RESUMEN
Pentavalent antimonial unresponsiveness is an emerging problem in endemic areas and information on factors which could modulate the transmission of drug-resistant phenotypes and parasites during life cycle are warranted. Using axenic amastigotes resistant to potassium antimonyl tartrate (Sb(III)) we investigated the modulation of antimonyl resistance during the in vitro life cycle. We assessed: (i) the stability of the drug-resistant phenotype during the in vitro life cycle; (ii) the transmission of drug-resistant clones when mixed with a wild-type clone at different susceptible/chemoresistant ratios (50/50,90/10,10/90) after one or two in vitro life cycles. We demonstrate that: (i) mutants which were 12,28,35 and 44 fold more resistant to Sb(III)-antimonial than their parental wild-type, were Glucantime Sb(V)-resistant when growing in THP-1 cells; (ii) the drug-resistant phenotype was partially retained during long-term in vitro culture (3 months) in drug free medium; (iii) the antimonyl-resistant phenotype was retained after one or more in vitro life cycles. However, when drug-resistant parasites were mixed with susceptible, mutants could not be detected in the resulting population, after one or two in vitro life cycles, whatever the initial wild-type/chemoresistant ratio. These results could be explained by the lower capacity of drug-resistant amastigotes to undergo the amastigote-promastigote differentiation process, leading probably to their sequential elimination during life cycle. Taken together, these observations demonstrate that different factors could modulate the transmission of Leishmania drug resistance during the parasite's life cycle.
Asunto(s)
Antimonio/farmacología , Antiprotozoarios/farmacología , Resistencia a Medicamentos , Leishmania infantum/efectos de los fármacos , Animales , Línea Celular , Enfermedades Endémicas , Humanos , Leishmania infantum/genética , Leishmania infantum/crecimiento & desarrollo , Leishmaniasis Visceral/parasitología , Estadios del Ciclo de Vida , Pruebas de Sensibilidad Parasitaria , FenotipoRESUMEN
We have recently characterized a novel Leishmania major gene encoding a polypeptide of 30 kDa that was homologous to mammalian ribosomal protein S3a and was named LmS3a-related protein (LmS3arp). The protein was found to be expressed by all the Leishmania species so far examined (L. infantum, L. amazonensis, and L. mexicana). In the present study we have extended our approach to the analysis of LmS3arp activity on T- and B-cell functions in a murine model. The results presented in this report show that LmS3arp plays a dual role in the regulation of T- and B-cell reactivity. Indeed, we found that injection of the LmS3arp recombinant protein (rLmS3arp) into BALB/c mice induces preferential activation of B cells, as shown by the following criteria: (i) increased expression of CD69 molecules on immunoglobulin M (IgM)-secreting spleen cells, (ii) a considerable increase of IgM-secreting B cells, and (iii) elevated levels of IgM antibodies in the sera of injected animals. Moreover, the IgM antibodies are not specific to the Leishmania antigens but preferentially recognize heterologous antigens like myosin, thyroglobulin, DNA, and keyhole limpet hemocyanin. Furthermore, the strong polyclonal expansion of nonspecific, non-parasite-directed B-cell clones induced by rLmS3arp is concomitant with a marked inhibition of T-cell proliferation. Analysis of cytokine production revealed a significant downregulation of gamma interferon, interleukin-2 (IL-2), and IL-12 secretion. Taken together, our data suggest that rLmS3arp, through direct or indirect action toward B and T cells and cytokine secretion, could participate in the immunoregulatory processes that play a role in the balance of the Th1 and Th2 immune response.
