RESUMEN
Toxoplasma gondii, an obligate intracellular parasite of the phylum Apicomplexa, can cause severe disease in humans with an immature or suppressed immune system. The outcome of Toxoplasma infection is highly dependent on the strain type, as are many of its in vitro growth properties. Here we use genetic crosses between type II and III lines to show that strain-specific differences in the modulation of host cell transcription are mediated by a putative protein kinase, ROP16. Upon invasion by the parasite, this polymorphic protein is released from the apical organelles known as rhoptries and injected into the host cell, where it ultimately affects the activation of signal transducer and activator of transcription (STAT) signalling pathways and consequent downstream effects on a key host cytokine, interleukin (IL)-12. Our findings provide a new mechanism for how an intracellular eukaryotic pathogen can interact with its host and reveal important differences in how different Toxoplasma lineages have evolved to exploit this interaction.
Asunto(s)
Regulación de la Expresión Génica , Fosfotransferasas/metabolismo , Proteínas Protozoarias/metabolismo , Toxoplasma/enzimología , Toxoplasma/fisiología , Toxoplasmosis/genética , Toxoplasmosis/parasitología , Animales , Núcleo Celular/metabolismo , Núcleo Celular/parasitología , Cruzamientos Genéticos , Femenino , Humanos , Interleucina-12/metabolismo , Masculino , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT6/metabolismo , Transducción de Señal , Toxoplasma/clasificación , Toxoplasma/genética , Transcripción Genética/genéticaRESUMEN
Toxoplasma gondii is a protozoan pathogen that produces severe disease in humans and animals. This obligate intracellular parasite provides an excellent model for the study of how such pathogens are able to invade, survive, and replicate intracellularly. DNA encoding chloramphenicol acetyltransferase was introduced into T. gondii and transiently expressed with the use of three vectors based on different Toxoplasma genes. The ability to introduce genes and have them efficiently and faithfully expressed is an essential tool for understanding the structure-function relation of genes and their products.
Asunto(s)
Expresión Génica , Genes Protozoarios , Toxoplasma/genética , Transfección , Animales , Células Cultivadas , Cloranfenicol O-Acetiltransferasa/genética , Vectores Genéticos , HumanosRESUMEN
A system for stable transformation of Toxoplasma gondii tachyzoites was developed that exploited the susceptibility of Toxoplasma to chloramphenicol. Introduction of the chloramphenicol acetyltransferase (CAT) gene fused to Toxoplasma flanking sequences followed by chloramphenicol selection resulted in parasites stably expressing CAT. A construct incorporating the tandemly repeated gene, B1, targeted efficiently to its homologous chromosomal locus. Knockout of the single-copy gene, ROP1, was also successful. Stable transformation should permit the identification and analysis of Toxoplasma genes important in the interaction of this opportunistic parasite with its host.
Asunto(s)
Cloranfenicol O-Acetiltransferasa/genética , Genes Protozoarios , Toxoplasma/genética , Transformación Genética , Animales , Cloranfenicol/farmacología , Resistencia a Medicamentos , Marcadores Genéticos , Familia de Multigenes , Plásmidos , Recombinación Genética , Toxoplasma/efectos de los fármacos , TransfecciónRESUMEN
Toxoplasma gondii is a common human pathogen causing serious, even fatal, disease in the developing fetus and in immunocompromised patients. Despite its ability to reproduce sexually and its broad geographic and host range, Toxoplasma has a clonal population structure comprised principally of three lines. We have analyzed 15 polymorphic loci in the archetypal type I, II, and III strains and found that polymorphism was limited to, at most, two rather than three allelic classes and no polymorphism was detected between alleles in strains of a given type. Multilocus analysis of 10 nonarchetypal isolates likewise clustered the vast majority of alleles into the same two distinct ancestries. These data strongly suggest that the currently predominant genotypes exist as a pandemic outbreak from a genetic mixing of two discrete ancestral lines. To determine if such mixing could lead to the extreme virulence observed for some strains, we examined the F(1) progeny of a cross between a type II and III strain, both of which are relatively avirulent in mice. Among the progeny were recombinants that were at least 3 logs more virulent than either parent. Thus, sexual recombination, by combining polymorphisms in two distinct and competing clonal lines, can be a powerful force driving the natural evolution of virulence in this highly successful pathogen.
