Rebeca Manning Cela - Academia.edu (original) (raw)
Papers by Rebeca Manning Cela
Synanthropic triatomines in Hidalgo state, Mexico: Spatial-temporal distribution, domestic transmission cycle, and natural infection with Trypanosoma cruzi
Acta Tropica
Additional file 1: Figure S1. of Transcription of Leishmania major U2 small nuclear RNA gene is directed by extragenic sequences located within a tRNA-like and a tRNA-Ala gene
Sequence alignment of U2 snRNA genes from trypanosomatids. Sequences were obtained from the TriTr... more Sequence alignment of U2 snRNA genes from trypanosomatids. Sequences were obtained from the TriTrypDB databases of the following species of trypanosomatids: L. major Friedlin (Lmj), L. arabica LEM1108 (Lar), L. tarentolae Parrot-TarII (Ltr), L. infantum JPCM5 (Lin), L. amazonensis MHOMBR/71973/M2269 (Laz), L. donovani BPK282A1 (Ldn), L. aethiopica L147 (Lae), L. mexicana U1103 (Lmx), L. panamensis MHOM/COL/81/L13 (Lpn), L. braziliensis M2904 (Lbr), L. enriettii LEM3045 (Len), E. monterogeii LV88 (Emg), C. fasciculata Cf-Cl (Cfs), L. pyrrhocoris H10 (Lpy), L. seymouri ATCC 30220 (Lsy), L. collosoma (unspecified strain) [39] (Lcl), T. cruzi CL Brener Non-Esmeraldo-like, copy of the chromosome 23 (Tcr), T. vivax Y486 (Tvx), T. congolense IL3000 (Tcn), T. evansi STIB 805 (Tev) and T. brucei DAL972 (Tbr). The alignment was obtained with the DNAMAN software, and manually corrected. Arrows indicate the identified pseudouridines (Ψ) in the L. major U2 snRNA (this work). BPRR and Sm sites ar...
Additional file 2: Figure S2. of Transcription of Leishmania major U2 small nuclear RNA gene is directed by extragenic sequences located within a tRNA-like and a tRNA-Ala gene
Northern blot analysis of the tagged U2 snRNA. The experiment was performed with total RNA from c... more Northern blot analysis of the tagged U2 snRNA. The experiment was performed with total RNA from cells transfected with construct 1 (pComp), construct 6 (pBS + 6/+12), the unrelated vector pLMRIB or mock-transfected cells. The probe was the oligonucleotide LmjU2tag-Rev, which specifically recognizes the tag sequence. (TIF 807 kb)
Additional file 4: Figure S4. of Transcription of Leishmania major U2 small nuclear RNA gene is directed by extragenic sequences located within a tRNA-like and a tRNA-Ala gene
Predicted secondary structure of the tRNA-Ala (panel a) and the tRNA-like associated to the L. ma... more Predicted secondary structure of the tRNA-Ala (panel a) and the tRNA-like associated to the L. major U2 snRNA gene (panel b). The minimum free energy structures are shown. The color scale indicates low (blue) to high (red) probabilities of base pairing. (PDF 355 kb)
Research Article Identification of Protein Complex Associated with LYT1 of
Copyright © 2013 C. Lugo-Caballero et al. This is an open access article distributed under the Cr... more Copyright © 2013 C. Lugo-Caballero et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. To carry out the intracellular phase of its life cycle, Trypanosoma cruzimust infect a host cell. Although a few molecules have been reported to participate in this process, one known protein is LYT1, which promotes lysis under acidic conditions and is involved in parasite infection and development. Alternative transcripts from a single LYT1 gene generate two proteins with differential functions and compartmentalization. Single-gene products targeted to more than one location can interact with disparate proteins that might affect their function and targeting properties. The aim of this work was to study the LYT1 interaction map using coimmunoprecipitation assays with transgenic parasites expressing LYT1 products fused to GFP. We d...
BioMed Research International, 2019
Leishmania major, a protozoan parasite that diverged early from the main eukaryotic lineage, exhi... more Leishmania major, a protozoan parasite that diverged early from the main eukaryotic lineage, exhibits unusual mechanisms of gene expression. Little is known in this organism about the transcription factors involved in the synthesis of tRNA, 5S rRNA, and snRNAs, transcribed by RNA Polymerase III (Pol III). Here we identify and characterize the TFIIIB subunit Bdp1 in L. major (LmBdp1). Bdp1 plays key roles in Pol III transcription initiation in other organisms, as it participates in Pol III recruitment and promoter opening. In silico analysis showed that LmBdp1 contains the typical extended SANT domain as well as other Bdp1 conserved regions. Nevertheless, LmBdp1 also displays distinctive features, including the presence of only one aromatic residue in the N-linker region. We were not able to produce null mutants of LmBdp1 by homologous recombination, as the obtained double replacement cell line contained an extra copy of LmBdp1, indicating that LmBdp1 is essential for the viability o...
