RESUMEN
Sporulation in Dictyostelium fruiting bodies evolved from amoebozoan encystation with both being induced by cAMP acting on PKA, but with downstream components still being unknown. Using tagged mutagenesis to find missing pathway components, we identified a sporeless mutant defective in a nuclear protein, SpaA. Expression of prespore genes was strongly reduced in spaA- cells, while expression of many spore stage genes was absent. Chromatin immunoprecipitation (ChIP) of a SpaA-YFP gene fusion showed that (pre)spore gene promoters bind directly to SpaA, identifying SpaA as a transcriptional regulator. SpaA dependent spore gene expression required PKA in vivo and was stimulated in vitro by the membrane-permeant PKA agonist 8Br-cAMP. The PKA agonist also promoted SpaA binding to (pre)spore promoters, placing SpaA downstream of PKA. Sequencing of SpaA-YFP ChIPed DNA fragments revealed that SpaA binds at least 117 (pre)spore promoters, including those of other transcription factors that activate some spore genes. These factors are not in turn required for spaA expression, identifying SpaA as the major trancriptional inducer of sporulation.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Dictyostelium/crecimiento & desarrollo , Esporas Protozoarias/crecimiento & desarrollo , Factores de Transcripción/metabolismo , Inmunoprecipitación de Cromatina , Análisis Mutacional de ADN , ADN Protozoario/metabolismo , Dictyostelium/enzimología , Dictyostelium/genética , Dictyostelium/metabolismo , Eliminación de Gen , Perfilación de la Expresión Génica , Regiones Promotoras Genéticas , Unión Proteica , Esporas Protozoarias/enzimología , Esporas Protozoarias/genética , Esporas Protozoarias/metabolismo , Factores de Transcripción/genéticaRESUMEN
OBJECTIVE: To determine whether Tai Chi Chuan or ballroom dancing promotes better performance with respect to postural balance, gait, and postural transfer among elderly people. METHODS: We evaluated 76 elderly individuals who were divided into two groups: the Tai Chi Chuan Group and the Dance Group. The subjects were tested using the NeuroCom Balance Master¯ force platform system with the following protocols: static balance tests (the Modified Clinical Tests of Sensory Interaction on Balance and Unilateral Stance) and dynamic balance tests (the Walk Across Test and Sit-to-stand Transfer Test). RESULTS: In the Modified Clinical Test of Sensory Interaction on Balance, the Tai Chi Chuan Group presented a lower sway velocity on a firm surface with open and closed eyes, as well as on a foam surface with closed eyes. In the Modified Clinical Test of Sensory Interaction on Unilateral Stance, the Tai Chi Chuan Group presented a lower sway velocity with open eyes, whereas the Dance Group presented a lower sway velocity with closed eyes. In the Walk Across Test, the Tai Chi Chuan Group presented faster walking speeds than those of the Dance Group. In the Sit-to-stand Transfer Test, the Tai Chi Chuan Group presented shorter transfer times from the sitting to the standing position, with less sway in the final standing position. CONCLUSION: The elderly individuals who practiced Tai Chi Chuan had better bilateral balance with eyes open on both types of surfaces compared with the Dance Group. The Dance Group had better unilateral postural balance with eyes closed. The Tai Chi Chuan Group had faster walking speeds, shorter transfer times, and better postural balance in the final standing position during the Sit-to-stand Test. .
Asunto(s)
/metabolismo , AMP Cíclico/metabolismo , Dictyostelium/enzimología , Dictyostelium/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Proteínas Protozoarias/metabolismo , /genética , Dictyostelium/crecimiento & desarrollo , Dictyostelium/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Ácido Fólico/farmacología , /deficiencia , /genética , /metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/deficiencia , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Mutación , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteínas Protozoarias/genética , Transducción de Señal , Esporas Protozoarias/enzimología , Esporas Protozoarias/genética , Complejo Vitamínico B/farmacologíaRESUMEN
A new species of Didymium (Myxomycetes), D. xerophilum, is described, and some details of its life cycle are provided. The new species was collected during studies of arid areas of Argentina and Peru. It can be distinguished by the persistent funnel-shaped invagination of the peridium, the top of which appears as a deep umbilicus in closed sporothecae, and the calcareous hypothallus shared among several sporocarps. This combination of characters, with a circumscissile dehiscence of the sporotheca and a cream stalk packed with rhombic lime crystals, is unknown in other described species. Morphology was examined with scanning electron microscopy and light microscopy, and micrographs of relevant details are included here. Phylogenetic analysis with 18S rDNA sequences of different species of Didymium supports the distinct identity of this new species. Some collections of this myxomycete were made at up to 4600 m, an altitude almost unknown for this group of microorganisms.