Asunto(s)
Antígenos de Superficie/inmunología , Linfocitos B/inmunología , Leishmania major/inmunología , Activación de Linfocitos/inmunología , Proteínas Protozoarias/inmunología , Proteínas Ribosómicas/inmunología , Linfocitos T/inmunología , Animales , Antígenos CD/biosíntesis , Antígenos de Diferenciación de Linfocitos T/biosíntesis , Biomarcadores , Citocinas/biosíntesis , Regulación hacia Abajo , Inmunoglobulina M/biosíntesis , Lectinas Tipo C , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Bazo/citología , Bazo/inmunologíaAsunto(s)
Citocinas/biosíntesis , Citocinas/genética , Fibroblastos/citología , Regulación de la Expresión Génica/inmunología , Macrófagos/citología , Proteínas Protozoarias/biosíntesis , Proteínas Protozoarias/genética , Regulación hacia Arriba/inmunología , Animales , División Celular/genética , División Celular/inmunología , Línea Celular , Fibroblastos/enzimología , Fibroblastos/inmunología , Glutatión Transferasa/biosíntesis , Glutatión Transferasa/genética , Glutatión Transferasa/fisiología , Macrófagos/enzimología , Macrófagos/inmunología , Ratones , Proteínas Protozoarias/fisiología , Trypanosoma cruzi/enzimologíaRESUMEN
We have previously identified a Trypanosoma cruzi cDNA encoding a protein named Tc52 sharing structural and functional properties with the thioredoxin and glutaredoxin protein family involved in thiol-disulphide redox reactions. Furthermore, we reported that Tc52 also plays a role in T. cruzi-associated immunosuppression observed during Chagas' disease. Moreover, Tc52 gene targeting deletion strategy allowed us to demonstrate that monoallelic disruption of Tc52 resulted in the alteration of the metacyclogenesis process and the production of less virulent parasites. Sequence analysis of a 7358 bp genomic fragment containing the Tc52 encoding gene revealed two additional open reading frames (ORF-A and C). The ORFs are likely to have protein coding function by a number of criteria, including reverse transcriptase polymerase chain reaction (RT-PCR), Western blot and immunofluorescence analyses. The deduced amino-acid (aa) sequence of the ORF-A localized upstream of the Tc52 gene revealed that it contains within its N-terminus (aa 1 to 170) four RGG boxes known to act as RNA binding motifs in some proteins that interact with RNA, interspersed with a high density of glycine with regular spacing of tryptophan (WX(9-10)) in which X is often a glycine. Moreover, the C-terminal part of the ORF-C (aa 253-289) contains a motif that is strikingly similar (7-35% identity, 14-46% similarity over 28aa) to a short sequence (RNP1) comprising the consensus sequence RNA binding domain (CS-RBD) found in a number of proteins that interact with RNA. The aa sequence from the ORF-C localized downstream of the Tc52 gene showed significant homology to human adenosine deaminase acting on RNA (hADAT1) that specifically deaminates adenosine 37 to inosine in eukaryotic tRNA(Ala) and to its homologue yeast protein (Tad1p) (22-25% identity and an additional 38-40% similarity over 177aa). Moreover, highly similar motifs of the deaminase domain are present in the T. cruzi ORF-C. Furthermore, the 5' flanking regions of the genes contained repeat TATA and CAAT nucleotide sequences which resemble the motifs found upstream of the transcription initiation sites in eukaryotic promoters. Therefore, the characterization of novel T. cruzi genes encoding proteins which show similarity to components of RNA processing reactions provides new tools to investigate the gene expression regulation in these parasitic organisms. Moreover, our recent findings on the Tc52 encoding gene underline the interest of genetic manipulation of T. cruzi, not only making it possible to use more closely an in vitro approach to find out how genes function, but also to obtain 'attenuated' strains that could be used in the development of vaccinal strategies.
