Autodisplay of an avidin with biotin-binding activity on the surface of Escherichia coli.

OBJECTIVES: To display a recombinant avidin fused to the autotransporter ShdA to bind biotinylated molecules on the surface of Escherichia coli. RESULTS: Two chimeric protein constructs containing avidin fused to the autotransporter ShdA were expressed on the surface of Escherichia coli DH5α. One fusion protein contained 476 amino acids of the ShdA α and ß domains, whereas the second consisted of a 314 amino acid from α and truncated ß domains. Protein production was verified by SDS-PAGE using an antibody to the molecular FLAG-tag. The surface display of the avidin-shdA fusion protein was confirmed by confocal microscopy and flow cytometry analysis, and the biotin-binding activity was evaluated by fluorescence microscopy and flow cytometry using biotin-4-fluorescein and biotinylated-ovalbumin (OVA). CONCLUSIONS: Expression of a recombinant avidin with biotin-binding activity on the surface of E. coli was achieved using the autotransporter ShdA. This system is an alternative to bind biotinylated molecules to E. coli.

BRF1, a subunit of RNA polymerase III transcription factor TFIIIB, is essential for cell growth of Trypanosoma brucei.

RNA polymerase III (Pol III) synthesizes small RNA molecules that are essential for cell viability. Accurate initiation of transcription by Pol III requires general transcription factor TFIIIB, which is composed of three subunits: TFIIB-related factor BRF1, TATA-binding protein and BDP1. Here we report the molecular characterization of BRF1 in Trypanosoma brucei (TbBRF1), a parasitic protozoa that shows distinctive transcription characteristics. In silico analysis allowed the detection in TbBRF1 of the three conserved domains located in the N-terminal region of all BRF1 orthologues, namely a zinc ribbon motif and two cyclin repeats. Homology modelling suggested that, similarly to other BRF1 and TFIIB proteins, the TbBRF1 cyclin repeats show the characteristic structure of five α-helices per repeat, connected by a short random-coiled linker. As expected for a transcription factor, TbBRF1 was localized in the nucleus. Knock-down of TbBRF1 by RNA interference (RNAi) showed that this protein is essential for the viability of procyclic forms of T. brucei, since ablation of TbBRF1 led to growth arrest of the parasites. Nuclear run-on and quantitative real-time PCR analyses demonstrated that transcription of all the Pol III-dependent genes analysed was reduced, at different levels, after RNAi induction.

Flagellar motility of Trypanosoma cruzi epimastigotes.

The hemoflagellate Trypanosoma cruzi is the causative agent of American trypanosomiasis. Despite the importance of motility in the parasite life cycle, little is known about T. cruzi motility, and there is no quantitative description of its flagellar beating. Using video microscopy and quantitative vectorial analysis of epimastigote trajectories, we find a forward parasite motility defined by tip-to-base symmetrical flagellar beats. This motion is occasionally interrupted by base-to-tip highly asymmetric beats, which represent the ciliary beat of trypanosomatid flagella. The switch between flagellar and ciliary beating facilitates the parasite's reorientation, which produces a large variability of movement and trajectories that results in different distance ranges traveled by the cells. An analysis of the distance, speed, and rotational angle indicates that epimastigote movement is not completely random, and the phenomenon is highly dependent on the parasite behavior and is characterized by directed and tumbling parasite motion as well as their combination, resulting in the alternation of rectilinear and intricate motility paths.

Improved method for in vitro secondary amastigogenesis of Trypanosoma cruzi: morphometrical and molecular analysis of intermediate developmental forms.

Trypanosoma cruzi undergoes a biphasic life cycle that consists of four alternate developmental stages. In vitro conditions to obtain a synchronic transformation and efficient rates of pure intermediate forms (IFs), which are indispensable for further biochemical, biological, and molecular studies, have not been reported. In the present study, we established an improved method to obtain IFs from secondary amastigogenesis. During the transformation kinetics, we observed progressive decreases in the size of the parasite body, undulating membrane and flagellum that were concomitant with nucleus remodeling and kinetoplast displacement. In addition, a gradual reduction in parasite movement and acquisition of the amastigote-specific Ssp4 antigen were observed. Therefore, our results showed that the in vitro conditions used obtained large quantities of highly synchronous and pure IFs that were clearly distinguished by morphometrical and molecular analyses. Obtaining these IFs represents the first step towards an understanding of the molecular mechanisms involved in amastigogenesis.

LYT1 protein is required for efficient in vitro infection by Trypanosoma cruzi.

Trypanosoma cruzi invasion of host cells involves several discrete steps: attachment, parasite internalization mediated by recruitment and fusion of host cell lysosomes, and escape from the parasitophorous vacuole to liberate amastigotes to multiply freely in the cytosol. This report describes the initial characterization of the LYT1 gene and the demonstration that the gene product is involved in cell lysis and infectivity. Mutational analysis demonstrated that deletion of LYT1 resulted in attenuation of infection, which was associated with diminished hemolytic activity. Reintroduction of LYT1 restored infectivity in null mutants, confirming the critical role of LYT1 in infection. Additionally, in vitro stage transition experiments with LYT1-deficient lines showed that these parasites converted to extracellular amastigote-like cells and metacyclic trypomastigotes more rapidly than wild-type parasites, suggesting that the diminished infectivity was not a result of the LYT1 deficiency that affected the parasite's ability to complete the life cycle.