Asunto(s)
Mixomicetos/clasificación , Mixomicetos/aislamiento & purificación , Altitud , Argentina , Datos de Secuencia Molecular , Mixomicetos/genética , Mixomicetos/crecimiento & desarrollo , Perú , Filogenia , Esporas Protozoarias/clasificación , Esporas Protozoarias/enzimología , Esporas Protozoarias/genética , Esporas Protozoarias/aislamiento & purificaciónRESUMEN
Cyclic AMP (cAMP) is an important intracellular signaling molecule for many G protein-mediated signaling pathways but the specificity of cAMP signaling in cells with multiple signaling pathways is not well-understood. In Dictyostelium, at least two different G protein signaling pathways, mediated by the Gα2 and Gα4 subunits, are involved with cAMP accumulation, spore production, and chemotaxis and the stimulation of these pathways results in the activation of ERK2, a mitogen-activated protein kinase that can down regulate the cAMP-specific phosphodiesterase RegA. The regA gene was disrupted in gα2(−) and gα4(−) cells to determine if the absence of this phosphodiesterase rescues the development of these G protein mutants as it does for erk2(−) mutants. There gA(−) mutation had no major effects on developmental morphology but enriched the distribution of the Gα mutant cells to the prespore/prestalk border in chimeric aggregates. The loss of RegA function had no effect on Gα4- mediated folate chemotaxis. However, the regA gene disruption in gα4(−) cells, but not in gα2(−) cells, resulted in a substantial rescue and acceleration of spore production. This rescue in sporulation required cell autonomous signaling because the precocious sporulation could not be induced through intercellular signaling in chimeric aggregates. However, intercellular signals from regA(−) strains increased the expression of the prestalk gene ecmB and accelerated the vacuolization of stalk cells. Intercellular signaling from the gα4(−)regA(−) strain did not induce ecmA gene expression indicating cell-type specificity in the promotion of prestalk cell development. regA gene disruption in a Gα4(HC) (Gα4 overexpression) strain did not result in precocious sporulation or stalk cell development indicating that elevated Gα4 subunit expression can mask regA(−) associated phenotypes even when provided with wild-type intercellular signaling. These findings indicate that the Gα2 and Gα4-mediated pathways provide different contributions to the development of spores and stalk cells and that the absence of RegA function can bypass some but not all defects in G protein regulated spore development.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , AMP Cíclico/metabolismo , Dictyostelium/enzimología , Dictyostelium/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Proteínas Protozoarias/metabolismo , 3',5'-AMP Cíclico Fosfodiesterasas/genética , Dictyostelium/crecimiento & desarrollo , Dictyostelium/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Ácido Fólico/farmacología , Subunidad alfa de la Proteína de Unión al GTP Gi2/deficiencia , Subunidad alfa de la Proteína de Unión al GTP Gi2/genética , Subunidad alfa de la Proteína de Unión al GTP Gi2/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/deficiencia , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Mutación , Proteínas Protozoarias/genética , Transducción de Señal , Esporas Protozoarias/enzimología , Esporas Protozoarias/genética , Complejo Vitamínico B/farmacologíaRESUMEN
Amoebas survive environmental stress by differentiating into encapsulated cysts. As cysts, pathogenic amoebas resist antibiotics, which particularly counteracts treatment of vision-destroying Acanthamoeba keratitis. Limited genetic tractability of amoeba pathogens has left their encystation mechanisms unexplored. The social amoeba Dictyostelium discoideum forms spores in multicellular fruiting bodies to survive starvation, while other dictyostelids, such as Polysphondylium pallidum can additionally encyst as single cells. Sporulation is induced by cAMP acting on PKA, with the cAMP phosphodiesterase RegA critically regulating cAMP levels. We show here that RegA is deeply conserved in social and pathogenic amoebas and that deletion of the RegA gene in P. pallidum causes precocious encystation and prevents cyst germination. We heterologously expressed and characterized Acanthamoeba RegA and performed a compound screen to identify RegA inhibitors. Two effective inhibitors increased cAMP levels and triggered Acanthamoeba encystation. Our results show that RegA critically regulates Amoebozoan encystation and that components of the cAMP signalling pathway could be effective targets for therapeutic intervention with encystation.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Amoeba/enzimología , AMP Cíclico/metabolismo , Proteínas Protozoarias/metabolismo , 3',5'-AMP Cíclico Fosfodiesterasas/genética , Acanthamoeba/enzimología , Acanthamoeba/fisiología , Amoeba/fisiología , Secuencia de Bases , Dictyostelium/enzimología , Dictyostelium/fisiología , Datos de Secuencia Molecular , Proteínas Protozoarias/clasificación , Proteínas Protozoarias/genética , Esporas Protozoarias/enzimología , Esporas Protozoarias/metabolismoRESUMEN