Biochemical and Biophysical Research Communications, 2016
The role and regulation of actin in Trypanosoma cruzi and other related parasites is largely unkn... more The role and regulation of actin in Trypanosoma cruzi and other related parasites is largely unknown. Based on early genome analysis, it was proposed that there was a reduced dependency on the actomyosin system in the trypanosomatid parasites. However, more recent studies have extended the set of potential actin regulatory proteins, particularly for T. cruzi. One of the identified actin-binding proteins in trypanosomatids is profilin. In other systems, it is capable of simultaneously binding both monomeric actin and several actin-regulatory factors. Hence, the study of profilin and its ligands may help to identify novel pathways in which actin is involved. In T. cruzi, profilin is encoded by a single copy gene. In this work, we demonstrated that this gene is constitutively expressed in both insect and mammalian stages of the parasite, and that the protein is diffusely distributed. Furthermore, we identified some of its potential ligands by LC-MS using GST-profilin pull-down assays of parasite's protein extracts. Many of them were trypanosomatid specific proteins with unknown functions, although proteins from the carbohydrate metabolism, and two metallopeptidases were also detected. As expected, known ligands of profilin in other organisms were identified, including actin, the microtubule components, and the elongation factor 1-alpha. Our work suggests that profilin and the actin system may be regulated by unknown factors and participate in novel biological processes.
Molecular characterization of 5S ribosomal RNA genes and transcripts in the protozoan parasiteLeishmania major
Parasitology, 2016
SUMMARYEukaryotic 5S rRNA, synthesized by RNA polymerase III (Pol III), is an essential component... more SUMMARYEukaryotic 5S rRNA, synthesized by RNA polymerase III (Pol III), is an essential component of the large ribosomal subunit. Most organisms contain hundreds of 5S rRNA genes organized into tandem arrays. However, the genome of the protozoan parasiteLeishmania majorcontains only 11 copies of the 5S rRNA gene, which are interspersed and associated with other Pol III-transcribed genes. Here we report that, in general, the number and order of the 5S rRNA genes is conserved between different species ofLeishmania. While in most organisms 5S rRNA genes are normally associated with the nucleolus, combined fluorescentin situhybridization and indirect immunofluorescence experiments showed that 5S rRNA genes are mainly located at the nuclear periphery inL. major. Similarly, the tandemly repeated 5S rRNA genes inTrypanosoma cruziare dispersed throughout the nucleus. In contrast, 5S rRNA transcripts inL. majorwere localized within the nucleolus, and scattered throughout the cytoplasm, where...
Protist, 2016
Little is known about nucleosome structure and epigenetic regulation of transcription of rRNA gen... more Little is known about nucleosome structure and epigenetic regulation of transcription of rRNA genes in early-branched eukaryotes. Here we analyze the chromatin architecture and distribution of some histone modifications in the rRNA genes in the parasitic protozoon Leishmania major. Southern blots of MNase-partially-digested chromatin with DNA probes spanning the whole rRNA gene repeat showed that the intergenic spacer presents a tight nucleosomal structure, whereas the promoter region is practically devoid of nucleosomes. Intermediate levels of nucleosomes were found in the rRNA coding regions. ChIP assays allowed us to determine that H3K14ac, H3K23ac and H3K27ac, epigenetics marks that are generally associated with activation of transcription, are enriched in the promoter region. In contrast, H4K20me3, which is generally related to transcriptional silencing, was absent from the promoter region and intergenic spacer and enriched in the coding region. Interestingly, the distribution pattern for H3K9me3, generally linked to heterochromatin formation, was very similar to the distribution observed with the euchromatin marks, suggesting that this modification could be involved in transcriptional activation of rRNA genes in L. major.
PLOS ONE, 2015
The present work is aimed at characterizing the motility of parasite T. cruzi in its epimastigote... more The present work is aimed at characterizing the motility of parasite T. cruzi in its epimastigote form. To that end, we recorded the trajectories of two strains of this parasite (a wildtype strain and a stable transfected strain, which contains an ectopic copy of LYT1 gene and whose motility is known to be affected). We further extracted parasite trajectories from the recorded videos, and statistically analysed the following trajectory-step features: step length, angular change of direction, longitudinal and transverse displacements with respect to the previous step, and mean square displacement. Based on the resulting observations, we developed a mathematical model to simulate parasite trajectories. The fact that the model predictions closely match most of the experimentally observed parasite-trajectory characteristics, allows us to conclude that the model is an accurate description of T. cruzi motility.
