Ribossômico/genética , Vesícula Biliar/parasitologia , Myxozoa/anatomia & histologia , Myxozoa/classificação , Myxozoa/genética , Filogenia , Subunidades Ribossômicas Menores/genética , Estações do Ano , Tunísia/epidemiologia
5.
RNA ; 26(9): 1268-1282, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32467310

RESUMO

PPR proteins are a diverse family of RNA binding factors found in all Eukaryotic lineages. They perform multiple functions in the expression of organellar genes, mostly on the post-transcriptional level. PPR proteins are also significant determinants of evolutionary nucleo-organellar compatibility. Plant PPR proteins recognize their RNA substrates using a simple modular code. No target sequences recognized by animal or yeast PPR proteins were identified prior to the present study, making it impossible to assess whether this plant PPR code is conserved in other organisms. Dmr1p (Ccm1p, Ygr150cp) is a S. cerevisiae PPR protein essential for mitochondrial gene expression and involved in the stability of 15S ribosomal RNA. We demonstrate that in vitro Dmr1p specifically binds a motif composed of multiple AUA repeats occurring twice in the 15S rRNA sequence as the minimal 14 nt (AUA)4AU or longer (AUA)7 variant. Short RNA fragments containing this motif are protected by Dmr1p from exoribonucleolytic activity in vitro. Presence of the identified motif in mtDNA of different yeast species correlates with the compatibility between their Dmr1p orthologs and S. cerevisiae mtDNA. RNA recognition by Dmr1p is likely based on a rudimentary form of a PPR code specifying U at every third position, and depends on other factors, like RNA structure.


Assuntos
Proteínas Mitocondriais/genética , Motivos de Nucleotídeos/genética , RNA Ribossômico/genética , RNA/genética , Subunidades Ribossômicas Menores/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Expressão Gênica/genética , Mitocôndrias/genética , Ribossomos/genética
6.
Mol Phylogenet Evol ; 149: 106839, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32325195

RESUMO

Alveolates are a major supergroup of eukaryotes encompassing more than ten thousand free-living and parasitic species, including medically, ecologically, and economically important apicomplexans, dinoflagellates, and ciliates. These three groups are among the most widespread eukaryotes on Earth, and their environmental success can be linked to unique innovations that emerged early in each group. Understanding the emergence of these well-studied and diverse groups and their innovations has relied heavily on the discovery and characterization of early-branching relatives, which allow ancestral states to be inferred with much greater confidence. Here we report the phylogenomic analyses of 313 eukaryote protein-coding genes from transcriptomes of three members of one such group, the colponemids (Colponemidia), which support their monophyly and position as the sister lineage to all other known alveolates. Colponemid-related sequences from environmental surveys and our microscopical observations show that colponemids are not common in nature, but they are diverse and widespread in freshwater habitats around the world. Studied colponemids possess two types of extrusive organelles (trichocysts or toxicysts) for active hunting of other unicellular eukaryotes and potentially play an important role in microbial food webs. Colponemids have generally plesiomorphic morphology and illustrate the ancestral state of Alveolata. We further discuss their importance in understanding the evolution of alveolates and the origin of myzocytosis and plastids.


Assuntos
Alveolados/classificação , Comportamento Predatório/fisiologia , Alveolados/genética , Alveolados/ultraestrutura , Animais , Biodiversidade , Geografia , Filogenia , Subunidades Ribossômicas Menores/genética
7.
Parasitol Res ; 119(3): 893-901, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31938889

RESUMO

We surveyed introduced yellow perch Perca flavescens (Mitchill, 1814) from the Willamette River, OR, USA, to determine if these fish have co-introduced myxosporean parasites. Mature parasite myxospores were observed in brains of 3/19 fish, and were morphologically and molecularly consistent with Myxobolus neurophilus (Guilford 1963), a parasite known from yellow perch in their native range. We identified another Myxobolus species from the gill filaments of 1/22 fish. The spores from the gill filaments were oval-shaped, 11.7 (10.7-12.3) µm long × 8.6 (7.7-9.0) µm wide × 5.2 (4.6-5.6) µm thick, with two oval-shaped polar capsules 5.7 (5.1-6.5) µm × 2.7 (2.4-3.2) µm, each containing a polar tubule with 8-9 turns. Small-subunit ribosomal DNA sequences from each of four plasmodia were identical, and 4.0% different (over 1800 nucleotides) from the closest known myxosporeans. Interestingly, these sequences had overlapping peaks in their chromatograms, which suggested that DNA from multiple species was present. Hence, we isolated and sequenced three individual myxospores and found that they too had mixed chromatograms, which indicated presence of at least two sequence types of small-subunit ribosomal DNA in each spore (GenBank accession MK592012, MK592013), a rare character among described myxosporeans. The spore morphology, morphometry, tissue tropism, and DNA sequence supported a diagnosis of a novel species, Myxobolus doubleae n. sp. This parasite is unknown from yellow perch in its native range, despite extensive historical surveys, which suggests that introduced yellow perch might have acquired an endemic Myxobolus species via spillback from another fish host.