In Dictyostelium discoideum, AprA and CfaD are secreted proteins that inhibit cell proliferation. We found that the proliferation of cells lacking CnrN, a phosphatase and tensin homolog (PTEN)-like phosphatase, is not inhibited by exogenous AprA and is increased by exogenous CfaD. The expression of CnrN in cnrN cells partially rescues these altered sensitivities, suggesting that CnrN is necessary for the ability of AprA and CfaD to inhibit proliferation. Cells lacking CnrN accumulate normal levels of AprA and CfaD. Like cells lacking AprA and CfaD, cnrN cells proliferate faster and reach a higher maximum cell density than wild type cells, tend to be multinucleate, accumulate normal levels of mass and protein per nucleus, and form less viable spores. When cnrN cells expressing myc-tagged CnrN are stimulated with a mixture of rAprA and rCfaD, levels of membrane-associated myc-CnrN increase. AprA also causes chemorepulsion of Dictyostelium cells, and CnrN is required for this process. Combined, these results suggest that CnrN functions in a signal transduction pathway downstream of AprA and CfaD mediating some, but not all, of the effects of AprA and CfaD.
Asunto(s)
Núcleo Celular/genética , Quimiotaxis/genética , Dictyostelium/genética , Fosfohidrolasa PTEN/genética , Proteínas Protozoarias/genética , Esporas Protozoarias/genética , Recuento de Células , Núcleo Celular/efectos de los fármacos , Núcleo Celular/enzimología , Proliferación Celular/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Dictyostelium/efectos de los fármacos , Dictyostelium/enzimología , Regulación de la Expresión Génica , Fosfohidrolasa PTEN/deficiencia , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Esporas Protozoarias/efectos de los fármacos , Esporas Protozoarias/enzimologíaRESUMEN
Signaling pathways controlled by reversible protein phosphorylation (catalyzed by kinases and phosphatases) in the malaria parasite Plasmodium are of great interest, for both increased understanding of parasite biology and identification of novel drug targets. Here, we report a functional analysis in Plasmodium of an ancient bacterial Shewanella-like protein phosphatase (SHLP1) found only in bacteria, fungi, protists, and plants. SHLP1 is abundant in asexual blood stages and expressed at all stages of the parasite life cycle. shlp1 deletion results in a reduction in ookinete (zygote) development, microneme formation, and complete ablation of oocyst formation, thereby blocking parasite transmission. This defect is carried by the female gamete and can be rescued by direct injection of mutant ookinetes into the mosquito hemocoel, where oocysts develop. This study emphasizes the varied functions of SHLP1 in Plasmodium ookinete biology and suggests that it could be a novel drug target for blocking parasite transmission.
Asunto(s)
Fosfoproteínas Fosfatasas/metabolismo , Plasmodium berghei/enzimología , Proteínas Protozoarias/metabolismo , Secuencia de Aminoácidos , Eliminación de Gen , Células Germinativas/enzimología , Datos de Secuencia Molecular , Fosfoproteínas Fosfatasas/genética , Plasmodium berghei/crecimiento & desarrollo , Plasmodium berghei/patogenicidad , Proteínas Protozoarias/genética , Esporas Protozoarias/enzimología , Esporas Protozoarias/crecimiento & desarrollo , Virulencia/genética , Cigoto/enzimologíaRESUMEN
Ribonucleotide reductases (RNRs) catalyze the only pathway for de novo synthesis of deoxyribonucleotides needed for DNA replication and repair. The vast majority of eukaryotes encodes only a class I RNR, but interestingly some eukaryotes, including the social amoeba Dictyostelium discoideum, encode both a class I and a class II RNR. The amino acid sequence of the D. discoideum class I RNR is similar to other eukaryotic RNRs, whereas that of its class II RNR is most similar to the monomeric class II RNRs found in Lactobacillus spp. and a few other bacteria. Here we report the first study of RNRs in a eukaryotic organism that encodes class I and class II RNRs. Both classes of RNR genes were expressed in D. discoideum cells, although the class I transcripts were more abundant and strongly enriched during mid-development compared with the class II transcript. The quaternary structure, allosteric regulation, and properties of the diiron-oxo/radical cofactor of D. discoideum class I RNR are similar to those of the mammalian RNRs. Inhibition of D. discoideum class I RNR by hydroxyurea resulted in a 90% reduction in spore formation and decreased the germination viability of the surviving spores by 75%. Class II RNR could not compensate for class I inhibition during development, and an excess of vitamin B12 coenzyme, which is essential for class II activity, did not improve spore formation. We suggest that class I is the principal RNR during D. discoideum development and growth and is important for spore formation, possibly by providing dNTPs for mitochondrial replication.