Asunto(s)
Recombinación Genética , Toxoplasma/genética , Toxoplasma/patogenicidad , Toxoplasmosis Animal/parasitología , Toxoplasmosis/parasitología , Alelos , Animales , Secuencia de Bases , Cruzamientos Genéticos , Genes Protozoarios , Variación Genética , Genotipo , Humanos , Intrones , Dosificación Letal Mediana , Ratones , Ratones Endogámicos CBA , Datos de Secuencia Molecular , Polimorfismo Genético , Polimorfismo de Nucleótido Simple , Toxoplasma/clasificación , Toxoplasma/aislamiento & purificación , Virulencia/genéticaRESUMEN
Toxoplasma gondii is an obligate intracellular pathogen within the phylum Apicomplexa. This protozoan parasite is one of the most widespread, with a broad host range including many birds and mammals and a geographic range that is nearly worldwide. While infection of healthy adults is usually relatively mild, serious disease can result in utero or when the host is immunocompromised. This sophisticated eukaryote has many specialized features that make it well suited to its intracellular lifestyle. In this review, we describe the current knowledge of how the asexual tachyzoite stage of Toxoplasma attaches to, invades, replicates in, and exits the host cell. Since this process is closely analogous to the way in which viruses reproduce, we refer to it as the Toxoplasma "lytic cycle."
Asunto(s)
Toxoplasma/fisiología , Toxoplasma/patogenicidad , Animales , Humanos , Estadios del Ciclo de Vida , Reproducción , Toxoplasma/crecimiento & desarrollo , Toxoplasmosis/fisiopatología , Toxoplasmosis Animal/fisiopatologíaRESUMEN
Introns are almost always present in ribosomal protein genes, even in organisms in which introns are rare. Although trans spliced, the trypanosome ribosomal protein gene S14 apparently does not have cis introns, which supports the notion that such introns are absent in this organism.
Asunto(s)
Genes , Intrones , Proteínas Ribosómicas/genética , Trypanosoma/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Escherichia coli/genética , Exones , Humanos , Datos de Secuencia Molecular , Saccharomyces cerevisiae/genética , Homología de Secuencia de Ácido Nucleico , Transcripción GenéticaRESUMEN
Maturation of mRNA precursors in trypanosomes involves an apparent trans splicing event in which a 39-nucleotide miniexon sequence, common to all trypanosome mRNAs, is joined to the 5' end of a protein-coding exon. We demonstrate that the processing machinery responsible for the maturation of tubulin mRNA precursors in Trypanosoma brucei can be disrupted by heat shock. This results in an accumulation of polycistronic RNA species and a decrease in the abundance of branched splicing intermediates. At normal temperatures, tubulin polycistronic transcripts were also detected and were shown in pulse-chase experiments to be abundantly synthesized and very rapidly turned over. These results, combined with results of the heat shock experiments, suggest that these polycistronic transcripts are the precursors of the (monocistronic) tubulin mRNAs of trypanosomes.
Asunto(s)
Genes , Proteínas de Choque Térmico/genética , Precursores del ARN/genética , ARN Mensajero/genética , Transcripción Genética , Trypanosoma brucei brucei/genética , Tubulina (Proteína)/genética , Animales , Calor , Precursores del ARN/biosíntesis , ARN Mensajero/biosíntesisRESUMEN
Most, if not all, trypanosome mRNAs have the same 35-base sequence at their 5' terminus which is derived from a short RNA (medRNA) probably by the process of trans-splicing. It is of interest, evolutionarily and mechanistically, to determine the chemical structure of the 5' terminus of the precursor (medRNA) and product (mRNA). We demonstrate here that the cap structure of both is most probably 7-methylguanosine in a 5',5' triphosphate linkage, consistent with a precursor/product relationship.