Asunto(s)
Genes Protozoarios/genética , Proteínas Protozoarias/genética , Edición de ARN , Trypanosoma cruzi/genética , Adenosina Desaminasa/genética , Adenosina Desaminasa/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Núcleo Celular/metabolismo , Clonación Molecular , ADN Protozoario/química , ADN Protozoario/genética , Técnica del Anticuerpo Fluorescente , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Proteínas Protozoarias/metabolismo , Proteínas de Unión al ARN/genética , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Transcripción GenéticaAsunto(s)
Acetilcisteína/farmacología , Enfermedad de Chagas/tratamiento farmacológico , Enfermedad de Chagas/inmunología , Glutatión/farmacología , Animales , Enfermedad de Chagas/sangre , Glutatión/análogos & derivados , Glutatión/sangre , Tolerancia Inmunológica/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Bazo/inmunología , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/inmunologíaRESUMEN
Following purification by affinity chromatography, a Leishmania major S-hexylglutathione- binding protein of molecular mass 66kDa was isolated. The immune serum against the parasite 66kDa polypeptide when used to screen a L. major cDNA library could identify clones encoding for the human v-fos transformation effector homologue, namely ribosomal protein S3a, and thus was named LmS3a-related protein (LmS3arp). A 1027bp cDNA fragment was found to contain the entire parasite gene encoding for a highly basic protein of 30kDa calculated molecular mass sharing homology to various ribosomal S3a proteins from different species. Using computer methods for a multiple alignment and sequence motif search, we found that LmS3arp shares a sequence homology to class theta glutathione S-transferase mainly in a segment containing critical residues involved in glutathione binding. These new findings are discussed in the light of recent published data showing multiple function(s) of the ribosomal proteins S3a.
Asunto(s)
Antígenos de Protozoos/genética , Antígenos de Superficie/genética , Genes Protozoarios/genética , Leishmania major/genética , Proteínas Protozoarias , Proteínas Ribosómicas/genética , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Antígenos de Protozoos/inmunología , Antígenos de Protozoos/metabolismo , Secuencia de Bases , Western Blotting , Línea Celular , Clonación Molecular , ADN Complementario/química , ADN Complementario/genética , ADN Complementario/inmunología , Electroforesis en Gel de Poliacrilamida , Técnica del Anticuerpo Fluorescente , Biblioteca de Genes , Glutatión/metabolismo , Leishmania major/química , Leishmania major/crecimiento & desarrollo , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Pruebas de Precipitina , Unión Proteica , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Radioisótopos de AzufreRESUMEN
We have identified previously a Trypanosoma cruzi gene encoding a protein named Tc52 sharing structural and functional properties with the thioredoxin and glutaredoxin protein family involved in thiol-disulphide redox reactions. Furthermore, we have reported that Tc52 also played a role in T. cruzi-associated immunosuppression observed during Chagas' disease. In an effort to understand further the biological role of Tc52, we used a gene-targeted deletion strategy to create T. cruzi mutants. Although T. cruzi tolerates deletion of one wild-type Tc52 allele, deletion of both genes is a lethal event, indicating that at least one active Tc52 gene is required for parasite survival. Monoallelic disruption of Tc52 (Tc52+/-) resulted in the production of T. cruzi lines that express less Tc52 mRNA and produced lower amounts of Tc52 protein compared with wild-type cells. In axenic cultures, growth rates of epimastigote forms bearing an interrupted allele were not different from those of wild-type parasites. Furthermore, monoallelic disruption of the Tc52 gene did not modify the growth rate of epimastigotes or their sensitivity to inhibition by benznidazole and nifurtimox, the two drugs used to treat Chagasic patients. Moreover, the antimonial drug SbIII, which is known, at least in Leishmania parasites, to be conjugated to a thiol and extruded by an ATP-coupled pump, had a similar effect on wild-type and mutant parasites, being equally sensitive. Hence, parasite drug sensitivity was also observed in clones overexpressing the Tc52 protein as well as in those carrying an antisense plasmid construct. Surprisingly, a significant impairment of the ability of epimastigotes carrying a Tc52 single gene replacement or antisense construct to differentiate into metacyclic trypomastigotes and to proliferate in vitro and in vivo was observed, whereas no significant enhancement of these biological properties was seen in the case of parasites that overexpress Tc52 protein. Moreover, functional complementation of Tc52+/- single mutant or selection of antisense revertant clones demonstrated that the phenotype observed is a direct consequence of Tc52 gene manipulation. Taken together, these results may suggest that Tc52 could participate among other factors in the phenotypic expression of T. cruzi virulence.