Asunto(s)
Dictyostelium/enzimología , Proteínas Protozoarias/metabolismo , Ribonucleótido Reductasas/metabolismo , Regulación Alostérica , Complejos de Coordinación/química , Citidina Difosfato/química , Dictyostelium/genética , Dictyostelium/fisiología , Inhibidores Enzimáticos/farmacología , Radicales Libres/química , Expresión Génica , Regulación Enzimológica de la Expresión Génica , Guanosina Difosfato/química , Hierro/química , Cinética , Filogenia , Proteínas Protozoarias/genética , Ribonucleótido Reductasas/antagonistas & inhibidores , Ribonucleótido Reductasas/química , Ribonucleótido Reductasas/genética , Espectrofotometría Ultravioleta , Esporas Protozoarias/enzimología , Esporas Protozoarias/genética , Tirosina/químicaRESUMEN
A key challenge in the successful treatment of Acanthamoeba infections is its ability to transform into a dormant cyst form that is resistant to physiological conditions and pharmacological therapies, resulting in recurrent infections. The carbohydrate linkage analysis of cyst walls of Acanthamoeba castellanii showed variously linked sugar residues, including xylofuranose/xylopyranose, glucopyranose, mannopyranose, and galactopyranose. Here, it is shown that exogenous xylose significantly reduced A. castellanii differentiation in encystation assays (P < 0.05 using paired t test, one-tailed distribution). Using small interfering RNA (siRNA) probes against xylose isomerase and cellulose synthase, as well as specific inhibitors, the findings revealed that xylose isomerase and cellulose synthase activities are crucial in the differentiation of A. castellanii. Inhibition of both enzymes using siRNA against xylose isomerase and cellulose synthase but not scrambled siRNA attenuated A. castellanii metamorphosis, as demonstrated by the arrest of encystation of A. castellanii. Neither inhibitor nor siRNA probes had any effect on the viability and extracellular proteolytic activities of A. castellanii.
Asunto(s)
Acanthamoeba castellanii/enzimología , Acanthamoeba castellanii/crecimiento & desarrollo , Isomerasas Aldosa-Cetosa/antagonistas & inhibidores , Glucosiltransferasas/antagonistas & inhibidores , ARN Interferente Pequeño/metabolismo , Esporas Protozoarias/enzimología , Esporas Protozoarias/crecimiento & desarrollo , Isomerasas Aldosa-Cetosa/genética , Silenciador del Gen , Glucosiltransferasas/genética , ARN Interferente Pequeño/genéticaRESUMEN
BACKGROUND: Regulation of surface antigenic variation in Giardia lamblia is controlled post-transcriptionally by an RNA-interference (RNAi) pathway that includes a Dicer-like bidentate RNase III (gDicer). This enzyme, however, lacks the RNA helicase domain present in Dicer enzymes from higher eukaryotes. The participation of several RNA helicases in practically all organisms in which RNAi was studied suggests that RNA helicases are potentially involved in antigenic variation, as well as during Giardia differentiation into cysts. RESULTS: An extensive in silico analysis of the Giardia genome identified 32 putative Super Family 2 RNA helicases that contain almost all the conserved RNA helicase motifs. Phylogenetic studies and sequence analysis separated them into 22 DEAD-box, 6 DEAH-box and 4 Ski2p-box RNA helicases, some of which are homologs of well-characterized helicases from higher organisms. No Giardia putative helicase was found to have significant homology to the RNA helicase domain of Dicer enzymes. Additionally a series of up- and down-regulated putative RNA helicases were found during encystation and antigenic variation by qPCR experiments. Finally, we were able to recognize 14 additional putative helicases from three different families (RecQ family, Swi2/Snf2 and Rad3 family) that could be considered DNA helicases. CONCLUSIONS: This is the first comprehensive analysis of the Super Family 2 helicases from the human intestinal parasite G. lamblia. The relative and variable expression of particular RNA helicases during both antigenic variation and encystation agrees with the proposed participation of these enzymes during both adaptive processes. The putatives RNA and DNA helicases identified in this early-branching eukaryote provide initial information regarding the biological role of these enzymes in cell adaptation and differentiation.
