Norbel Galanti

Chromosome specific markers reveal conserved linkage groups in spite of extensive chromosomal size variation in Trypanosoma cruzi

The karyotypes of three cloned stocks, CL Brener (CL), CA I/72 (CA) and Sylvio X10/7 (X10), of Trypanosoma cruzi were studied by pulsed-field gel electrophoresis followed by ethidium bromide staining and hybridization with 35 different probes, 30 of which identified single chromosomes. The chromosome-specific probes identified between 26 and 31 chromosomal bands in the three cloned stocks, corresponding to 20 unique chromosomes in CL and 19 in CA and X10. Considering the DNA content of the parasite, it was predicted that the markers recognise at least half of all T. cruzi chromosomes. A majority of identified chromosomes showed large differences in size among different strains, in some cases by up to 50%. Interestingly, CL had in general larger chromosomes than the two other studied cloned stocks. Several of the markers showed linkage and nine different linkage groups were identified, each comprising 2-4 markers. The linkage between the markers was maintained in 8 of the 9 linkage groups when a panel comprising 26 different T. cruzi strains representing major T. cruzi populations was tested. One linkage group was found to be maintained in some strains but not in others. This result shows that chromosomal rearrangements occur in the T. cruzi genome, albeit with a low frequency. Repetitive DNA, both non-coding and in one case coding, was more abundant in the cloned stock CL Brener than in CA and X10. The information presented will make it possible to select chromosomes for the construction of physical chromosomal maps required for the T. cruzi genome project.

A gene family encoding heterogeneous histone H1 proteins in Trypanosoma cruzi.

A gene family encoding a set of histone H1 proteins in Trypanosoma cruzi is described. The sequence of 3 genomic and 4 cDNA clones revealed the presence of several motifs characteristic of histone H1, although heterogeneity at the polypeptide level was evident. The clones encode histone H1 proteins of an unusually small size (74-97 amino acids), which lack the globular domain found in histone H1 of higher eukaryotes. All histone H1 mRNAs from T. cruzi are polyadenylated, although no typical polyadenylation signal was found. Furthermore, the genes encoding the histone H1 proteins in T. cruzi are found in a tandem array containing 15-20 gene copies per haploid genome. This tandem array is located on a large chromosome of 2.2 Mb.

Histone genes in trypanosomatids.

Histone genes in Trypanosomatids are of considerable interest because these flagellates do not condense their chromatin during mitosis. In contrast to higher eukaryotes, histone genes in Trypanosomatids are found on separate chromosomes, and their transcripts are polyadenylated. Sequence similarity of Trypanosomatid core histones with those of higher eukaryotes is found predominantly in the globular region; the N-terminal is highly divergent. Finally, in general, Trypanosomatid histones H1 are of low molecular weight, bearing closest homology to the C-terminal region of the higher eukaryote histones H1. These features constitute interesting targets for a rational approach to the study of these protozoa, as discussed here by Norbel Galanti and colleagues.

Productive infection of Piscirickettsia salmonis in macrophages and monocyte-like cells from rainbow trout, a possible survival strategy.

Piscirickettsia salmonis is the etiologic agent of the salmonid rickettsial septicemia (SRS), an endemic disease which causes significant losses in salmon production. This intracellular bacterium is normally cultured in salmonid epithelial cell lines inducing characteristic cytopathic effects (CPEs). In this study we demonstrate that P. salmonis is able to infect, survive, replicate, and propagate in the macrophages/monocytes cell line RTS11 derived from rainbow trout spleen, without inducing the characteristic CPEs and the host cells showing the same expression levels as non‐infected control cell. On the other hand, bacteria were capable of expressing specific proteins within infected cells. Infected macrophages cease proliferation and a fraction of them detached from the plate, transform to non‐adhesive, monocyte‐like cells with proliferative activity. Productive infection of P. salmonis into salmonid macrophage/monocyte cells in culture provides an excellent model for the study of host–pathogen interactions, almost unknown in the case of P. salmonis. Our results suggest that the infection of cells from the salmonid innate immune system without inducing an important cell death response should lead to the persistence of the bacteria and consequently their dissemination to other tissues, favoring the evasion of the first line of defense against pathogens. J. Cell. Biochem. 108: 631–637, 2009.

