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1.
Biol Open ; 10(9)2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34590698

RESUMO

Most intracellular pathogens replicate in a vacuole to avoid the defense system of the host. A few pathogens recruit host mitochondria around those vacuoles, but the molecules responsible for mitochondrial recruitment remain unidentified. It is only in the apicomplexan parasite Toxoplasma gondii, that mitochondrial association factor 1b (MAF1b) has been identified as an association factor for host mitochondria. Here, we show that rhoptry kinase family protein 39 (ROP39) induces host mitochondrial recruitment in T. gondii. We found that the abundance of ROP39 was increased on host mitochondria extracted from human foreskin fibroblasts (HFFs) infected with T. gondii. ROP39 expressed exogenously in HFFs localized on host mitochondria, indicating that it has the potential to bind to host mitochondria without assistance from other parasite factors. Confocal microscopy revealed that ROP39 colocalized with host mitochondria on the membrane of parasitophorous vacuoles, in which the parasites reside. Moreover, we observed about a 10% reduction in the level of mitochondrial association in rop39-knockout parasites compared with a parental strain.

2.
Nat Commun ; 12(1): 4498, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301931

RESUMO

In animal germlines, PIWI proteins and the associated PIWI-interacting RNAs (piRNAs) protect genome integrity by silencing transposons. Here we report the extensive sequence and quantitative correlations between 2',3'-cyclic phosphate-containing RNAs (cP-RNAs), identified using cP-RNA-seq, and piRNAs in the Bombyx germ cell line and mouse testes. The cP-RNAs containing 5'-phosphate (P-cP-RNAs) identified by P-cP-RNA-seq harbor highly consistent 5'-end positions as the piRNAs and are loaded onto PIWI protein, suggesting their direct utilization as piRNA precursors. We identified Bombyx RNase Kappa (BmRNase κ) as a mitochondria-associated endoribonuclease which produces cP-RNAs during piRNA biogenesis. BmRNase κ-depletion elevated transposon levels and disrupted a piRNA-mediated sex determination in Bombyx embryos, indicating the crucial roles of BmRNase κ in piRNA biogenesis and embryonic development. Our results reveal a BmRNase κ-engaged piRNA biogenesis pathway, in which the generation of cP-RNAs promotes robust piRNA production.


Assuntos
Endorribonucleases/genética , Perfilação da Expressão Gênica/métodos , Proteínas de Insetos/genética , RNA Interferente Pequeno/genética , RNA/genética , Animais , Sequência de Bases , Bombyx , Linhagem Celular , Endorribonucleases/metabolismo , Feminino , Proteínas de Insetos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Mutação , Ácidos Fosfatídicos/química , RNA/química , RNA/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , RNA-Seq/métodos , Testículo/metabolismo
3.
PLoS One ; 15(2): e0227749, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32012177

RESUMO

Toxoplasma gondii is classified into 16 haplogroups based on a worldwide genotyping study of the parasite. However, only a few isolates from Japan were included in this analysis. To conduct more precise genotyping of T. gondii, we examined the genotypes of Japanese isolates in this study. DNA sequences of 6 loci were determined in 17 Japanese isolates and compared with those of strains of 16 haplogroups. As a result, Japanese isolates were classified into four groups. We investigated the virulence of some Japanese isolates and found a highly virulent strain in mice, comparable to that of RH strain, although this Japanese isolate was sister to strains of haplogroup 2, which show moderate virulence in mice. We further investigated whether this high virulence isolate had different virulence mechanism and strategy to adapt to Japanese host from other strains by comparing the virulence-related genes, ROP5, 18 and the immunomodulatory gene, ROP16 of the isolate with those of archetypical strains (GT1, ME49 and VEG). This analysis indicated the high virulence of the isolate in mice was partly explained by gene sequences of ROP5 and ROP16. These findings lead to the elucidation of biodiversity of T. gondii and have potential to optimize the diagnostic protocol.


Assuntos
Variação Genética , Toxoplasma/genética , Toxoplasmose Animal/genética , Toxoplasmose/genética , Alelos , Animais , Genótipo , Humanos , Japão , Camundongos , Filogenia , Proteínas Tirosina Quinases/genética , Proteínas de Protozoários/genética , Toxoplasma/patogenicidade , Toxoplasmose/parasitologia , Toxoplasmose Animal/parasitologia , Virulência/genética
5.
Cell Syst ; 8(4): 302-314.e8, 2019 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-30981730