Asunto(s)
Variación Antigénica , ADN Helicasas/metabolismo , Giardia lamblia/enzimología , Giardia lamblia/crecimiento & desarrollo , ARN Helicasas/metabolismo , Esporas Protozoarias/crecimiento & desarrollo , Biología Computacional , ADN Helicasas/genética , Genoma de Protozoos/genética , Giardia lamblia/genética , ARN Helicasas/genética , Esporas Protozoarias/enzimologíaRESUMEN
BACKGROUND: Oxygen sensing is a near universal signaling modality that, in eukaryotes ranging from protists such as Dictyostelium and Toxoplasma to humans, involves a cytoplasmic prolyl 4-hydroxylase that utilizes oxygen and α-ketoglutarate as potentially rate-limiting substrates. A divergence between the animal and protist mechanisms is the enzymatic target: the animal transcriptional factor subunit hypoxia inducible factor-α whose hydroxylation results in its poly-ubiquitination and proteasomal degradation, and the protist E3SCF ubiquitin ligase subunit Skp1 whose hydroxylation might control the stability of other proteins. In Dictyostelium, genetic studies show that hydroxylation of Skp1 by PhyA, and subsequent glycosylation of the hydroxyproline, is required for normal oxygen sensing during multicellular development at an air/water interface. Because it has been difficult to detect an effect of hypoxia on Skp1 hydroxylation itself, the role of Skp1 modification was investigated in a submerged model of Dictyostelium development dependent on atmospheric hyperoxia. RESULTS: In static isotropic conditions beneath 70-100% atmospheric oxygen, amoebae formed radially symmetrical cyst-like aggregates consisting of a core of spores and undifferentiated cells surrounded by a cortex of stalk cells. Analysis of mutants showed that cyst formation was inhibited by high Skp1 levels via a hydroxylation-dependent mechanism, and spore differentiation required core glycosylation of Skp1 by a mechanism that could be bypassed by excess Skp1. Failure of spores to differentiate at lower oxygen correlated qualitatively with reduced Skp1 hydroxylation. CONCLUSION: We propose that, in the physiological range, oxygen or downstream metabolic effectors control the timing of developmental progression via activation of newly synthesized Skp1.
Asunto(s)
Dictyostelium/crecimiento & desarrollo , Oxígeno/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Protozoarias/metabolismo , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Dictyostelium/enzimología , Dictyostelium/fisiología , Glicosilación , Hidroxilación , Oxígeno/fisiología , Procolágeno-Prolina Dioxigenasa/metabolismo , Esporas Protozoarias/citología , Esporas Protozoarias/enzimología , Esporas Protozoarias/fisiologíaRESUMEN
Genes for mannitol-metabolizing enzymes, mannitol phosphate dehydrogenase (MPDH) and mannitol dehydrogenase (MDH), have been recently identified in the genome of Acanthamoeba castellanii and their potential role in stress tolerance was proposed. Using qRT-PCR, comparison has been made of mRNA levels of the enzymes for mannitol metabolism at various time intervals during the stress defence reactions of encystation and pseudocyst formation. Gradual decrease of both enzymes during encystation and slight increases at the beginning of pseudocyst formation were observed. Detailed analysis of mRNA sequences of the two genes revealed similarities with various alcohol dehydrogenases rather than mannitol dehydrogenases. Our results indicate there is probably no protective role for mannitol in Acanthamoeba as no mannitol was detected using HILIC ESI MS, in any Acanthamoeba life cycle stage. Possible misinterpretation of previously published sequences as encoding enzymes of the mannitol metabolic pathway is discussed.