Asunto(s)
Alelos , Proteínas Protozoarias/fisiología , Trypanosoma cruzi/crecimiento & desarrollo , Secuencia de Aminoácidos , Animales , Antimonio/farmacología , Secuencia de Bases , ADN Protozoario , Eliminación de Gen , Macrófagos , Metales Pesados , Datos de Secuencia Molecular , Nifurtimox/farmacología , Nitroimidazoles/farmacología , Oligonucleótidos Antisentido , Fenotipo , Proteínas Protozoarias/biosíntesis , Proteínas Protozoarias/genética , Transcripción Genética , Tripanocidas/farmacología , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/genética , Trypanosoma cruzi/fisiologíaRESUMEN
In previous studies we have characterized several Leishmania major polypeptides and showed that one member of this group (LmSIR2rp) shared significant homology to silent information regulator 2 (SIR2) of Saccharomyces cerevisiae, a protein playing a role in both telomeric and mating type loci repression in these organisms. In the present study, by using molecular and immunological approaches, we could identify LmSIR2rp homologues in different Leishmania species and developmental stages (e.g. logarithmic (LP) and stationary phase promastigotes (SP) and amastigotes). The reactive antigen was also detected in Trypanosoma cruzi extracts. Surprisingly, immunofluorescence assays revealed that LmSIR2rp is associated mainly with cytoplasmic granules of different sizes and numbers depending on the life stage of the parasite used. No reactivity was observed in the nucleus, in agreement with the Western blot showing an absence of immunoreactivity of anti-LmSIR2rp immune serum against parasite nuclear extracts. Furthermore, immunoprecipitation of [35S]methionine-labeled promastigote antigens after pulse chase experiments, using anti-LmSIR2rp fusion protein antibodies, showed that the protein is among parasite excreted-secreted antigens (ESA). Moreover, immunofluorescence assays conducted with short time incubations of either purified LmSIR2rp or viable promastigotes with murine macrophages, revealed that LmSIR2rp could be bound to the macrophage surface. The unexpected cytoplasmic localization of LmSIR2rp and its presence in ESA may suggest a new mode of action for silent information regulatory factor homologues.
Asunto(s)
Antígenos de Protozoos/análisis , Proteínas de Unión al ADN/análisis , Proteínas Fúngicas/análisis , Histona Desacetilasas , Leishmania major/inmunología , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae , Transactivadores/análisis , Animales , Western Blotting , Técnica del Anticuerpo Fluorescente , Peso Molecular , Pruebas de Precipitina , Homología de Secuencia de Aminoácido , Sirtuina 2 , SirtuinasRESUMEN
Reports of a wide variation in space and time of the frequency of autogeny in Aedes (Ochlerotatus) detritus (Haliday, 1833) of the Camargue (Delta of the Rhône, Bouches-du-Rhône, Gard, France) led us to make an enzymatic analysis of male and female adults from different larval biotopes. The study showed the existence of two genetically distinct, sympatric populations which are morphologically indistinguishable. By diagnostic enzymes monomorph Got-2, Gpd, the grouping of the individual into two subgroups satisfies a Hardy-Weinberg equilibrium for the polymorphic enzymes. It is concluded that Ae. detritus is composed of a complex of two sibling species provisionally designated by the letters A (Got-2RR, GpdCC) and B (Got-2LL, GpdBB). In the present article, we retrace the history of the binomen Ae. detritus since the original description (sub nom. Culex detritus) to the split into the detritus complex. Certain ecophysiological (steno-eurygamy) and chorological (bioclimatic gradients N-S) criteria show that the sibling species B should be assigned to the taxon described in UK (Holywood, County Down, Ireland) by A.H. Haliday. Species A is here named Aedes (Ochlerotatus) Coluzzii n. sp.
Asunto(s)
Aedes/clasificación , Aedes/anatomía & histología , Animales , Femenino , Larva/anatomía & histología , Larva/clasificación , Masculino , FilogeniaRESUMEN
The wing morphometry of a laboratory-bred population of Phlebotomus perniciosus was studied using a semi-automatic, measurement system. The methods used were found to be precise, reproducible and valid for morphometrical studies. Each of several, different wing indices was found to be similar whether the wing was mounted in Hoyer's medium, Euparal or chloral balm. All samples showed similar differences between the wings of males and females, the wings of the males being smaller than those of the females.