RESUMO

Gram-negative bacteria are intrinsically resistant to drugs because of their double-membrane envelope structure that acts as a permeability barrier and as an anchor for efflux pumps. Antibiotics are blocked and expelled from cells and cannot reach high-enough intracellular concentrations to exert a therapeutic effect. Efforts to target one membrane protein at a time have been ineffective. Here, we show that m1G37-tRNA methylation determines the synthesis of a multitude of membrane proteins via its control of translation at proline codons near the start of open reading frames. Decreases in m1G37 levels in Escherichia coli and Salmonella impair membrane structure and sensitize these bacteria to multiple classes of antibiotics, rendering them incapable of developing resistance or persistence. Codon engineering of membrane-associated genes reduces their translational dependence on m1G37 and confers resistance. These findings highlight the potential of tRNA methylation in codon-specific translation to control the development of multi-drug resistance in Gram-negative bacteria.


Assuntos
Farmacorresistência Bacteriana , Proteínas de Escherichia coli/genética , Processamento Pós-Transcricional do RNA , RNA de Transferência/metabolismo , tRNA Metiltransferases/genética , Antibacterianos/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Escherichia coli , Proteínas de Escherichia coli/metabolismo , Metilação , RNA de Transferência/genética , Salmonella , Transcriptoma , tRNA Metiltransferases/metabolismo
6.
Nucleic Acids Res ; 46(7): e37, 2018 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-29361055

RESUMO

Active tRNAs are extensively post-transcriptionally modified, particularly at the wobble position 34 and the position 37 on the 3'-side of the anticodon. The 5-carboxy-methoxy modification of U34 (cmo5U34) is present in Gram-negative tRNAs for six amino acids (Ala, Ser, Pro, Thr, Leu and Val), four of which (Ala, Ser, Pro and Thr) have a terminal methyl group to form 5-methoxy-carbonyl-methoxy-uridine (mcmo5U34) for higher reading-frame accuracy. The molecular basis for the selective terminal methylation is not understood. Many cmo5U34-tRNAs are essential for growth and cannot be substituted for mutational analysis. We show here that, with a novel genetic approach, we have created and isolated mutants of Escherichia coli tRNAPro and tRNAVal for analysis of the selective terminal methylation. We show that substitution of G35 in the anticodon of tRNAPro inactivates the terminal methylation, whereas introduction of G35 to tRNAVal confers it, indicating that G35 is a major determinant for the selectivity. We also show that, in tRNAPro, the terminal methylation at U34 is dependent on the primary m1G methylation at position 37 but not vice versa, indicating a hierarchical ranking of modifications between positions 34 and 37. We suggest that this hierarchy provides a mechanism to ensure top performance of a tRNA inside of cells.


Assuntos
Anticódon/genética , Conformação de Ácido Nucleico , RNA de Transferência de Prolina/genética , RNA de Transferência/genética , Sequência de Bases , Códon/genética , Escherichia coli/genética , Metilação , RNA Bacteriano/genética , Uridina/análogos & derivados , Uridina/genética
7.
Parasitol Int ; 67(1): 47-58, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28344153

RESUMO

Cytokinins are plant hormones that are involved in regulation of cell proliferation, cell cycle progression, and cell and plastid development. Here, we show that the apicomplexan parasites Toxoplasma gondii and Plasmodium berghei, an opportunistic human pathogen and a rodent malaria agent, respectively, produce cytokinins via a biosynthetic pathway similar to that in plants. Cytokinins regulate the growth and cell cycle progression of T. gondii by mediating expression of the cyclin gene TgCYC4. A natural form of cytokinin, trans-zeatin (t-zeatin), upregulated expression of this cyclin, while a synthetic cytokinin, thidiazuron, downregulated its expression. Immunofluorescence microscopy and quantitative PCR analysis showed that t-zeatin increased the genome-copy number of apicoplast, which are non-photosynthetic plastid, in the parasite, while thidiazuron led to their disappearance. Thidiazuron inhibited growth of T. gondii and Plasmodium falciparum, a human malaria parasite, suggesting that thidiazuron has therapeutic potential as an inhibitor of apicomplexan parasites.