Asunto(s)
Acanthamoeba castellanii/enzimología , Manitol Deshidrogenasas/metabolismo , Manitol/metabolismo , Proteínas Protozoarias/metabolismo , Acanthamoeba castellanii/metabolismo , Acanthamoeba castellanii/fisiología , Secuencia de Aminoácidos , Metabolismo de los Hidratos de Carbono , Secuencia Conservada , Regulación Enzimológica de la Expresión Génica , Manitol Deshidrogenasas/genética , Anotación de Secuencia Molecular , Datos de Secuencia Molecular , Proteínas Protozoarias/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Esporas Protozoarias/enzimología , Estrés Fisiológico , Transcripción GenéticaRESUMEN
Giardia intestinalis is considered an early-branching eukaryote and is therefore a valuable model for studying primordial cellular processes. This work reports the characterization of the ubiquitin-activating enzyme (E1) during growth and different stages of trophozoite differentiation into cysts. We found that in Giardia E1 expression (both at mRNA and protein levels) is regulated during encystation. The enzyme is proteolytically processed mainly into two fragments of 68kDa (N-terminal) and 47kDa (C-terminal). This phenomenon has not been described for any other E1. In trophozoites, this enzyme localized at spots within the cytoplasm as detected by using polyclonal antibodies against either E1 N- or C-terminal fragments. This pattern changed during encystation into a diffuse localization throughout the cytoplasm of encysting cells. E1 localizes in mature cysts at cytoplasmic spots and in the cyst wall. Our antisense silencing experiments suggested that E1 is an essential gene for parasite viability. On the other hand, E1 over-expression greatly increased the encystation rate, indicating a relationship between E1 and Giardia differentiation.
Asunto(s)
Giardia lamblia/enzimología , Giardia lamblia/crecimiento & desarrollo , Enzimas Activadoras de Ubiquitina/metabolismo , Animales , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Ratones , Ratones Endogámicos BALB C , Viabilidad Microbiana , Proteolisis , Esporas Protozoarias/enzimología , Esporas Protozoarias/crecimiento & desarrollo , Trofozoítos/enzimología , Trofozoítos/crecimiento & desarrolloRESUMEN
The genome of Dictyostelium contains two novel hybrid-type polyketide synthases (PKSs) known as 'Steely'; the Steely enzyme is formed by the fusion of type I and type III PKSs. One of these enzymes, SteelyB, is known to be responsible for the production of the stalk cell-inducing factor DIF-1 in vivo. On the other hand, the product(s) and expression pattern of SteelyA are not clearly understood, because there are two different reports associated with the in vitro products of SteelyA and its expression pattern. To solve this problem, we first examined the expression pattern using two different primer sets and found that it was quite similar to that shown in the dictyExpress database. stlA expression peaked at approximately 3 h and declined, but showed a small peak around the end of development. Next, we examined the in vivo product of SteelyA using a stlA null mutant and found that the mutant lacked 4-methyl-5-pentylbenzene-1,3-diol (MPBD). This null mutant showed aberrant, glassy sori, and most of the cells in the sori remained amoeba-like without a cell wall. This defect was restored by adding 200 nM of MPBD to the agar. These results indicate that SteelyA produces MPBD in vivo and induces spore maturation.
Asunto(s)
Dictyostelium/enzimología , Sintasas Poliquetidas/metabolismo , Proteínas Protozoarias/metabolismo , Resorcinoles/metabolismo , Esporas Protozoarias/crecimiento & desarrollo , Dictyostelium/genética , Dictyostelium/crecimiento & desarrollo , Dictyostelium/metabolismo , Sintasas Poliquetidas/genética , Proteínas Protozoarias/genética , Esporas Protozoarias/enzimología , Esporas Protozoarias/genética , Esporas Protozoarias/metabolismoRESUMEN
Entamoeba histolytica forms chitin-walled cysts during encystation process, where formation of the cyst wall needs not only chitin synthase but also chitinase. During excystation, quadruplet amoebae emerge from the chitin-walled cysts by dissolving the wall, so that chitinase may be necessary for excystation process as well. There is, however, no report on chitinase expression during excystation. In this study, we used Entamoeba invadens, a reptilian amoeba, as a model for encystation and excystation of E. histolytica, and studied chitinase mRNA expression in those processes. Although expression of three E. invadens chitinases designated EiChit1, EiChit2, and EiChit3 during encystation has been reported, we identified another enzyme named as EiChit4 in the E. invadens genome database. Therefore, we investigated the primary structure and mRNA expression of these four chitinases of Ei in the excystation as well as the encystation by real-time reverse transcription polymerase chain reaction (RT-PCR). Like EiChit1, EiChit4 had an 8 × Cys chitin-binding domain (CBD) and a hydrophilic spacer between the CBD and catalytic domain, and was also closer to EiChit1 than EiChit2 and EiChit3 in the phylogenetic tree. During encystation, the expression of all four chitinases increased in the early phase; the increase in EiChit1 and EiChit4 was much higher than in EiChit2 and EiChit3. Then, the expression of all four chitinases sharply decreased in the later phase. In cysts, EiChit1 was most abundantly expressed and EiChit4 was at a lower level, while the expressions of EiChit2 and EiChit3 were virtually absent. Following the induction of excystation, mRNA levels of EiChit1 and EiChit4 in cysts 5 h after induction were significantly lower than those in cysts before induction, while those of EiChit2 and EiChit3 were remarkably higher than before induction. The mRNAs of only EiChit2 and EiChit3 remarkably increased when the excystation was induced in the presence of cytochalasin D. These data demonstrate different structures and expressions of four chitinases in the differentiation of E. invadens.