Identification of Protein Complex Associated with LYT1 of Trypanosoma cruzi
BioMed Research International, 2013
To carry out the intracellular phase of its life cycle,Trypanosoma cruzimust infect a host cell. ... more To carry out the intracellular phase of its life cycle,Trypanosoma cruzimust infect a host cell. Although a few molecules have been reported to participate in this process, one known protein is LYT1, which promotes lysis under acidic conditions and is involved in parasite infection and development. Alternative transcripts from a singleLYT1gene generate two proteins with differential functions and compartmentalization. Single-gene products targeted to more than one location can interact with disparate proteins that might affect their function and targeting properties. The aim of this work was to study the LYT1 interaction map using coimmunoprecipitation assays with transgenic parasites expressing LYT1 products fused to GFP. We detected several proteins of sizes from 8 to 150 kDa that bind to LYT1 with different binding strengths. By MS-MS analysis, we identified proteins involved in parasite infectivity (trans-sialidase), development (kDSPs and histones H2A and H2B), and motility and...
Journal of Biomedicine and Biotechnology, 2010
The parasitesLeishmaniaspp.,Trypanosoma brucei,andTrypanosoma cruziare the trypanosomatid protozo... more The parasitesLeishmaniaspp.,Trypanosoma brucei,andTrypanosoma cruziare the trypanosomatid protozoa that cause the deadly human diseases leishmaniasis, African sleeping sickness, and Chagas disease, respectively. These organisms possess unique mechanisms for gene expression such as constitutive polycistronic transcription of protein-coding genes and trans-splicing. Little is known about either the DNA sequences or the proteins that are involved in the initiation and termination of transcription in trypanosomatids.In silicoanalyses of the genome databases of these parasites led to the identification of a small number of proteins involved in gene expression. However, functional studies have revealed that trypanosomatids have more general transcription factors than originally estimated. Many posttranslational histone modifications, histone variants, and chromatin modifying enzymes have been identified in trypanosomatids, and recent genome-wide studies showed that epigenetic regulation m...
Journal of Biomedicine and Biotechnology, 2010
Trypanosoma cruziundergoes a biphasic life cycle that consists of four alternate developmental st... more Trypanosoma cruziundergoes 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...
Journal of Biomedicine and Biotechnology, 2012
The hemoflagellateTrypanosoma cruziis the causative agent of American trypanosomiasis. Despite th... more The hemoflagellateTrypanosoma cruziis the causative agent of American trypanosomiasis. Despite the importance of motility in the parasite life cycle, little is known aboutT. cruzimotility, 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 characte...
Experimental Parasitology, 2010
Trypanosoma cruzi is an obligate intracellular parasite that infects phagocytic and non-phagocyti... more Trypanosoma cruzi is an obligate intracellular parasite that infects phagocytic and non-phagocytic mammalian cells by a complex process that appears to involve several discrete steps. Even though the infection process was described many years ago, the molecular mechanisms involved remain poorly understood. As fluorescent proteins have proven to be excellent tools for live-cell imaging, we used EGFPand DsRed1-1-transfected trypomastigotes, amastigotes and epimastigotes to study the infection process in living cells. Contrary to what has been reported, our results showed that epimastigotes are as infective as trypomastigotes and amastigotes. Besides, differences in replication, differentiation and parasite release times were observed among the stages. Our results suggest that the different developmental stages use distinct attachment and invasion mechanisms. We propose that fluorescent-based plasmid expression systems are good models for studying the infection process of intracellular microorganisms and could offers insights about the molecular mechanisms involved.
Archives of Medical Research, 2006
Background. Trypanosoma cruzi, the causative agent of Chagas disease, exhibits a complex life cyc... more Background. Trypanosoma cruzi, the causative agent of Chagas disease, exhibits a complex life cycle that is accompanied by the stage-specific gene expression. At the molecular level, very little is known about gene regulation in trypanosomes. Complex gene organizations coupled with polycistronic transcription units make the analysis of regulated gene expression difficult in trypanosomes. The ubiquitin genes of T. cruzi are a good example of this complexity. They are organized as a single cluster containing five ubiquitin fusion (FUS) and five polyubiquitin (PUB) genes that are polycistronically transcribed but expressed differently in response to developmental and environmental changes. Methods. Gene replacements were used to study FUS and PUB gene expression at different stages of growth and at different points in the life cycle of T. cruzi. Results. Based on the levels of reporter gene expression, it was determined that FUS1 expression was downregulated as the parasites approached stationary phase, whereas PUB12.5 polyubiquitin gene expression increased. Conversely, FUS1 expression increases when epimastigotes and amastigotes differentiate into trypomastigotes, whereas the expression of PUB12.5 decreases when epimastigotes differentiate into amastigotes and trypomastigotes. Conclusions. Although the level of CAT activity in logarithmic growing epimastigotes is six-to seven-fold higher when the gene was expressed from the FUS1 locus than when expressed from the PUB12.5 locus, the rate of transcription from the two loci was the same implying that post-transcriptional mechanisms play a dominant role in the regulation of gene expression.