Assuntos
Doenças dos Peixes/parasitologia , Myxobolus/isolamento & purificação , Doenças Parasitárias em Animais/parasitologia , Percas/parasitologia , Animais , DNA de Protozoário/genética , DNA Ribossômico/genética , Brânquias/parasitologia , Filogenia , Subunidades Ribossômicas Menores/genética , Rios/parasitologia , Esporos de Protozoários
8.
Parasitol Res ; 119(1): 85-96, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31768684

RESUMO

Examination of 35 barramundi (Lates calcarifer) from aquaculture cages in Setiu Wetland, Malaysia, revealed a single fish infected with three Henneguya spp. (Cnidaria: Myxosporea). Characterization of the infections using tissue tropism, myxospore morphology and morphometry and 18S rDNA sequencing supported description of three new species: Henneguya setiuensis n. sp., Henneguya voronini n. sp. and H. calcarifer n. sp. Myxospores of all three species had typical Henneguya morphology, with two polar capsules in the plane of the suture, an oval spore body, smooth valve cell surfaces, and two caudal appendages. Spores were morphometrically similar, and many dimensions overlapped, but H. voronini n. sp. had shorter caudal appendages compared with H. calcarifer n. sp. and H. setiuensis n. sp. Gross tissue tropism distinguished the muscle parasite H. calcarifer n. sp. from gill parasites H. setiuensis n. sp. and H. voronini n. sp.; and these latter two species were further separable by fine-scale location of developing plasmodia, which were intra-lamellar for H. setiuensis n. sp. and basal to the filaments for H. voronini n. sp. small subunit ribosomal DNA sequences distinguished all three species: the two gill species H. setiuensis n. sp. and H voronini n. sp. were only 88% similar (over 1708 bp), whereas the muscle species H. calcarifer n. sp. was most similar to H. voronini n. sp. (98% over 1696 bp). None of the three novel species was more than 90% similar to any known myxosporean sequence in GenBank. Low infection prevalence of these myxosporeans and lack of obvious tissue pathology from developing plasmodia suggested none of these parasites are currently a problem for barramundi culture in Setiu Wetland; however additional surveys of fish, particularly at different times of the year, would be informative for better risk assessment.


Assuntos
Doenças dos Peixes/parasitologia , Myxozoa/classificação , Myxozoa/isolamento & purificação , Doenças Parasitárias em Animais/diagnóstico , Doenças Parasitárias em Animais/parasitologia , Perciformes/parasitologia , Animais , Aquicultura , Cnidários/classificação , DNA Ribossômico/genética , Doenças dos Peixes/diagnóstico , Peixes , Brânquias/parasitologia , Malásia , Filogenia , RNA Ribossômico 18S/genética , Subunidades Ribossômicas Menores/genética , Esporos/genética , Áreas Alagadas
9.
Parasitol Res ; 119(1): 243-248, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31754858

RESUMO

Cryptosporidiosis has been reported as an important cause of neonatal diarrhea and mortality in cattle, sheep, and other ruminants, but its impact on alpaca health has not been studied thoroughly. In this study, we have determined the prevalence and evaluated the role of cryptosporidiosis as a risk factor for diarrhea occurrence in newborn alpacas. During the calving season (January-March) of 2006, stool specimens (N = 1312) were collected from 24 herds of newborn alpacas in Puno and Cuzco, departments that account for the largest populations of alpacas in Peru. All the specimens were microscopically screened for Cryptosporidium spp. using the acid-fast technique. The association between Cryptosporidium detection and diarrhea was analyzed using χ2 test and generalized lineal model. Cryptosporidium species were determined by PCR-RFLP analysis of the small subunit rRNA gene. Cryptosporidium oocysts were detected in 159 of 1312 (12.4%) newborn alpacas. Results of the analyses demonstrated that crypstosporidiosis was significantly associated with diarrhea (PR = 3.84; CI95% 2.54-5.81; p < 0.0001). Only Cryptosporidium parvum was detected in the 153 Cryptosporidium-infected animals. Thus, there is an association of C. parvum infection with diarrhea in neonatal alpacas.