Assuntos
Ciclo Celular/efeitos dos fármacos , Citocininas/farmacologia , Plasmodium berghei/enzimologia , Plasmodium berghei/fisiologia , Toxoplasma/efeitos dos fármacos , Toxoplasma/fisiologia , Citocininas/metabolismo , Compostos de Fenilureia/farmacologia , Reguladores de Crescimento de Plantas/metabolismo , Reguladores de Crescimento de Plantas/farmacologia , Plasmodium berghei/crescimento & desenvolvimento , Tiadiazóis/farmacologia , Toxoplasma/crescimento & desenvolvimento
8.
Enzymes ; 41: 89-115, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28601227

RESUMO

TrmD is an S-adenosyl methionine (AdoMet)-dependent methyl transferase that synthesizes the methylated m1G37 in tRNA. TrmD is specific to and essential for bacterial growth, and it is fundamentally distinct from its eukaryotic and archaeal counterpart Trm5. TrmD is unusual by using a topological protein knot to bind AdoMet. Despite its restricted mobility, the TrmD knot has complex dynamics necessary to transmit the signal of AdoMet binding to promote tRNA binding and methyl transfer. Mutations in the TrmD knot block this intramolecular signaling and decrease the synthesis of m1G37-tRNA, prompting ribosomes to +1-frameshifts and premature termination of protein synthesis. TrmD is unique among AdoMet-dependent methyl transferases in that it requires Mg2+ in the catalytic mechanism. This Mg2+ dependence is important for regulating Mg2+ transport to Salmonella for survival of the pathogen in the host cell. The strict conservation of TrmD among bacterial species suggests that a better characterization of its enzymology and biology will have a broad impact on our understanding of bacterial pathogenesis.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Metilação , Processamento Pós-Transcricional do RNA , RNA de Transferência/química , RNA de Transferência/metabolismo , tRNA Metiltransferases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Especificidade por Substrato
9.
Parasitol Int ; 66(5): 588-592, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28495225

RESUMO

The domestic pigeon, Columba livia domestica, is reared for meat production, as a pet, or for racing. Few reports have characterized the parasitic protists from the genus Isospora isolated from Columbiformes. We detected Isospora-like oocysts from C. livia reared for racing. The oocyst contained two sporocysts, and each sporocyst included four sporozoites. The sporulated oocysts (n=4) were spherical; their mean diameters were 25.6 (24.0-27.2)×24.7 (23.4-26.0) µm. Micropyles, polar granules, and oocyst residuum were absent. The mean length and width of the sporocysts (n=8) were 19.5 (18.5-20.5) and 11.2 (10.2-12.1) µm, respectively. Stieda and sub-Stieda bodies were observed. Single-oocyst PCR revealed two different 18S rRNA gene sequences and one 28S rRNA gene sequence in a single oocyst of Isospora sp. Based on a phylogenetic analysis of the 18S rRNA gene, the two sequences made a group which fell within a cluster of known avian Isospora species. A tree based on the 28S rRNA gene sequence indicated that sequences from the pigeon Isospora sp. fell within a cluster of avian Isospora species. Both trees failed to clarify the phylogenetic relationships among the avian Isospora species due to limited resolution. Because the morphological description of Isospora sp. is based on only four oocysts, Isospora sp. is not proposed as a novel species here. This is the first description of Isospora sp. isolated from the domestic pigeon C. livia.


Assuntos
Doenças das Aves/parasitologia , Columbidae/parasitologia , Isospora/genética , Isosporíase/veterinária , Animais , DNA de Protozoário/genética , Fezes/parasitologia , Isospora/citologia , Isospora/isolamento & purificação , Isosporíase/parasitologia , Oocistos/citologia , Oocistos/genética , Oocistos/isolamento & purificação , Filogenia , Reação em Cadeia da Polimerase , RNA Ribossômico 18S/genética , RNA Ribossômico 28S/genética , Esporozoítos/citologia , Esporozoítos/genética , Esporozoítos/isolamento & purificação
10.
Parasitol Int ; 65(5 Pt A): 378-88, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27217289

RESUMO

Host cell microdomains are involved in the attachment, entry, and replication of intracellular microbial pathogens. Entry into the host cell of Toxoplasma gondii and the subsequent survival of this protozoan parasite are tightly coupled with the proteins secreted from organelle called rhoptry. The rhoptry proteins are rapidly discharged into clusters of vesicles, called evacuoles, which are then delivered to parasitophorous vacuoles (PVs) or nucleus. In this study, we examined the roles of two host cell microdomain components, cholesterol and glycosylphosphatidylinositol (GPI), in evacuole formation. The acute depletion of cholesterol from the host cell plasma membrane blocked evacuole formation but not invasion. Whereas the lack of host cell GPI also altered evacuole formation but not invasion, instead inducing excess evacuole formation. The latter effect was not influenced by the evacuole-inhibiting effects of host cell cholesterol depletion, indicating the independent roles of host GPI and cholesterol in evacuole formation. In addition, the excess formation of evacuoles resulted in the enhanced recruitment of host mitochondria and endoplasmic reticulum to PVs, which in turn stimulated the growth of the parasite.