Asunto(s)
Phlebotomus/anatomía & histología , Alas de Animales/anatomía & histología , Animales , Biometría/métodos , Femenino , Masculino , Análisis MultivarianteRESUMEN
This study presents the results of the isoenzymatic characterization of 21 strains of Leishmania of sandfly (P. perniciosus) origin from the Torvizcón area. It forms an integral part of a larger eco-epidemiological study of the Alpujarras (Granada province, Southern Spain). The strains analysed were shown to belong to the L. infantum complex based on the results of 15 enzymes. The electrophoretic profiles for the enzymes MDH, G6PD and NP1 have permitted the identification of four zymodemes: GR-1 (5 strains), GR-2 (2 strains), GR-3 (13 strains) and GR-7 (1 strain); only one of these zymodemes, GR-1, was found in the Torvizcón area in the vertebrate host (man and dog). This is the first time zymodeme GR-7 has been described.
Asunto(s)
Insectos Vectores/parasitología , Isoenzimas/análisis , Leishmania infantum/clasificación , Phlebotomus/parasitología , Animales , Electroforesis en Gel de Almidón , Femenino , Leishmania infantum/enzimología , Leishmania infantum/aislamiento & purificación , EspañaAsunto(s)
Animales de Laboratorio/genética , Phlebotomus/genética , Polimorfismo Genético , Animales , Animales de Laboratorio/clasificación , Animales de Laboratorio/parasitología , Electroforesis , Estudios de Evaluación como Asunto , Genotipo , Isoenzimas , Leishmaniasis/parasitología , Fenotipo , Phlebotomus/clasificación , Phlebotomus/parasitología , Vigilancia de la PoblaciónRESUMEN
Ecoepidemiological analysis of a Moroccan focus of leishmaniasis caused by Leishmania tropica revealed considerable enzymatic diversity. Seven zymodemes belonging to the complex were identified in 149 strains isolated from humans, dogs, and the vector Phlebotomus sergenti. Three distinct subgroups were identifiable, two of which were in turn, composed of three "small variant" zymodemes. The diversity appears to be related to the age of the focus, which may have allowed colonization by zymodemes of different geographic origins. Diversification into "small variants" is apparently the result of recent mutation, possibly associated with genetic exchange.
Asunto(s)
Isoenzimas/análisis , Leishmania tropica/clasificación , Animales , Perros , Electroforesis en Gel de Almidón , Variación Genética , Humanos , Leishmania tropica/enzimología , Leishmania tropica/genética , Marruecos , Phlebotomus/parasitología , Polimorfismo GenéticoRESUMEN
In a Moroccan focus of cutaneous leishmaniasis caused by Leishmania tropica, 7,907 female sandflies captured with CDC traps were dissected from summer to autumn 1989. Among species of the genus Phlebotomus, only P. sergenti harbored promastigotes. Eighty-nine strains belonging to the complex L. tropica were isolated. The frequency of vector infection was zero in June, rose to 1.3% in August, and reached 9.9% in October, which indicates that the period of high risk is at the end of the hot season. Out of 89 strains isolated, 74 were completely typed and corresponded to the following four zymodemes: MON-102 (one strain), MON-107 (56 strains), MON-122 (two strains), and MON-123 (15 strains). Only the first two were observed in humans. The distribution of zymodemes MON-102 and MON-107 was very different in humans, dogs, and the vector. In one of the sites surveyed, which was strongly dominated by MON-107, the absence of human cases involving this zymodeme suggests the existence of a wild reservoir.