Vet. Méx, 2012
Resumen LYT1 es una molécula con actividad lítica en condiciones ácidas, que según se demostró ge... more Resumen LYT1 es una molécula con actividad lítica en condiciones ácidas, que según se demostró genéticamente, participa en el proceso de infección y transición de estadio de T. cruzi. Su diferente funcionalidad es resultado de la producción de dos proteínas, obtenidas por trans-empalme alternativo, que contienen una secuencia de secreción y una nuclear (LYT1s) o únicamente la secuencia nuclear (LYT1n). Para evaluar la localización de los diferentes productos de LYT1, se analizaron parásitos transgénicos que expresan la ...
Journal of Eukaryotic Microbiology, 1994
ABSTRACT. Parasitic amebas propagate among hosts through cysts, the resistant forms in their life... more ABSTRACT. 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 stag...
BFA-sensitive and insensitive exocytic pathways in Entamoeba histolytica trophozoites: their relationship to pathogenesis
Cellular Microbiology, 2003
Entamoeba histolytica manifests its pathogenicity through several cellular processes triggered by... more Entamoeba histolytica manifests its pathogenicity through several cellular processes triggered by external stimuli that activate signal transduction pathways. The intense secretory activity resulting from stimulation is not correlated with a typical endoplasmic reticulum (ER) or Golgi organization, and little is known in this parasite about endocytic/exocytic pathways. The interactions of trophozoites with fibronectin (FN) and cultured mammalian cells, which elicit secretory activities, were chosen to study mechanisms that regulate cytoplamic traffic. Results showed that Brefeldin A (BFA) induced redistribution of the vesicular network recognized by antibodies against amoebic proteins PDI and ERD2. Furthermore, BFA diminished traffic to the plasma membrane of the beta1 integrin-like FN receptor and the heavy subunit of the Gal/GalNAc lectin, required for adhesion to FN and target cells, respectively. However, BFA did not prevent thiol-proteinase secretion or inhibit the traffic of de novo synthesized proteinases. These data suggest that two distinct transport systems occur in E. histolytica, one similar to classical membrane protein transport and another independent of BFA and inducible by external stimuli. Actin-myosin contractility of the cortical cytoskeleton seems necessary for the final release of exported proteinases and the proper function of the surface proteins involved in adhesion.
Signal Transduction in Entamoeba histolytica Induced By Interaction with Fibronectin
Archives of Medical Research, 2002
Interaction of Entamoeba histolytica trophozoites with extracellular matrix (ECM) proteins activa... more Interaction of Entamoeba histolytica trophozoites with extracellular matrix (ECM) proteins activates signaling pathways through G-protein-coupled receptors. Increments of adenylyl cyclase activity and cAMP produce a striking reorganization of actin into structures that apparently facilitate adhesive, locomotive, and secretory activities. The reorganization of actin is induced by phosphorylation of actin-associated proteins by diverse kinases activated during the signaling process. Although cAMP-dependent kinases have not yet been identified in this parasite, the activation of the adenylyl cyclase route and its effects on particular motility-related functions strongly suggest their presence. Phosphokinase A (PKA) was detected by phosphorylation of the specific substrate, kemptide, its further activation by cAMP, and its inhibition by H89. The catalytic subunit of the enzyme was identified by immunofluorescence microscopy and by immunoprecipitation. Adhesion and damage to cultured cells were monitored by FN-binding and cytotoxicity assays. A cAMP-dependent kinase activated by effectors and agonists of adenylyl cyclase and also during interaction of trophozoites with fibronectin (FN) was found. The enzyme is associated with small granules in the cytoplasm and upon activation, a fraction of its catalytic subunit with an Mr of 100 kDa was translocated to the nucleus, while another fraction was aggregated into big clusters. Activity and translocation were blocked by H89, a specific inhibitor of PKA. Trophozoites stimulated by dBcAMP or forskolin-formed lamellae and restructured actin, but no significant increase in their adhesion to FN was observed and only showed 10% stimulus in their capacity to damage target cells. Treatment with H89 decreased adhesion to 40% and caused 80% inhibition in cell damage. These amebas showed altered organization of the actin structures induced by dBcAMP or FN. Our results support previous suggestions concerning the participation of PKA in the response elicited by the interaction of E. histolytica trophozoites with ECM proteins. They also indicate that adhesion and secretion in conjunction with motile activities are related to invasion processes.