Asunto(s)
Quitinasas/biosíntesis , Quitinasas/genética , Entamoeba/enzimología , Entamoeba/crecimiento & desarrollo , ARN Mensajero/biosíntesis , Esporas Protozoarias/enzimología , Esporas Protozoarias/crecimiento & desarrollo , Secuencia de Aminoácidos , Análisis por Conglomerados , ADN Protozoario/química , ADN Protozoario/genética , Entamoeba/genética , Entamoeba/metabolismo , Regulación de la Expresión Génica , Datos de Secuencia Molecular , Filogenia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Esporas Protozoarias/genética , Esporas Protozoarias/metabolismoRESUMEN
Metalloproteinases (MMP) belong to the family of cation dependent endopeptidases that degrade matrices at physiological pH and to cleave extracellular matrix proteins. They play an important role in diverse physiological and pathological processes; not only there diverse types of MMP differ in structure and functionally, but also their enzymatic activity is regulated at multiple levels. Trying to shed some light over the processes that govern the pathology of African Trypanosomiasis, the aim of the present study was to examine the proteolytic activity of the crude trypanosome protein extract obtained from the bloodstream forms of Trypanosoma brucei brucei parasites. We hereby report the partial biochemical characterization of a neutral Trypanosoma brucei-metalloproteinase that displays marked proteolytic activities on gelatin and casein, with a molecular mass of approximately 40 kDa, whose activity is strongly dependent of pH and temperature. Furthermore, we show that this activity can be inhibited by classical MMP inhibitors such as EDTA, EGTA, phenantroline, and also by tetracycline and derivatives. This study has a relevant role in the search for new therapeutical targets, for the use of metalloproteinases inhibitors as treatment strategies, or as enhancement to trypanocidal drugs used in the treatment of the disease.
Asunto(s)
Metaloproteasas/química , Trypanosoma brucei brucei/enzimología , Animales , Cromatografía por Intercambio Iónico , Precursores Enzimáticos/metabolismo , Concentración de Iones de Hidrógeno , Metaloproteasas/aislamiento & purificación , Metaloproteasas/metabolismo , Ratones , Ratones Endogámicos BALB C , Esporas Protozoarias/enzimología , TemperaturaRESUMEN
Microsporidia are fungal-like unicellular eukaryotes which develop as obligate intracellular parasites. They differentiate into resistant spores that are protected by a thick cell wall composed of glycoproteins and chitin. Despite an extensive description of the fibrillar structure of this wall, very little is known about its protein components and deposit mechanisms. In this study on the human pathogen Encephalitozoon cuniculi, we identify by mass spectrometry the target of polyclonal antibodies previously raised against a 33-kDa protein located at the outer face of the parasite plasma membrane. This 254-amino acid protein is encoded by the ECU11_0510 open reading frame and presents two isoforms of 33 and 55 kDa. Sequence analysis supports an assignment to the polysaccharide deacetylase family with a suspected chitin deacetylase activity (EcCDA). As demonstrated by TEM studies, EcCDA is present at the plasma membrane of the early stages of E. cuniculi life-cycle. At the sporoblast stage, the enzyme accumulates especially in paramural bodies which are convolutions of the plasma membrane opened to the wall. The identification of an EcCDA homologue in the insect parasite Antonospora locustae (ex Nosema locustae) suggests a widespread distribution of this enzyme among Microsporidia. This characterization of a new microsporidian surface protein creates new perspectives to understand spore wall formation and spore resistance.