Assuntos
Colesterol/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Interações Hospedeiro-Parasita , Microdomínios da Membrana/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/crescimento & desenvolvimento , Toxoplasmose/patologia , Animais , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Toxoplasma/metabolismo , Vacúolos/metabolismo
11.
Parasitol Int ; 65(4): 319-22, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27021922

RESUMO

Neospora caninum causes abortion and stillbirth in cattle. Identification of effective drugs against this parasite remains a challenge. Previous studies have suggested that disruption of abscisic acid (ABA)-mediated signaling in apicomplexan parasites such as Toxoplasma gondii offers a new drug target. In this study, the ABA inhibitor, fluridone (FLU), was evaluated for its action against N. caninum. Production of endogenous ABA within N. caninum was confirmed by ultra-performance liquid chromatography-tandem quadruple mass spectrometry. Subsequently, FLU treatment efficacy was assessed using in vitro. Results revealed that FLU inhibited the growth of N. caninum and T. gondii in vitro (IC50 143.1±43.96µM and 330.6±52.38µM, respectively). However, FLU did not affect parasite replication at 24h post-infection, but inhibited egress of N. caninum thereafter. To evaluate the effect of FLU in vivo, N. caninum-infected mice were treated with FLU for 15days. FLU treatment appeared to ameliorate acute neosporosis induced by lethal parasite challenge. Together, our data shows that ABA might control egress in N. caninum. Therefore, FLU has potential as a candidate drug for the treatment of acute neosporosis.


Assuntos
Ácido Abscísico/antagonistas & inibidores , Doenças dos Bovinos/tratamento farmacológico , Coccidiose/tratamento farmacológico , Neospora/efeitos dos fármacos , Reguladores de Crescimento de Plantas/antagonistas & inibidores , Complicações na Gravidez/veterinária , Piridonas/farmacologia , Animais , Bovinos , Doenças dos Bovinos/parasitologia , Coccidiose/parasitologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Gravidez , Organismos Livres de Patógenos Específicos , Natimorto/veterinária
12.
PLoS One ; 10(10): e0140559, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26466097

RESUMO

The apicomplexan parasite Toxoplasma gondii produces the plant hormone abscisic acid, but it is unclear if phytohormones are produced by the malaria parasite Plasmodium spp., the most important parasite of this phylum. Here, we report detection of salicylic acid, an immune-related phytohormone of land plants, in P. berghei ANKA and T. gondii cell lysates. However, addition of salicylic acid to P. falciparum and T. gondii culture had no effect. We transfected P. falciparum 3D7 with the nahG gene, which encodes a salicylic acid-degrading enzyme isolated from plant-infecting Pseudomonas sp., and established a salicylic acid-deficient mutant. The mutant had a significantly decreased concentration of parasite-synthesized prostaglandin E2, which potentially modulates host immunity as an adaptive evolution of Plasmodium spp. To investigate the function of salicylic acid and prostaglandin E2 on host immunity, we established P. berghei ANKA mutants expressing nahG. C57BL/6 mice infected with nahG transfectants developed enhanced cerebral malaria, as assessed by Evans blue leakage and brain histological observation. The nahG-transfectant also significantly increased the mortality rate of mice. Prostaglandin E2 reduced the brain symptoms by induction of T helper-2 cytokines. As expected, T helper-1 cytokines including interferon-γ and interleukin-2 were significantly elevated by infection with the nahG transfectant. Thus, salicylic acid of Plasmodium spp. may be a new pathogenic factor of this threatening parasite and may modulate immune function via parasite-produced prostaglandin E2.


Assuntos
Imunidade/efeitos dos fármacos , Malária Cerebral/imunologia , Malária Cerebral/parasitologia , Reguladores de Crescimento de Plantas/farmacologia , Plasmodium berghei/metabolismo , Ácido Salicílico/farmacologia , Animais , Animais Geneticamente Modificados , Citocinas/sangue , Citocinas/metabolismo , Feminino , Humanos , Malária Cerebral/metabolismo , Malária Cerebral/mortalidade , Camundongos , Plasmodium falciparum/genética , Plasmodium falciparum/imunologia , Prostaglandinas/sangue , Prostaglandinas/metabolismo
13.
Vet Parasitol ; 196(1-2): 184-8, 2013 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-23601844