Synanthropic triatomines in Hidalgo state, Mexico: Spatial-temporal distribution, domestic transmission cycle, and natural infection with Trypanosoma cruzi
Acta Tropica
Additional file 1: Figure S1. of Transcription of Leishmania major U2 small nuclear RNA gene is directed by extragenic sequences located within a tRNA-like and a tRNA-Ala gene
Sequence alignment of U2 snRNA genes from trypanosomatids. Sequences were obtained from the TriTr... more Sequence alignment of U2 snRNA genes from trypanosomatids. Sequences were obtained from the TriTrypDB databases of the following species of trypanosomatids: L. major Friedlin (Lmj), L. arabica LEM1108 (Lar), L. tarentolae Parrot-TarII (Ltr), L. infantum JPCM5 (Lin), L. amazonensis MHOMBR/71973/M2269 (Laz), L. donovani BPK282A1 (Ldn), L. aethiopica L147 (Lae), L. mexicana U1103 (Lmx), L. panamensis MHOM/COL/81/L13 (Lpn), L. braziliensis M2904 (Lbr), L. enriettii LEM3045 (Len), E. monterogeii LV88 (Emg), C. fasciculata Cf-Cl (Cfs), L. pyrrhocoris H10 (Lpy), L. seymouri ATCC 30220 (Lsy), L. collosoma (unspecified strain) [39] (Lcl), T. cruzi CL Brener Non-Esmeraldo-like, copy of the chromosome 23 (Tcr), T. vivax Y486 (Tvx), T. congolense IL3000 (Tcn), T. evansi STIB 805 (Tev) and T. brucei DAL972 (Tbr). The alignment was obtained with the DNAMAN software, and manually corrected. Arrows indicate the identified pseudouridines (Ψ) in the L. major U2 snRNA (this work). BPRR and Sm sites ar...
Additional file 2: Figure S2. of Transcription of Leishmania major U2 small nuclear RNA gene is directed by extragenic sequences located within a tRNA-like and a tRNA-Ala gene
Northern blot analysis of the tagged U2 snRNA. The experiment was performed with total RNA from c... more Northern blot analysis of the tagged U2 snRNA. The experiment was performed with total RNA from cells transfected with construct 1 (pComp), construct 6 (pBS + 6/+12), the unrelated vector pLMRIB or mock-transfected cells. The probe was the oligonucleotide LmjU2tag-Rev, which specifically recognizes the tag sequence. (TIF 807 kb)
Additional file 4: Figure S4. of Transcription of Leishmania major U2 small nuclear RNA gene is directed by extragenic sequences located within a tRNA-like and a tRNA-Ala gene
Predicted secondary structure of the tRNA-Ala (panel a) and the tRNA-like associated to the L. ma... more Predicted secondary structure of the tRNA-Ala (panel a) and the tRNA-like associated to the L. major U2 snRNA gene (panel b). The minimum free energy structures are shown. The color scale indicates low (blue) to high (red) probabilities of base pairing. (PDF 355 kb)
Research Article Identification of Protein Complex Associated with LYT1 of
Copyright © 2013 C. Lugo-Caballero et al. This is an open access article distributed under the Cr... more Copyright © 2013 C. Lugo-Caballero et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. To carry out the intracellular phase of its life cycle, Trypanosoma cruzimust infect a host cell. Although a few molecules have been reported to participate in this process, one known protein is LYT1, which promotes lysis under acidic conditions and is involved in parasite infection and development. Alternative transcripts from a single LYT1 gene generate two proteins with differential functions and compartmentalization. Single-gene products targeted to more than one location can interact with disparate proteins that might affect their function and targeting properties. The aim of this work was to study the LYT1 interaction map using coimmunoprecipitation assays with transgenic parasites expressing LYT1 products fused to GFP. We d...
BioMed Research International, 2019
Leishmania major, a protozoan parasite that diverged early from the main eukaryotic lineage, exhi... more Leishmania major, a protozoan parasite that diverged early from the main eukaryotic lineage, exhibits unusual mechanisms of gene expression. Little is known in this organism about the transcription factors involved in the synthesis of tRNA, 5S rRNA, and snRNAs, transcribed by RNA Polymerase III (Pol III). Here we identify and characterize the TFIIIB subunit Bdp1 in L. major (LmBdp1). Bdp1 plays key roles in Pol III transcription initiation in other organisms, as it participates in Pol III recruitment and promoter opening. In silico analysis showed that LmBdp1 contains the typical extended SANT domain as well as other Bdp1 conserved regions. Nevertheless, LmBdp1 also displays distinctive features, including the presence of only one aromatic residue in the N-linker region. We were not able to produce null mutants of LmBdp1 by homologous recombination, as the obtained double replacement cell line contained an extra copy of LmBdp1, indicating that LmBdp1 is essential for the viability o...