Asunto(s)
Exones , Guanosina/análogos & derivados , Caperuzas de ARN , Empalme del ARN , ARN Mensajero/genética , Trypanosoma brucei brucei/genética , Animales , Guanosina/genéticaRESUMEN
The recent development of an efficient transfection system for the apicomplexan Toxoplasma gondii allows a comprehensive dissection of the elements involved in gene transcription in this obligate intracellular parasite. We demonstrate here that for the SAG1 gene, a stretch of six repeated sequences in the region 35 to 190 bp upstream of the first of two transcription start sites is essential for efficient and accurate transcription initiation. This repeat element shows characteristics of a selector in determining the position of the transcription start sites.
Asunto(s)
Antígenos de Protozoos/genética , Regulación de la Expresión Génica , Genes Protozoarios , Proteínas Protozoarias/genética , Toxoplasma/genética , Animales , Secuencia de Bases , Cartilla de ADN/química , ADN Protozoario/genética , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , ARN Mensajero/genética , Secuencias Repetitivas de Ácidos Nucleicos , Eliminación de Secuencia , Transcripción GenéticaRESUMEN
Within its intermediate host, Toxoplasma gondii switches between two forms: a rapidly replicating tachyzoite and an encysted bradyzoite. Bradyzoites persist within the host throughout its life, hidden from antimicrobial agents and the immune system. The signals that mediate switching are poorly understood. A gene trap was employed to isolate genes whose expression is up-regulated early in the switching of bradyzoites via the negative and positive selectable marker hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT). T. gondii was transfected with promoterless HXGPRT and negatively selected with 6-thioxanthine to inhibit the growth of tachyzoites expressing HXGPRT. The surviving tachyzoites were then induced for in vitro bradyzoite formation and treated with mycophenolic acid and xanthine to positively select for parasites in which the construct had integrated downstream of a bradyzoite-specific gene. Strains were checked for their ability to differentiate by using Dolichos biflorus agglutinin (a bradyzoite-specific lectin) and a monoclonal antibody against P36 (a bradyzoite-specific surface antigen). After differentiation, all gene-trapped clones had Dolichos immunofluorescence and all but one expressed P36. The sequences flanking the insertion site of this P36-negative strain were homologous to the Toxoplasma family of surface antigens, strongly suggesting that P36 is encoded by the disruptive gene. Genetic mapping and complementation of the P36-negative strain further indicated that the disrupted gene is P36. Reverse transcriptase PCR and S1 nuclease digestion were used to compare mRNA levels during the tachyzoite and bradyzoite stages. The presumptive P36 gene does not appear to regulate its mRNA levels between the two stages, indicating a posttranscriptional mechanism of regulation for early bradyzoite-specific genes.
Asunto(s)
ADN Protozoario/aislamiento & purificación , Regulación del Desarrollo de la Expresión Génica , Genes Protozoarios , Pentosiltransferasa/metabolismo , Toxoplasma/genética , Secuencia de Aminoácidos , Animales , Antígenos de Protozoos/genética , Antígenos de Superficie/genética , Glicoproteínas de Membrana/genética , Datos de Secuencia Molecular , Mutagénesis Insercional , Proteínas Protozoarias/genética , ARN Mensajero/análisis , Alineación de Secuencia , Regulación hacia ArribaRESUMEN
Toxoplasma gondii is an obligate intracellular pathogen within the phylum Apicomplexa. Invasion and egress by this protozoan parasite are rapid events that are dependent upon parasite motility and appear to be directed by fluctuations in intracellular [Ca(2+)]. Treatment of infected host cells with the calcium ionophore A23187 causes the parasites to undergo rapid egress in a process termed ionophore-induced egress (IIE). In contrast, when extracellular parasites are exposed to this ionophore, they quickly lose infectivity (termed ionophore-induced death [IID]). From among several Iie(-) mutants described here, two were identified that differ in several attributes, most notably in their resistance to IID. The association between the Iie(-) and Iid(-) phenotypes is supported by the observation that two-thirds of mutants selected as Iid(-) are also Iie(-). Characterization of three distinct classes of IIE and IID mutants revealed that the Iie(-) phenotype is due to a defect in a parasite-dependent activity that normally causes infected host cells to be permeabilized just prior to egress. Iie(-) parasites underwent rapid egress when infected cells were artificially permeabilized by a mild saponin treatment, confirming that this step is deficient in the Iie(-) mutants. A model is proposed that includes host cell permeabilization as a critical part of the signaling pathway leading to parasite egress. The fact that Iie(-) mutants are also defective in early stages of the lytic cycle indicates some commonality between these normal processes and IIE.