Assuntos
Camelídeos Americanos/parasitologia , Criptosporidiose/epidemiologia , Criptosporidiose/parasitologia , Cryptosporidium parvum/isolamento & purificação , Diarreia/veterinária , Animais , Animais Recém-Nascidos , Cryptosporidium parvum/classificação , Cryptosporidium parvum/citologia , Cryptosporidium parvum/genética , Diarreia/epidemiologia , Diarreia/parasitologia , Fezes/parasitologia , Oocistos/citologia , Peru/epidemiologia , Prevalência , Subunidades Ribossômicas Menores/genética , Fatores de Risco
10.
Int J Mol Sci ; 20(18)2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31510048

RESUMO

Several control mechanisms of eukaryotic gene expression target the initiation step of mRNA translation. The canonical translation initiation pathway begins with cap-dependent attachment of the small ribosomal subunit (SSU) to the messenger ribonucleic acid (mRNA) followed by an energy-dependent, sequential 'scanning' of the 5' untranslated regions (UTRs). Scanning through the 5'UTR requires the adenosine triphosphate (ATP)-dependent RNA helicase eukaryotic initiation factor (eIF) 4A and its efficiency contributes to the specific rate of protein synthesis. Thus, understanding the molecular details of the scanning mechanism remains a priority task for the field. Here, we studied the effects of inhibiting ATP-dependent translation and eIF4A in cell-free translation and reconstituted initiation reactions programmed with capped mRNAs featuring different 5'UTRs. An aptamer that blocks eIF4A in an inactive state away from mRNA inhibited translation of capped mRNA with the moderately structured ß-globin sequences in the 5'UTR but not that of an mRNA with a poly(A) sequence as the 5'UTR. By contrast, the nonhydrolysable ATP analogue ß,γ-imidoadenosine 5'-triphosphate (AMP-PNP) inhibited translation irrespective of the 5'UTR sequence, suggesting that complexes that contain ATP-binding proteins in their ATP-bound form can obstruct and/or actively block progression of ribosome recruitment and/or scanning on mRNA. Further, using primer extension inhibition to locate SSUs on mRNA ('toeprinting'), we identify an SSU complex which inhibits primer extension approximately eight nucleotides upstream from the usual toeprinting stop generated by SSUs positioned over the start codon. This '-8 nt toeprint' was seen with mRNA 5'UTRs of different length, sequence and structure potential. Importantly, the '-8 nt toeprint' was strongly stimulated by the presence of the cap on the mRNA, as well as the presence of eIFs 4F, 4A/4B and ATP, implying active scanning. We assembled cell-free translation reactions with capped mRNA featuring an extended 5'UTR and used cycloheximide to arrest elongating ribosomes at the start codon. Impeding scanning through the 5'UTR in this system with elevated magnesium and AMP-PNP (similar to the toeprinting conditions), we visualised assemblies consisting of several SSUs together with one full ribosome by electron microscopy, suggesting direct detection of scanning intermediates. Collectively, our data provide additional biochemical, molecular and physical evidence to underpin the scanning model of translation initiation in eukaryotes.


Assuntos
Regiões 5' não Traduzidas/genética , Biossíntese de Proteínas , Capuzes de RNA/genética , RNA Mensageiro/genética , Subunidades Ribossômicas Menores/genética , Trifosfato de Adenosina/metabolismo , Adenilil Imidodifosfato/metabolismo , Animais , Linhagem Celular Tumoral , Sistema Livre de Células , Fator de Iniciação 4F em Eucariotos/metabolismo , Camundongos , Modelos Genéticos , RNA Helicases/metabolismo , Subunidades Ribossômicas Menores/metabolismo , Ribossomos/genética , Ribossomos/metabolismo
11.
Nucleic Acids Res ; 47(15): 8301-8317, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31265110