Up-regulation of action mRNA and reorganization of the cytoskeleton in Entamoeba histolytica trophozoites.

Actin mRNA levels were measured in Entamoeba histolytica trophozoites after experimentally inducing changes in the organization of the cytoskeleton. The treatment of trophozoites with forskolin, N6,2'-O-dibutyryl-adenosine 3':5'-cyclic monophosphate, and phorbol myristate acetate induced the organization of actin into multiple dots and defined structures with a concomitant increase in F-actin content. Cytochalasin D elicited polarization of the structured actin and formation of aggregates, as well as an increment in F-actin. Simultaneously, up-regulation of actin mRNA levels was produced by all the drugs. De novo synthesis of actin mRNA, as measured by nuclear run-ons, showed increased transcription of actin mRNA. On the other hand, treatment of cells with actinomycin D blocked the elevation of actin mRNA synthesis induced by forskolin, dibutyryl cyclic AMP, or cytochalasin D whereas, the increment induced by PMA was not affected. These data indicate a regulatory control of actin mRNA synthesis at the transcriptional level by forskolin, dibutyryl cyclic AMP and cytochalasin D, and transcriptional as well as post-transcriptional controls by phorbol myristate acetate. The experiments presented here suggest the possibility that, regulation of actin mRNA transcription in E. histolytica trophozoites is linked to growth conditions, that are accompanied by reorganization of the actin cytoskeleton and thus, related to the motility and invasiveness of the parasite.

Fibronectin-induced intracellular calcium rise in Entamoeba histolytica trophozoites: effect on adhesion and the actin cytoskeleton.

The interaction of Entamoeba histolytica trophozoites with fibronectin (FN) promotes adhesion of the protein to the cells and its later degradation by locally released proteases. Binding to FN-covered surfaces induces, in addition, the formation of actin adhesion plates and focal contacts in the amebas. The signaling mechanisms underlying the response to FN are incompletely understood. In this paper we examined the modifications of cytosolic free calcium ([Ca2+]i) induced in the trophozoites by the interaction with FN and their effect on adhesion and the actin cytoskeleton organization. FN produced a sustained rise of [Ca2+]i that could be correlated to the incremented adhesion to FN-covered surfaces. Further increments in [Ca2+]i produced by Ca2+ ionophores A23187 or ionomycin significantly increased the adhesion of trophozoites, whereas depletion of cytoplasmic Ca2+, by treatment with the ionophores in the absence of external Ca2+ or using the chelator BAPTA/AM, blocked it almost completely. To study the role of internal calcium we used the plant lactone thapsigargin, which was found to produce a transient increase of [Ca2+]i but a low stimulatory effect on adhesion and the organization of actin plates. The shifting of soluble actin to the F-actin form and the stabilization of adhesion plates and focal contacts, seen as results, of the FN stimulus, were positively influenced by rises in [Ca2+]i and negatively affected by its decrement. Additional evidence for Ca2+ -mediated signaling in the response to FN was provided by the poor adhesion and defective actin plate organization observed in trophozoites treated with calmodulin antagonists. The results presented here suggest that FN action is mainly dependent on the influx of external Ca2+.

Actin mRNA levels and actin synthesis during the encystation of Entamoeba invadens.

Parasitic amebas propagate among hosts through cysts, the resistant forms in their life cycle. In spite of their key role in infection, little is known about the encystation process and the mechanisms involved in reaching this stage. Two features drastically affected by encystation are motility and cell shape, both of which are determined by the cytoskeleton, composed mainly of actin in these organisms. Therefore, we studied the occurrence and relative levels of actin and actin synthesis during encystation of Entamoeba invadens. Using a cDNA actin probe obtained from a library of E. histolytica and a monoclonal antibody against actin, we found that, while the total actin levels sharply decrease as encystation proceeds, the levels of actin mRNA are reduced only in mature cysts. Moreover, actin synthesis does not take place in precysts and the later stages of cyst formation. In contrast, the levels of other proteins remain stable in trophozoites, precysts and cysts, and stage specific peptides are actively synthesized in precysts. The results indicate the encystation is accompanied by a preferential down-regulation of actin synthesis and a decrease in actin levels. The reorganization of the cytoskeletion occurring as trophozoites transform into round, quiescent cells, could be a regulatory factor in the observed changes.

Heterogeneity of the ribosomal DNA episome in strains and species of Entamoeba.

Ribosomal DNA sequences in several species of the genus Entamoeba are highly repeated and display restriction fragment-length polymorphism (RFLP), which has been used to identify species and differentiate strains. However, the continuous variability of the non-transcribed repeat sequences in the ribosomal episome hinders an accurate typification. Looking for more reliable markers, we used DNA probes containing conserved sequences in the ribosomal episome--coding regions for the 16S and 5.8S rRNAs and transcribed spacers flanking the rDNA sequences, and the coding region for the 3' end of the 26S rRNA--to analyse hybridization patterns from five cloned pathogenic strains of Entamoeba histolytica, two strains of the also pathogenic Entamoeba invadens and the non-pathogenic Laredo strain of Entamoeba moshkovskii. Our results provide reliable bases for the differentiation of clones, strains and species of Entamoeba and the reconstruction of E. histolytica episomes. Differences in the number and length of rDNA-containing DNA fragments, previously observed by other investigators and confirmed by us, can be better defined by the present analysis.