Asunto(s)
Amidohidrolasas/fisiología , Pared Celular/fisiología , Encephalitozoon cuniculi/fisiología , Amidohidrolasas/genética , Amidohidrolasas/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Pared Celular/enzimología , Encephalitozoon cuniculi/enzimología , Encephalitozoon cuniculi/genética , Inmunohistoquímica , Isoenzimas/genética , Isoenzimas/aislamiento & purificación , Isoenzimas/fisiología , Microscopía Electrónica de Transmisión , Peso Molecular , Estructura Terciaria de Proteína , Alineación de Secuencia , Esporas Protozoarias/enzimologíaRESUMEN
Cyclic AMP metabolism is essential for the survival of the social amoebae Dictyostelium discoideum. Three distinct adenylyl cyclases are expressed and required for the normal development of this simple eukaryote. The adenylyl cyclase expressed during aggregation, ACA, is related to the mammalian and Drosophila G protein-coupled enzymes and is responsible for the synthesis of cAMP that is required for cell-cell signaling in early development. ACB harbors histidine kinase and response-regulator domains and is required for terminal differentiation. Finally, the adenylyl cyclase expressed during germination, ACG, acts as an osmosensor and is involved in controlling spore germination. Together, these enzymes generate the various levels of cAMP that are required for D. discoideum to transition from uni- to multi-cellularity. This review will highlight the properties of these enzymes and describe the signaling cascades that lead to their activation.
Asunto(s)
Adenilil Ciclasas/fisiología , Dictyostelium/enzimología , Adenilil Ciclasas/genética , Animales , Dictyostelium/fisiología , Proteínas de Unión al GTP/fisiología , Concentración Osmolar , Transducción de Señal/fisiología , Esporas Protozoarias/enzimología , Esporas Protozoarias/fisiologíaRESUMEN
UDPgalactose:polysaccharide galactosyl-transferase is the enzyme that is specifically localized in prespore cells of Dictyostelium discoideum and its activity sharply changes in response to differentiation and dedifferentiation. To clarify the nature of this enzyme, we first developed an improved assay method for the enzyme, and by using this method, we partially purified the enzyme through DEAE-sepharose, phenyl-sepharose and ATP-sepharose chromatography. The apparent molecular mass of the enzyme was ca. 200 KDa (by non-denaturing polyacrylamide gel gradient analysis) and the isoelectric point was around pH 7. The enzyme exhibited a hitherto undescribed property, that is the reaction proceeds faster at 0 degrees C than at 21 degrees C, with a smaller K(m) value and an unchanged V(max) value. This low-temperature resistant property of the enzyme is consistent with the previous observation (Maeda 1984, J. Cell Sci. 69, 159-165) that prespore differentiation is favored at low temperatures. The reaction appears to proceed in a double displacement manner. ATP reversibly inhibited the enzyme with a K(i) value of 2 mM, suggesting the possibility that ATP regulates its activity in vivo.
Asunto(s)
Dictyostelium/enzimología , Galactosiltransferasas/aislamiento & purificación , Galactosiltransferasas/metabolismo , Adenosina Trifosfato/farmacología , Animales , Cromatografía de Afinidad , Cromatografía por Intercambio Iónico , Inhibidores Enzimáticos/farmacología , Estabilidad de Enzimas , Galactosiltransferasas/química , Punto Isoeléctrico , Peso Molecular , Esporas Protozoarias/enzimología , TemperaturaRESUMEN
Microsporidia constitute a group of extremely specialized intracellular parasites that infect virtually all animals. They are highly derived, reduced fungi that lack several features typical of other eukaryotes, including canonical mitochondria, flagella, and peroxisomes. Consistent with the absence of peroxisomes in microsporidia, the recently completed genome of the microsporidian Encephalitozoon cuniculi lacks a gene for catalase, the major enzymatic marker for the organelle. We show, however, that the genome of the microsporidian Nosema locustae, in contrast to that of E. cuniculi, encodes a group II large-subunit catalase. Surprisingly, phylogenetic analyses indicate that the N. locustae catalase is not specifically related to fungal homologs, as one would expect, but is instead closely related to proteobacterial sequences. This finding indicates that the N. locustae catalase is derived by lateral gene transfer from a bacterium. The catalase gene is adjacent to a large region of the genome that appears to be far less compact than is typical of microsporidian genomes, a characteristic which may make this region more amenable to the insertion of foreign genes. The N. locustae catalase gene is expressed in spores, and the protein is detectable by Western blotting. This type of catalase is a particularly robust enzyme that has been shown to function in dormant cells, indicating that the N. locustae catalase may play some functional role in the spore. There is no evidence that the N. locustae catalase functions in a cryptic peroxisome.