RESUMO

The genus Cryptosporidium, which is an obligate intracellular parasite, infects various vertebrates and causes a diarrheal disease known as cryptosporidiosis. Wild rodents are naturally infected with zoonotic Cryptosporidium; thus, they are potential reservoirs of the parasites. Mice are common rodents frequently found in agricultural areas and have many opportunities to contact other wild animals, livestock, and humans. Irrespective of the potential risk, there are few epidemiologic studies of Cryptosporidium in wild mice because of their low economic importance and the difficulty in conducting surveys. Hence, the species and genotypes of Cryptosporidium in wild mice living around various areas remain unclear. We investigated the species and genotype distribution and prevalence of Cryptosporidium in the large Japanese field mouse (Apodemus speciosus) in an agricultural site in Osaki, Miyagi Prefecture, Japan. In total, 15 mice were captured and examined in this study. By microscopic analysis, only one mouse (JFM 3) was determined to be Cryptosporidium-positive, while the parasite were detected in four mice (JFM 3, 6, 10, and 15) by a molecular approach using partial SSU rRNA gene sequences. Based on nucleotide sequence and phylogenetic analysis, the Cryptosporidium isolates were identified as C. ubiquitum (from JFM 10) and C. muris (from JFM 3 and 6). In contrast, the Cryptosporidium in JFM 15 was not identified as a known species or genotype and is therefore proposed as a novel genotype; the Naruko genotype. More molecular data are necessary to elucidate the taxonomic identity of this novel Cryptosporidium genotype. The C. muris Japanese field mouse genotypes showed marked divergence compared to that in a previous report. The large Japanese field mouse might thus represent a reservoir of multiple Cryptosporidium spp.


Assuntos
Criptosporidiose/veterinária , Cryptosporidium/genética , Cryptosporidium/isolamento & purificação , Murinae , Animais , Criptosporidiose/epidemiologia , Criptosporidiose/parasitologia , Genótipo , Filogenia
14.
Vet Parasitol ; 187(1-2): 123-8, 2012 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-22265080

RESUMO

Cattle are major hosts of Cryptosporidium spp. Cryptosporidiosis in neonatal calves is associated with retarded growth, weight loss and calf mortality, and zoonotic infections in humans. In many areas, cow-calf glazing system is an important beef cattle rearing method with distinct advantages in terms of cost and the labor required. However, few epidemiologic studies of Cryptosporidium spp. have been conducted in this system, especially using molecular diagnostic tools. To understand the transmission of Cryptosporidium spp. in a grazing system, we followed cryptosporidiosis on a grazing farm in Osaki City, Miyagi Prefecture, in northwest Japan for one year. Fecal samples were collected from Japanese Black and Japanese Shorthorn cattle and examined by PCR-RFLP and sequence analyses. Of 113 fecal samples collected in October 2010, 23 (20%) were positive for Cryptosporidium, including 15 samples (13%) having C. bovis, 6 (5%) having C. ryanae, and 2 (2%) having mixed infections of both species. Additionally, C. bovis or C. ryanae was detected on all other sampling dates involving smaller numbers of animals. The infection rate of C. bovis was significantly different among age groups, and calve-to-calve infection might be the major route of cryptosporidiosis transmission in beef cattle. Interestingly, one animal had C. bovis infection or re-infection for one year. Our results suggest that C. bovis and C. ryanae are distributed in Japan, but might have low level of detection in grazing beef cattle.


Assuntos
Doenças dos Bovinos/parasitologia , Criptosporidiose/veterinária , Cryptosporidium/classificação , Cryptosporidium/genética , Envelhecimento , Criação de Animais Domésticos , Animais , Sequência de Bases , Bovinos , Doenças dos Bovinos/epidemiologia , Criptosporidiose/epidemiologia , Criptosporidiose/parasitologia , DNA de Protozoário/genética , Feminino , Japão/epidemiologia , Polimorfismo Genético
15.
J Vet Med Sci ; 72(12): 1647-9, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20689224

RESUMO

Cryptosporidium parvum HNJ-1 is widely used as a reference strain in Japan. In the present study, the parasite was subjected for further molecular analysis including transcribed ribosomal region (ITS rRNA), dihydrofolate reductase (DHFR) and surface glycoprotein (GP60) genes. Partial sequence analysis of these genes indicated extensive polymorphism in ITS region compared with relevant sequences of other Cryptosporidium parvum isolates. In addition, this strain was identified as C. parvum IIaA15G2R1 subtype, based on the sequence results of GP60 gene locus.


Assuntos
Cryptosporidium parvum/classificação , Cryptosporidium parvum/isolamento & purificação , Animais , Criptosporidiose/epidemiologia , Cryptosporidium parvum/genética , DNA de Protozoário/genética , DNA Espaçador Ribossômico/genética , Regulação da Expressão Gênica , Glicoproteínas/genética , Humanos , Japão/epidemiologia , Camundongos , Filogenia , Polimorfismo Genético , RNA de Protozoário/genética , Tetra-Hidrofolato Desidrogenase/genética
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