Asunto(s)
Calcimicina/farmacología , Calcio/metabolismo , Permeabilidad de la Membrana Celular , Ácido Egtácico/análogos & derivados , Ionóforos/farmacología , Toxoplasma/fisiología , Animales , Calcio/farmacología , Línea Celular , Quelantes/farmacología , Citoesqueleto/metabolismo , Resistencia a Medicamentos , Ácido Egtácico/farmacología , Fibroblastos/parasitología , Genes Reporteros , Interacciones Huésped-Parásitos , Humanos , Mutagénesis , Fenotipo , Saponinas/farmacología , Toxoplasma/genéticaRESUMEN
We have defined the genomic organization and genomic context of a Trypanosoma brucei brucei gene family encoding variant surface glycoproteins (VSGs). This gene family is neither tandemly repeated nor closely linked in the genome, and is not located on small or intermediate size chromosomes. Two dispersed repeated sequence elements, RIME-ingi and the upstream repeat sequence, are linked to members of this gene family; however, the upstream repeat sequences are closely linked only to the basic copy. In other isolates of T.b. brucei this gene family appears conserved with some variation; a restriction fragment length polymorphism found among these isolates suggests the hypothesis that VSG genes may occasionally be diploid. A model accounting for both the generation of dispersed families of VSG genes, and for the interstrain variability of VSG genes, is proposed.
Asunto(s)
Familia de Multigenes , Trypanosoma brucei brucei/genética , Glicoproteínas Variantes de Superficie de Trypanosoma/genética , Animales , Evolución Biológica , Southern Blotting , Cósmidos , ADN/genética , ADN/aislamiento & purificación , Biblioteca de Genes , Genoma , Recombinación Genética , Secuencias Repetitivas de Ácidos Nucleicos , Mapeo RestrictivoRESUMEN
We have used analysis of DNA sequence data from four members of a Trypanosoma brucei variant surface glycoprotein gene family to investigate the molecular basis of the generation of antigenic diversity in African trypanosomes. Among these four sequences we find the greatest similarity in the untranslated sequences immediately upstream from the coding region. A complex pattern of nucleic acid and predicted amino acid sequence divergence appears starting at the coding sequence. Two related but highly divergent hydrophobic leaders are associated with different members of this gene family; both forms of these hydrophobic leaders appear to exist in other isolates of T. b. brucei. We find conservative replacements in the first 120 predicted amino acid residues of the mature protein; the following 80 predicted residues show less conservative replacements, and we suggest that this region may be hypervariable and exposed to the aqueous environment.
Asunto(s)
Variación Antigénica , ADN Protozoario/genética , Familia de Multigenes , Trypanosoma brucei brucei/genética , Glicoproteínas Variantes de Superficie de Trypanosoma/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN , ADN Protozoario/aislamiento & purificación , Datos de Secuencia Molecular , Biosíntesis de Proteínas , Conformación Proteica , ARN Mensajero/genética , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , Trypanosoma brucei brucei/inmunologíaRESUMEN
In the course of our genetic studies on Toxoplasma gondii, it was discovered that one cosmid hybridized to a repetitive element. The hybridization pattern observed for the enzyme BglII indicated that this cosmid hybridized to a large number of discrete, but related elements. Four BglII fragments were subcloned from the cosmid, and each was shown to hybridize with all the others, as well as to numerous dispersed sequences in genomic DNA. Three subclones were sequenced in their entirety, and shown to contain fragments of the genes for cytochrome oxidase subunit I and apocytochrome b, complete and functional copies of which have been found in only mitochondrial genomes. All the subcloned fragments were bounded at both ends by a 91 base-pair sequence, which contains a site for BglII. This 91 base-pair sequence could be found as either a direct or inverted repeat. It was determined that the BglII elements are arrayed downstream from a single copy nuclear gene. Comparison of genomic and cosmid DNAs confirmed that the cosmid faithfully reflects the nuclear genome. Although the mitochondrial genome of Toxoplasma has not been characterized, these nuclear mitochondrial-like sequences appear to be internally rearranged with respect to known, functional mitochondrial genomes, and with respect to each other. The finding of short repeated sequences flanking these elements may be a clue to the mechanism of their dissemination.