Biochemical and Biophysical Research Communications, 2016
The role and regulation of actin in Trypanosoma cruzi and other related parasites is largely unkn... more The role and regulation of actin in Trypanosoma cruzi and other related parasites is largely unknown. Based on early genome analysis, it was proposed that there was a reduced dependency on the actomyosin system in the trypanosomatid parasites. However, more recent studies have extended the set of potential actin regulatory proteins, particularly for T. cruzi. One of the identified actin-binding proteins in trypanosomatids is profilin. In other systems, it is capable of simultaneously binding both monomeric actin and several actin-regulatory factors. Hence, the study of profilin and its ligands may help to identify novel pathways in which actin is involved. In T. cruzi, profilin is encoded by a single copy gene. In this work, we demonstrated that this gene is constitutively expressed in both insect and mammalian stages of the parasite, and that the protein is diffusely distributed. Furthermore, we identified some of its potential ligands by LC-MS using GST-profilin pull-down assays of parasite's protein extracts. Many of them were trypanosomatid specific proteins with unknown functions, although proteins from the carbohydrate metabolism, and two metallopeptidases were also detected. As expected, known ligands of profilin in other organisms were identified, including actin, the microtubule components, and the elongation factor 1-alpha. Our work suggests that profilin and the actin system may be regulated by unknown factors and participate in novel biological processes.
Molecular characterization of 5S ribosomal RNA genes and transcripts in the protozoan parasiteLeishmania major
Parasitology, 2016
SUMMARYEukaryotic 5S rRNA, synthesized by RNA polymerase III (Pol III), is an essential component... more SUMMARYEukaryotic 5S rRNA, synthesized by RNA polymerase III (Pol III), is an essential component of the large ribosomal subunit. Most organisms contain hundreds of 5S rRNA genes organized into tandem arrays. However, the genome of the protozoan parasiteLeishmania majorcontains only 11 copies of the 5S rRNA gene, which are interspersed and associated with other Pol III-transcribed genes. Here we report that, in general, the number and order of the 5S rRNA genes is conserved between different species ofLeishmania. While in most organisms 5S rRNA genes are normally associated with the nucleolus, combined fluorescentin situhybridization and indirect immunofluorescence experiments showed that 5S rRNA genes are mainly located at the nuclear periphery inL. major. Similarly, the tandemly repeated 5S rRNA genes inTrypanosoma cruziare dispersed throughout the nucleus. In contrast, 5S rRNA transcripts inL. majorwere localized within the nucleolus, and scattered throughout the cytoplasm, where...
Protist, 2016
Little is known about nucleosome structure and epigenetic regulation of transcription of rRNA gen... more Little is known about nucleosome structure and epigenetic regulation of transcription of rRNA genes in early-branched eukaryotes. Here we analyze the chromatin architecture and distribution of some histone modifications in the rRNA genes in the parasitic protozoon Leishmania major. Southern blots of MNase-partially-digested chromatin with DNA probes spanning the whole rRNA gene repeat showed that the intergenic spacer presents a tight nucleosomal structure, whereas the promoter region is practically devoid of nucleosomes. Intermediate levels of nucleosomes were found in the rRNA coding regions. ChIP assays allowed us to determine that H3K14ac, H3K23ac and H3K27ac, epigenetics marks that are generally associated with activation of transcription, are enriched in the promoter region. In contrast, H4K20me3, which is generally related to transcriptional silencing, was absent from the promoter region and intergenic spacer and enriched in the coding region. Interestingly, the distribution pattern for H3K9me3, generally linked to heterochromatin formation, was very similar to the distribution observed with the euchromatin marks, suggesting that this modification could be involved in transcriptional activation of rRNA genes in L. major.
PLOS ONE, 2015
The present work is aimed at characterizing the motility of parasite T. cruzi in its epimastigote... more The present work is aimed at characterizing the motility of parasite T. cruzi in its epimastigote form. To that end, we recorded the trajectories of two strains of this parasite (a wildtype strain and a stable transfected strain, which contains an ectopic copy of LYT1 gene and whose motility is known to be affected). We further extracted parasite trajectories from the recorded videos, and statistically analysed the following trajectory-step features: step length, angular change of direction, longitudinal and transverse displacements with respect to the previous step, and mean square displacement. Based on the resulting observations, we developed a mathematical model to simulate parasite trajectories. The fact that the model predictions closely match most of the experimentally observed parasite-trajectory characteristics, allows us to conclude that the model is an accurate description of T. cruzi motility.