RESUMO

Assembly factors provide speed and directionality to the maturation process of the 30S subunit in bacteria. To gain a more precise understanding of how these proteins mediate 30S maturation, it is important to expand on studies of 30S assembly intermediates purified from bacterial strains lacking particular maturation factors. To reveal the role of the essential protein Era in the assembly of the 30S ribosomal subunit, we analyzed assembly intermediates that accumulated in Era-depleted Escherichia coli cells using quantitative mass spectrometry, high resolution cryo-electron microscopy and in-cell footprinting. Our combined approach allowed for visualization of the small subunit as it assembled and revealed that with the exception of key helices in the platform domain, all other 16S rRNA domains fold even in the absence of Era. Notably, the maturing particles did not stall while waiting for the platform domain to mature and instead re-routed their folding pathway to enable concerted maturation of other structural motifs spanning multiple rRNA domains. We also found that binding of Era to the mature 30S subunit destabilized helix 44 and the decoding center preventing binding of YjeQ, another assembly factor. This work establishes Era's role in ribosome assembly and suggests new roles in maintaining ribosome homeostasis.


Assuntos
Proteínas de Escherichia coli/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Homeostase , RNA Ribossômico 16S/metabolismo , Proteínas de Ligação a RNA/metabolismo , Subunidades Ribossômicas Menores de Bactérias/metabolismo , Subunidades Ribossômicas Menores/metabolismo , Sequência de Bases , Sítios de Ligação , Microscopia Crioeletrônica , Proteínas de Escherichia coli/genética , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/genética , Conformação de Ácido Nucleico , Ligação Proteica , RNA Ribossômico 16S/química , RNA Ribossômico 16S/genética , Proteínas de Ligação a RNA/genética , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Menores/genética , Subunidades Ribossômicas Menores/ultraestrutura , Subunidades Ribossômicas Menores de Bactérias/genética , Subunidades Ribossômicas Menores de Bactérias/ultraestrutura
12.
Nucleic Acids Res ; 47(14): 7548-7563, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31188444

RESUMO

Ribosome biogenesis is an essential process in all living cells, which entails countless highly sequential and dynamic structural reorganization events. These include formation of dozens RNA helices through Watson-Crick base-pairing within ribosomal RNAs (rRNAs) and between rRNAs and small nucleolar RNAs (snoRNAs), transient association of hundreds of proteinaceous assembly factors to nascent precursor (pre-)ribosomes, and stable assembly of ribosomal proteins. Unsurprisingly, the largest group of ribosome assembly factors are energy-consuming proteins (NTPases) including 25 RNA helicases in budding yeast. Among these, the DEAH-box Dhr1 is essential to displace the box C/D snoRNA U3 from the pre-rRNAs where it is bound in order to prevent premature formation of the central pseudoknot, a dramatic irreversible long-range interaction essential to the overall folding of the small ribosomal subunit. Here, we report the crystal structure of the Dhr1 helicase module, revealing the presence of a remarkable carboxyl-terminal domain essential for Dhr1 function in ribosome biogenesis in vivo and important for its interaction with its coactivator Utp14 in vitro. Furthermore, we report the functional consequences on ribosome biogenesis of DHX37 (human Dhr1) mutations found in patients suffering from microcephaly and other neurological diseases.


Assuntos
RNA Helicases DEAD-box/química , Domínios Proteicos , Subunidades Ribossômicas Menores/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Pareamento de Bases , Sítios de Ligação/genética , Cristalografia por Raios X , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Modelos Moleculares , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , RNA Nucleolar Pequeno/genética , RNA Nucleolar Pequeno/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Menores/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
13.
FEBS J ; 286(21): 4245-4260, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31199072