Asunto(s)
Núcleo Celular , ADN Mitocondrial/genética , Genoma , Secuencias Repetitivas de Ácidos Nucleicos/genética , Toxoplasma/genética , Secuencia de Aminoácidos , Animales , Apoproteínas/genética , Secuencia de Bases , Núcleo Celular/química , Clonación Molecular , Cósmidos , Grupo Citocromo b/genética , Citocromos b , Complejo IV de Transporte de Electrones/genética , Herencia Extracromosómica , Datos de Secuencia Molecular , Homología de Secuencia de Ácido Nucleico , Toxoplasma/químicaRESUMEN
The large subunit ribosomal RNA (LSRNA) of Trypanosoma brucei is unusual in being cleaved at multiple sites to yield six stable fragments of RNA. We report here the complete nucleotide sequence of two regions of the ribosomal DNA repeat unit. The first sequence includes all of the processing sites involved in the generation of one of the small LSRNA fragments. The second region encodes the trypanosome 5.8 S RNA. By RNA sequencing and S1 nuclease mapping, we have identified the processing sites involved in the generation of both of these small RNAs. On the basis of predicted secondary structure models, we infer that all the cleavages apparently occur near the junction of single- and double-stranded regions. The sites involved in the novel LSRNA processing show a clear symmetry with respect to a conserved region of ten base-pairs. No such signals are evident for the processing sites that generate the 5.8 S RNA.
Asunto(s)
ARN Ribosómico/genética , Trypanosoma brucei brucei/genética , Animales , Secuencia de Bases , ADN Ribosómico , Genes , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Regiones Terminadoras GenéticasRESUMEN
We have constructed a genetic linkage map for the parasitic protozoan, Toxoplasma gondii, using randomly selected low copy number DNA markers that define restriction fragment length polymorphisms (RFLPs). The inheritance patterns of 64 RFLP markers and two phenotypic markers were analyzed among 19 recombinant haploid progeny selected from two parallel genetic crosses between PLK and CEP strains. In these first successful interstrain crosses, these RFLP markers segregated into 11 distinct genetic linkage groups that showed close correlation with physical linkage groups previously defined by molecular karyotype. Separate linkage maps, constructed for each of the 11 chromosomes, indicated recombination frequencies range from approximately 100 to 300 kb per centimorgan. Preliminary linkage assignments were made for the loci regulating sinefungin resistance (snf-1) on chromosome IX and adenine arabinoside (ara-1) on chromosome V by linkage to RFLP markers. Despite random segregation of separate chromosomes, the majority of chromosomes failed to demonstrate internal recombination events and in 3/19 recombinant progeny no intramolecular recombination events were detected. The relatively low rate of intrachromosomal recombination predicts that tight linkage for unknown genes can be established with a relatively small set of markers. This genetic linkage map should prove useful in mapping genes that regulate drug resistance and other biological phenotypes in this important opportunistic pathogen.
Asunto(s)
Toxoplasma/genética , Animales , Mapeo Cromosómico , ADN/genética , Resistencia a Medicamentos , Ligamiento Genético , Marcadores Genéticos , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
We have developed several protocols for the use of beta-galactosidase (betaGal) from Escherichia coli as a reporter enzyme in transfection studies of Toxoplasma gondii (Tg) and as a readily screenable marker for stable transformation. Three Tg expression vectors with different promoters driving lacZ were constructed and shown in transient transfections to differ in their relative expression levels. Using a fluorescent betaGal substrate, it was possible to detect enzymatic activity with as little as 50 ng of transfected lacZ-containing plasmid DNA. When stably transformed intracellular parasites were cultivated in microtiter plates in the presence of the color substrate, chorophenol red-beta-D-galactopyranoside (CPRG), the signal from as few as 400 Tg could be readily detected by eye. Using serial dilutions of transfected parasite cultures in the presence of CPRG, we were able to clone stably expressing betaGal-positive Tg without the need for another selectable marker. Such lacZ transgenics could also be visualized histochemically in the tissue of infected mice. Thus, the application of betaGal to studies on Tg provides not only a much needed second reporter for transient transfection, it also comprises a safe and sensitive marker for the generation and analysis of stably transfected parasites.