Asunto(s)
Insectos Vectores/parasitología , Leishmania tropica/aislamiento & purificación , Phlebotomus/parasitología , Animales , Femenino , Humanos , Leishmania tropica/clasificación , Marruecos , Estaciones del AñoRESUMEN
Ecdysteroid and juvenile hormone titers were determined in the whole body of females of Aedes detritus and A. caspius. Since both hormones were assayed from the same extract, this method allowed determination of their simultaneous variations during egg formation, i.e., from the time the females emerged until the onset of oviposition. A drastic hormonal increase was observed at the beginning of vitellogenesis. This increase occurred as two high and sharp peaks, the first of ecdysteroids and the second, which took place 8 hr later, of juvenile hormones. The two peaks together lasted less than 12 hr, with the highest level at about 3 X 10(-7) mumol/mg fresh tissue. After the juvenile hormone peak, the oocytes entered into stage III/b, the time at which the intensive phase of vitellin accumulation in the eggs begins.
Asunto(s)
Aedes/metabolismo , Ecdisterona/metabolismo , Hormonas Juveniles/metabolismo , Vitelogénesis , Animales , Femenino , Factores de TiempoRESUMEN
The dispersal of Phlebotomus ariasi was studied in mark-release-recapture experiments in the summer of 1980 in a valley on the north-eastern slopes of the Oiselette range in the Cévennes mountains, in the commune of Roquedur, Gard, 50 km north of Montpellier, France. More than 5,000 specimens of P. ariasi were marked with fluorescent powders and released in 9 batches at 3 different places. Seven batches were engorged females and two were unengorged females and males. From 1-29 days after release, 497 marked sandflies (approximately 9%) were recaptured by active searches with UV lamps or in 58 CDC light traps set up in groups of 4 or 5 at 12 recapture stations. Females released engorged generally remained within 250 m of the release point for the first eight days while the bloodmeal was being digested after which there was a tendency to disperse to distances greater than 350 m presumably in a search for oviposition sites or another bloodmeal. The furthest distance to which a female released engorged was shown to move was 925 m; it was caught 12 days after release. Some of the females released unfed quickly moved away from release points, sometimes to distances of 1,000 m or more. One of these was caught 68.5 hrs after release at a station 2,200 m from the release point. Male sandflies tended to stay near the point of release and were not recaptured at distances greater than 600 m. There was no evidence that the movement of the sandflies was assisted by wind. Observations on the dispersal of female sandflies confirm that leishmaniasis can be more widely spread than generally assumed by the movements of the vector.
Asunto(s)
Insectos Vectores/fisiología , Leishmaniasis Visceral/transmisión , Phlebotomus/fisiología , Animales , Ecología , Femenino , Alimentos , Francia , Humanos , Masculino , Tiempo (Meteorología)RESUMEN
The authors have made the first discovery of Phlebotomus mascittii in Spain. The species was caught in Catalonia in the provinces of Gerona and Barcelona. Altogether 7 individual sandflies (4 male, 3 female) were collected (using oiled paper traps) in four separate sub-mediterranean sites (between 650 and 780 m).
Asunto(s)
Phlebotomus/aislamiento & purificación , Animales , Femenino , Masculino , EspañaRESUMEN
The number of gonotrophic cycles of phlebotomine sandflies in a leishmaniasis focus in the Cévennes was determined by the examination of the ovaries. The method was to count the number of dilatations on the ovariolar stalks which showed how many times the females had laid eggs. It was found that females of P. ariasi undergo at least three gonotrophic cycles. From mid-June at the beginning of the season, until the middle of August the proportion of parous females was low. After this time until the end of the season in mid-September, the frequency of 2 parous and 3 parous individuals steadily rose. It is concluded that the end of the summer is the period of maximum risk for the transmission of leishmaniasis in this focus.
Asunto(s)
Leishmaniasis/epidemiología , Phlebotomus/fisiología , Animales , Ecología , Femenino , Francia , Humanos , Leishmaniasis/transmisión , Oviposición , Phlebotomus/anatomía & histología , Reproducción , Estaciones del AñoRESUMEN
Using two sibling species of the Aedes detritus (Haliday, 1833) complex, one autogenous (A) and the other anautogenous (B), the role played by the larval environment on vitellogenesis was confirmed. A poor larval diet reduced the fecondity equally of both autogenous and anautogenous females. With the latter however blood meal ensured the maturation of a not negligible number of ovocytes.