RESUMO

The Small Ribosomal Subunit Biogenesis GTPase A (RsgA) is a bacterial assembly factor involved in the late stages of the 30S subunit maturation. It is a multidomain GTPase in which the central circularly permutated GTPase domain is flanked by an OB domain and a Zn-binding domain. All three domains participate in the interaction with the 30S particle thus ensuring an efficient coupling between catalytic activity and biological function. In vivo studies suggested the relevance of rsgA in bacterial growth and cellular viability, but other pleiotropic roles of RsgA are also emerging. Here, we report the 3D structure of RsgA from Pseudomonas aeruginosa (PaRsgA) in the GDP-bound form. We also report a biophysical and biochemical characterization of the protein in both the GDP-bound and its nucleotide-free form. In particular, we report a kinetic analysis of the RsgA binding to GTP and GDP. We found that PaRsgA is able to bind both nucleotides with submicromolar affinity. The higher affinity towards GDP (KD  = 0.011 µm) with respect to GTP (KD  = 0.16 µm) is mainly ascribed to a smaller GDP dissociation rate. Our results confirm that PaRsgA, like most other GTPases, has a weak intrinsic enzymatic activity (kCAT  = 0.058 min-1 ). Finally, the biological role of RsgA in P. aeruginosa was investigated, allowing us to conclude that rsgA is dispensable for P. aeruginosa growth but important for drug resistance and virulence in an animal infection model. DATABASES: Coordinates and structure factors for the protein structure described in this manuscript have been deposited in the Protein Data Bank (https://www.rcsb.org) with the accession code 6H4D.


Assuntos
Farmacorresistência Bacteriana/genética , GTP Fosfo-Hidrolases/ultraestrutura , Pseudomonas aeruginosa/metabolismo , Subunidades Ribossômicas Menores/genética , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/metabolismo , Infecções Bacterianas/microbiologia , Sítios de Ligação , Escherichia coli/genética , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/genética , Guanosina Difosfato/química , Cinética , Conformação Molecular , Ligação Proteica/genética , Conformação Proteica , Pseudomonas aeruginosa/enzimologia , Subunidades Ribossômicas Menores/metabolismo , Subunidades Ribossômicas Menores/ultraestrutura
14.
Mol Cell Biol ; 39(16)2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31138664

RESUMO

Increased ribosomal biogenesis occurs during tissue hypertrophy, but whether ribosomal biogenesis is impaired during atrophy is not known. We show that hyperammonemia, which occurs in diverse chronic disorders, impairs protein synthesis as a result of decreased ribosomal content and translational capacity. Transcriptome analyses, real-time PCR, and immunoblotting showed consistent reductions in the expression of the large and small ribosomal protein subunits (RPL and RPS, respectively) in hyperammonemic murine skeletal myotubes, HEK cells, and skeletal muscle from hyperammonemic rats and human cirrhotics. Decreased ribosomal content was accompanied by decreased expression of cMYC, a positive regulator of ribosomal biogenesis, as well as reduced expression and activity of ß-catenin, a transcriptional activator of cMYC. However, unlike the canonical regulation of ß-catenin via glycogen synthase kinase 3ß (GSK3ß)-dependent degradation, GSK3ß expression and phosphorylation were unaltered during hyperammonemia, and depletion of GSK3ß did not prevent ammonia-induced degradation of ß-catenin. Overexpression of GSK3ß-resistant variants, genetic depletion of IκB kinase ß (IKKß) (activated during hyperammonemia), protein interactions, and in vitro kinase assays showed that IKKß phosphorylated ß-catenin directly. Overexpressing ß-catenin restored hyperammonemia-induced perturbations in signaling responses that regulate ribosomal biogenesis. Our data show that decreased protein synthesis during hyperammonemia is mediated via a novel GSK3ß-independent, IKKß-dependent impairment of the ß-catenin-cMYC axis.


Assuntos
Hiperamonemia/metabolismo , Subunidades Ribossômicas Menores/genética , Subunidades Ribossômicas Menores/metabolismo , beta Catenina/química , beta Catenina/genética , Animais , Linhagem Celular , Modelos Animais de Doenças , Fibrose , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Células HEK293 , Humanos , Hiperamonemia/genética , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Camundongos , Proteólise , Proteômica , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ratos , Análise de Sequência de RNA , Transdução de Sinais
15.
BMC Evol Biol ; 19(1): 78, 2019 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-30871462