Identification of Protein Complex Associated with LYT1 of Trypanosoma cruzi
BioMed Research International, 2013
To carry out the intracellular phase of its life cycle,Trypanosoma cruzimust infect a host cell. ... more To carry out the intracellular phase of its life cycle,Trypanosoma cruzimust infect a host cell. Although a few molecules have been reported to participate in this process, one known protein is LYT1, which promotes lysis under acidic conditions and is involved in parasite infection and development. Alternative transcripts from a singleLYT1gene generate two proteins with differential functions and compartmentalization. Single-gene products targeted to more than one location can interact with disparate proteins that might affect their function and targeting properties. The aim of this work was to study the LYT1 interaction map using coimmunoprecipitation assays with transgenic parasites expressing LYT1 products fused to GFP. We detected several proteins of sizes from 8 to 150 kDa that bind to LYT1 with different binding strengths. By MS-MS analysis, we identified proteins involved in parasite infectivity (trans-sialidase), development (kDSPs and histones H2A and H2B), and motility and...
Journal of Biomedicine and Biotechnology, 2010
The parasitesLeishmaniaspp.,Trypanosoma brucei,andTrypanosoma cruziare the trypanosomatid protozo... more The parasitesLeishmaniaspp.,Trypanosoma brucei,andTrypanosoma cruziare the trypanosomatid protozoa that cause the deadly human diseases leishmaniasis, African sleeping sickness, and Chagas disease, respectively. These organisms possess unique mechanisms for gene expression such as constitutive polycistronic transcription of protein-coding genes and trans-splicing. Little is known about either the DNA sequences or the proteins that are involved in the initiation and termination of transcription in trypanosomatids.In silicoanalyses of the genome databases of these parasites led to the identification of a small number of proteins involved in gene expression. However, functional studies have revealed that trypanosomatids have more general transcription factors than originally estimated. Many posttranslational histone modifications, histone variants, and chromatin modifying enzymes have been identified in trypanosomatids, and recent genome-wide studies showed that epigenetic regulation m...
Journal of Biomedicine and Biotechnology, 2010
Trypanosoma cruziundergoes a biphasic life cycle that consists of four alternate developmental st... more Trypanosoma cruziundergoes 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...
Journal of Biomedicine and Biotechnology, 2012
The hemoflagellateTrypanosoma cruziis the causative agent of American trypanosomiasis. Despite th... more The hemoflagellateTrypanosoma cruziis the causative agent of American trypanosomiasis. Despite the importance of motility in the parasite life cycle, little is known aboutT. cruzimotility, 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 characte...
Experimental Parasitology, 2010
Trypanosoma cruzi is an obligate intracellular parasite that infects phagocytic and non-phagocyti... more Trypanosoma cruzi is an obligate intracellular parasite that infects phagocytic and non-phagocytic mammalian cells by a complex process that appears to involve several discrete steps. Even though the infection process was described many years ago, the molecular mechanisms involved remain poorly understood. As fluorescent proteins have proven to be excellent tools for live-cell imaging, we used EGFPand DsRed1-1-transfected trypomastigotes, amastigotes and epimastigotes to study the infection process in living cells. Contrary to what has been reported, our results showed that epimastigotes are as infective as trypomastigotes and amastigotes. Besides, differences in replication, differentiation and parasite release times were observed among the stages. Our results suggest that the different developmental stages use distinct attachment and invasion mechanisms. We propose that fluorescent-based plasmid expression systems are good models for studying the infection process of intracellular microorganisms and could offers insights about the molecular mechanisms involved.
Archives of Medical Research, 2006
Background. Trypanosoma cruzi, the causative agent of Chagas disease, exhibits a complex life cyc... more Background. Trypanosoma cruzi, the causative agent of Chagas disease, exhibits a complex life cycle that is accompanied by the stage-specific gene expression. At the molecular level, very little is known about gene regulation in trypanosomes. Complex gene organizations coupled with polycistronic transcription units make the analysis of regulated gene expression difficult in trypanosomes. The ubiquitin genes of T. cruzi are a good example of this complexity. They are organized as a single cluster containing five ubiquitin fusion (FUS) and five polyubiquitin (PUB) genes that are polycistronically transcribed but expressed differently in response to developmental and environmental changes. Methods. Gene replacements were used to study FUS and PUB gene expression at different stages of growth and at different points in the life cycle of T. cruzi. Results. Based on the levels of reporter gene expression, it was determined that FUS1 expression was downregulated as the parasites approached stationary phase, whereas PUB12.5 polyubiquitin gene expression increased. Conversely, FUS1 expression increases when epimastigotes and amastigotes differentiate into trypomastigotes, whereas the expression of PUB12.5 decreases when epimastigotes differentiate into amastigotes and trypomastigotes. Conclusions. Although the level of CAT activity in logarithmic growing epimastigotes is six-to seven-fold higher when the gene was expressed from the FUS1 locus than when expressed from the PUB12.5 locus, the rate of transcription from the two loci was the same implying that post-transcriptional mechanisms play a dominant role in the regulation of gene expression.