Asunto(s)
Regulación de la Expresión Génica , Toxoplasma/genética , beta-Galactosidasa/genética , Animales , Secuencia de Bases , Biomarcadores , Escherichia coli/enzimología , Histocitoquímica , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Transfección/métodosRESUMEN
The synthesis of most, if not all, mRNA in trypanosomes involves the splicing in trans of two RNA molecules. One of these includes the common 5' mini-exon or spliced-leader sequence, while the other contains the complete protein-coding exon sequence for a given gene. We have recently shown that trans-splicing of tubulin transcripts is disrupted by heat shock, whereas trans-splicing of Hsp70 mRNA continues unabated, despite the increased expression of this latter gene [Muhich and Boothroyd, Mol. Cell Biol. 8 (1988) 3837-3846]. In this report we show that, in addition to being polyadenylated, the dimeric beta-alpha tubulin transcripts which accumulate as a result of heat shock have a correctly processed 5'-end (through mini-exon addition), and that the extent of disruption is proportional to the degree of heat shock. We further show that mRNA levels for two evolutionarily-conserved heat-shock proteins, Hsp70 and Hsp85, are increased under the splicing-disruptive conditions of heat shock.
Asunto(s)
Proteínas de Choque Térmico/genética , Calor , Empalme del ARN , ARN Mensajero/genética , Trypanosoma brucei brucei/genética , Tubulina (Proteína)/genética , Animales , Secuencia de Bases , Exones , Regulación de la Expresión Génica , Proteínas de Choque Térmico/biosíntesis , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Homología de Secuencia de Ácido Nucleico , Trypanosoma brucei brucei/metabolismo , Trypanosoma cruzi/genética , Tubulina (Proteína)/biosíntesisRESUMEN
The complete nucleotide sequence of cDNA coding for the structural capsid polypeptides of foot-and-mouth disease virus (FMDV) (strain A(10)61) has been determined. Portions of the flanking sequence coding for the nonstructural proteins p20a and p52 are also provided. The three larger structural polypeptides VP1, VP2 and VP3 have unmodified Mrs of 23248, 24649 and 24213, respectively. The size of the smaller polypeptide, VP4, can only be estimated at 7360 because the 5'-limit of its coding region is not yet known with certainty. The sequence data for VP1 (the major immunising antigen) and the amino-terminal quarter of p52 are compared with the data of Kurz et al. (Nucl. Acids Res. 9 (1981) 1919-1931) for a different serotype (O1K). This shows that variation is much greater in the region coding for VP1 than in that coding for p52. This is reflected in the level of amino acid sequence variation predicted for the two proteins. Analysis of relative codon usage reveals a strong bias in favour of C and G over U and A in the third base position. The dinucleotide frequencies show a bias against A-U and U-A, and for A-C and C-A.
Asunto(s)
Aphthovirus/genética , Cápside/genética , ADN Recombinante/análisis , Genes Virales , Proteínas Virales/genética , Secuencia de Bases , ADN/análisis , ADN Bacteriano/genética , Escherichia coli/genética , ARN Viral/genéticaRESUMEN
We have studied the fundamentals of gene expression in the protozoan parasite Toxoplasma gondii by analyzing, in detail, the genes encoding alpha- and beta-tubulin. Southern analysis and quantitation studies reveal that, unlike in other organisms studied thus far, both these genes are present as single copies in the haploid Toxoplasma genome. Sequencing of these genes indicates that they both contain multiple introns with conserved 5' and 3' splice site signals. We have found that HeLa cell nuclear extracts are able to accurately splice a Toxoplasma pre-mRNA construct. We have mapped, for the alpha-tubulin gene, the exact site of transcription initiation and the approximate site of poly A addition by primer extension and RNase protection assays. Trans-splicing, as demonstrated in the Kinetoplastida, is not involved in the formation of the mature alpha-tubulin transcript in T. gondii.