RESUMO

BACKGROUND: Dictyostelid cellular slime molds (dictyostelids) are microscopic throughout their entire life cycle. The vegetative phase consists of single-celled amoeboid forms which live in the soil/leaf litter microhabitat of fields and forests along with animal dung, where they feed upon bacteria and other microbes, grow, and multiply until the available food supply is exhausted. When this happens, the amoeboid forms aggregate together in large numbers to form multi-celled pseudoplasmodia, which then give rise to fruiting bodies (sorocarps) that consist of supportive stalks and unwalled sori containing propagative spores. RESULTS: Dictyostelium purpureum var. pseudosessile, a new variant of dictyostelid, is described herein, based on morphological features and molecular data. This new variant was isolated from soil samples collected in two tropical areas of China. The complete spore-to-spore life cycle of this species, which required 50 h, including spore germination, myxamoebae, cell aggregation, pseudoplasmodium, and sorocarp formation, was documented. Descriptions and illustrations are provided for this species based on our collections. Data from ontogeny, morphology and phylogeny analyses (SSU) of D. purpureum var. pseudosessile confirm that it is a Group 4 species according to the newly proposed classification of dictyostelids. CONCLUSIONS: Our results suggest that the violet sori, widens at the midpoint of sorophore and simple recurved sorophore bases represent the prominent features for the new variant D. purpureum var. pseudosessile. The latter is a Group 4 species now known from two tropical areas of China where dictyostelids remains understudied.


Assuntos
Dictyostelium/classificação , Clima Tropical , Animais , China , Dictyostelium/genética , Dictyostelium/crescimento & desenvolvimento , Estágios do Ciclo de Vida , Filogenia , RNA Ribossômico/genética , Subunidades Ribossômicas Menores/genética
16.
PLoS Genet ; 14(11): e1007818, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30475795

RESUMO

No-go Decay (NGD) is a process that has evolved to deal with stalled ribosomes resulting from structural blocks or aberrant mRNAs. The process is distinguished by an endonucleolytic cleavage prior to degradation of the transcript. While many of the details of the pathway have been described, the identity of the endonuclease remains unknown. Here we identify residues of the small subunit ribosomal protein Rps3 that are important for NGD by affecting the cleavage reaction. Mutation of residues within the ribosomal entry tunnel that contact the incoming mRNA leads to significantly reduced accumulation of cleavage products, independent of the type of stall sequence, and renders cells sensitive to damaging agents thought to trigger NGD. These phenotypes are distinct from those seen in combination with other NGD factors, suggesting a separate role for Rps3 in NGD. Conversely, ribosomal proteins ubiquitination is not affected by rps3 mutations, indicating that upstream ribosome quality control (RQC) events are not dependent on these residues. Together, these results suggest that Rps3 is important for quality control on the ribosome and strongly supports the notion that the ribosome itself plays a central role in the endonucleolytic cleavage reaction during NGD.


Assuntos
Estabilidade de RNA , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Menores/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Endorribonucleases/genética , Endorribonucleases/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Genes Fúngicos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Elongação Traducional da Cadeia Peptídica , Conformação Proteica , RNA Fúngico/genética , RNA Mensageiro/genética , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , Subunidades Ribossômicas Menores/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Ubiquitinação
17.
Plant Physiol ; 177(4): 1539-1554, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29914890

RESUMO

Chloroplast ribosomes, which originated from cyanobacteria, comprise a large subunit (50S) and a small subunit (30S) containing ribosomal RNAs (rRNAs) and various ribosomal proteins. Genes for many chloroplast ribosomal proteins, as well as proteins with auxiliary roles in ribosome biogenesis or functioning, reside in the nucleus. Here, we identified Arabidopsis (Arabidopsis thaliana) CHLOROPLAST RIBOSOME ASSOCIATED (CRASS), a member of the latter class of proteins, based on the tight coexpression of its mRNA with transcripts for nucleus-encoded chloroplast ribosomal proteins. CRASS was acquired during the evolution of embryophytes and is localized to the chloroplast stroma. Loss of CRASS results in minor defects in development, photosynthetic efficiency, and chloroplast translation activity under controlled growth conditions, but these phenotypes are greatly exacerbated under stress conditions induced by the translational inhibitors lincomycin and chloramphenicol or by cold treatment. The CRASS protein comigrates with chloroplast ribosomal particles and coimmunoprecipitates with the 16S rRNA and several chloroplast ribosomal proteins, particularly the plastid ribosomal proteins of the 30S subunit (PRPS1 and PRPS5). The association of CRASS with PRPS1 and PRPS5 is independent of rRNA and is not detectable in yeast two-hybrid experiments, implying that either CRASS interacts indirectly with PRPS1 and PRPS5 via another component of the small ribosomal subunit or that it recognizes structural features of the multiprotein/rRNA particle. CRASS plays a role in the biogenesis and/or stability of the chloroplast ribosome that becomes critical under certain stressful conditions when ribosomal activity is compromised.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Transporte/metabolismo , Cloroplastos/metabolismo , Resposta ao Choque Frio/fisiologia , Biossíntese de Proteínas , Subunidades Ribossômicas Menores/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Transporte/genética , Cloroplastos/genética , Resposta ao Choque Frio/genética , Embriófitas/genética , Regulação da Expressão Gênica de Plantas , Imunoprecipitação , Plantas Geneticamente Modificadas , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , Subunidades Ribossômicas Menores/genética
18.
Mol Cell ; 70(1): 83-94.e7, 2018 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-29625040