Vet. Méx, 2012
Resumen LYT1 es una molécula con actividad lítica en condiciones ácidas, que según se demostró ge... more Resumen LYT1 es una molécula con actividad lítica en condiciones ácidas, que según se demostró genéticamente, participa en el proceso de infección y transición de estadio de T. cruzi. Su diferente funcionalidad es resultado de la producción de dos proteínas, obtenidas por trans-empalme alternativo, que contienen una secuencia de secreción y una nuclear (LYT1s) o únicamente la secuencia nuclear (LYT1n). Para evaluar la localización de los diferentes productos de LYT1, se analizaron parásitos transgénicos que expresan la ...
Journal of Eukaryotic Microbiology, 1994
ABSTRACT. Parasitic amebas propagate among hosts through cysts, the resistant forms in their life... more ABSTRACT. 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 stag...
BFA-sensitive and insensitive exocytic pathways in Entamoeba histolytica trophozoites: their relationship to pathogenesis
Cellular Microbiology, 2003
Entamoeba histolytica manifests its pathogenicity through several cellular processes triggered by... more Entamoeba histolytica manifests its pathogenicity through several cellular processes triggered by external stimuli that activate signal transduction pathways. The intense secretory activity resulting from stimulation is not correlated with a typical endoplasmic reticulum (ER) or Golgi organization, and little is known in this parasite about endocytic/exocytic pathways. The interactions of trophozoites with fibronectin (FN) and cultured mammalian cells, which elicit secretory activities, were chosen to study mechanisms that regulate cytoplamic traffic. Results showed that Brefeldin A (BFA) induced redistribution of the vesicular network recognized by antibodies against amoebic proteins PDI and ERD2. Furthermore, BFA diminished traffic to the plasma membrane of the beta1 integrin-like FN receptor and the heavy subunit of the Gal/GalNAc lectin, required for adhesion to FN and target cells, respectively. However, BFA did not prevent thiol-proteinase secretion or inhibit the traffic of de novo synthesized proteinases. These data suggest that two distinct transport systems occur in E. histolytica, one similar to classical membrane protein transport and another independent of BFA and inducible by external stimuli. Actin-myosin contractility of the cortical cytoskeleton seems necessary for the final release of exported proteinases and the proper function of the surface proteins involved in adhesion.
Signal Transduction in Entamoeba histolytica Induced By Interaction with Fibronectin
Archives of Medical Research, 2002
Interaction of Entamoeba histolytica trophozoites with extracellular matrix (ECM) proteins activa... more Interaction of Entamoeba histolytica trophozoites with extracellular matrix (ECM) proteins activates signaling pathways through G-protein-coupled receptors. Increments of adenylyl cyclase activity and cAMP produce a striking reorganization of actin into structures that apparently facilitate adhesive, locomotive, and secretory activities. The reorganization of actin is induced by phosphorylation of actin-associated proteins by diverse kinases activated during the signaling process. Although cAMP-dependent kinases have not yet been identified in this parasite, the activation of the adenylyl cyclase route and its effects on particular motility-related functions strongly suggest their presence. Phosphokinase A (PKA) was detected by phosphorylation of the specific substrate, kemptide, its further activation by cAMP, and its inhibition by H89. The catalytic subunit of the enzyme was identified by immunofluorescence microscopy and by immunoprecipitation. Adhesion and damage to cultured cells were monitored by FN-binding and cytotoxicity assays. A cAMP-dependent kinase activated by effectors and agonists of adenylyl cyclase and also during interaction of trophozoites with fibronectin (FN) was found. The enzyme is associated with small granules in the cytoplasm and upon activation, a fraction of its catalytic subunit with an Mr of 100 kDa was translocated to the nucleus, while another fraction was aggregated into big clusters. Activity and translocation were blocked by H89, a specific inhibitor of PKA. Trophozoites stimulated by dBcAMP or forskolin-formed lamellae and restructured actin, but no significant increase in their adhesion to FN was observed and only showed 10% stimulus in their capacity to damage target cells. Treatment with H89 decreased adhesion to 40% and caused 80% inhibition in cell damage. These amebas showed altered organization of the actin structures induced by dBcAMP or FN. Our results support previous suggestions concerning the participation of PKA in the response elicited by the interaction of E. histolytica trophozoites with ECM proteins. They also indicate that adhesion and secretion in conjunction with motile activities are related to invasion processes.