RESUMO

Growing resistance of pathogenic bacteria and shortage of antibiotic discovery platforms challenge the use of antibiotics in the clinic. This threat calls for exploration of unconventional sources of antibiotics and identification of inhibitors able to eradicate resistant bacteria. Here we describe a different class of antibiotics, odilorhabdins (ODLs), produced by the enzymes of the non-ribosomal peptide synthetase gene cluster of the nematode-symbiotic bacterium Xenorhabdus nematophila. ODLs show activity against Gram-positive and Gram-negative pathogens, including carbapenem-resistant Enterobacteriaceae, and can eradicate infections in animal models. We demonstrate that the bactericidal ODLs interfere with protein synthesis. Genetic and structural analyses reveal that ODLs bind to the small ribosomal subunit at a site not exploited by current antibiotics. ODLs induce miscoding and promote hungry codon readthrough, amino acid misincorporation, and premature stop codon bypass. We propose that ODLs' miscoding activity reflects their ability to increase the affinity of non-cognate aminoacyl-tRNAs to the ribosome.


Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Proteínas de Bactérias/biossíntese , DNA Bacteriano/genética , Infecções por Klebsiella/tratamento farmacológico , Subunidades Ribossômicas Menores/efeitos dos fármacos , Xenorhabdus/metabolismo , Aminoaciltransferases/genética , Aminoaciltransferases/metabolismo , Animais , Antibacterianos/metabolismo , Bactérias/genética , Bactérias/metabolismo , Proteínas de Bactérias/genética , Sítios de Ligação , Modelos Animais de Doenças , Feminino , Células Hep G2 , Humanos , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Masculino , Camundongos Endogâmicos ICR , Biossíntese de Proteínas/efeitos dos fármacos , Subunidades Ribossômicas Menores/genética , Subunidades Ribossômicas Menores/metabolismo
20.
J Eukaryot Microbiol ; 65(4): 484-504, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29316045

RESUMO

The about 1,000 species of tintinnid ciliates are identified and classified almost exclusively based on their lorica features, although the shortcomings of this structure are well-known, e.g. causing uncertain species limitations and nonmonophyletic taxa. Hence, the present redescription of Tintinnopsis everta Kofoid and Campbell, 1929 considers not only the lorica characteristics, but focuses on cell and genetic features. The species is redescribed from the North Atlantic and adjacent sea areas, namely the east coast of the USA, using live observation, protargol-stained material, scanning electron microscopy, and genetic analyses. The main stages of cell division are described, and the species' phylogenetic relationships are inferred from morphological data and the small subunit ribosomal RNA gene sequence. The estimates of its biogeographical distribution and autecology are based on a literature survey. The species is characterised by a complex somatic ciliary pattern with a unique position of the posterior kinety and a conspicuously large distance between the somatic ciliary fields and the collar membranelles. The phylogenetic relationships of Tintinnopsis everta vary in the molecular trees depending on the algorithms used and are, therefore, regarded as unresolved. Nevertheless, the new kind of complex somatic ciliary pattern distinctly contributes to a better understanding of the tintinnid biodiversity and evolution and provides features for a future split of the nonmonophyletic genus Tintinnopsis.


Assuntos
Alveolados/classificação , Alveolados/isolamento & purificação , Alveolados/genética , Alveolados/crescimento & desenvolvimento , Biodiversidade , DNA de Protozoário/genética , DNA Ribossômico/genética , Filogenia , Subunidades Ribossômicas Menores/genética , Rios/parasitologia , Água do Mar/parasitologia
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