H 1 histone and histone variants in Trypanosoma cruzi

Trypanosoma cruzi chromatin is not condensed in chromosomes during mitosis. In previous studies a characteristic H 1 was not found in SDS or in acid-urea-PAGE. Consequently, it was proposed that the particular behavior of T. cruzi chromatin in dividing cells was due to the absence of an H 1 histone. In the present work, histones from this parasite were systematically characterized by spectrofluorometric analysis, amino acid composition, PAGE in one and in two dimensions, differential extraction with PCA and TCA, immunological cross-reactivity with antisera, and immunoblotting. We conclude that T. cruzi contains all five histones, H 1 presenting solubility and immunological properties similar to those in other species, but with a particular electrophoretic mobility in Triton-PAGE. Thus an explanation other than the absence of H 1 should be offered in order to understand the behavior of T. cruzi chromatin during mitosis. Moreover, histone variants were described by two-dimensional PAGE. The presence of histone variants suggests that they may participate in the regulation of cell proliferation and differentiation of this parasite, as it has been postulated for higher eukaryotes.

Cellular DNA replication is independent of the synthesis or accumulation of ribosomal RNA.

We have used an antibody against RNA polymerase I to investigate the role of rRNA synthesis and/or accumulation in the control of cell proliferation. The antibody was microinjected directly into the nuclei of quiescent Swiss 3T3 cells that were subsequently stimulated with serum. Under the experimental conditions used, the microinjection of the antibody against RNA polymerase I (RNA pol I) caused a 50-70% decrease in nucleolar RNA synthesis that lasted for at least 17 h, a greater than 90% inhibition in the accumulation of nucleolar RNA, and a 70% inhibition in the accumulation of total cellular RNA. A control IgG, similarly microinjected into Swiss 3T3 cells had no inhibitory effect on either the synthesis or accumulation of nucleolar and cellular RNA. Despite the dramatic effect on the synthesis and accumulation of ribosomal RNA (rRNA) the antibody against RNA (rRNA) the antibody against RNA pol I was totally ineffective in inhibiting the entry into S phase of serum-stimulated Swiss 3T3 cells. Cells depleted of cellular RNA by metaphase arrest also entered S phase with subnormal amounts of cellular RNA. The results of these experiments clearly indicate that a normal rate of nucleolar RNA synthesis, and a normal rate of accumulation of total cellular RNA are not a prerequisite for the entry of cells into S phase.

Piscirickettsia salmonis induces apoptosis in macrophages and monocyte-like cells from rainbow trout

Piscirickettsia salmonis is the etiologic agent of the salmonid rickettsial septicemia (SRS) which causes significant losses in salmon production in Chile and other and in other regions in the southern hemisphere. As the killing of phagocytes is an important pathogenic mechanism for other bacteria to establish infections in vertebrates, we investigated whether P. salmonis kills trout macrophages by apoptosis. Apoptosis in infected macrophages was demonstrated by techniques based on morphological changes and host cell DNA fragmentation. Transmission electron microcopy showed classic apoptotic characteristics and terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling showed fragmented DNA. Programmed cell death type I was further confirmed by increased binding of annexin V to externalized phosphatidylserine in infected macrophages. Moreover, significant increases of caspase 3 activation were detected in infected cells and treatment with caspase inhibitor caused a decrease in levels of apoptosis. This is the first evidence that P. salmonis induces cell death in trout macrophages. This could lead to bacterial survival and evasion of the host immune response and play an important role in the establishment of infection in the host. J. Cell. Biochem. 110: 468–476, 2010.

Apoptosis as a possible mechanism of infertility in Echinococcus granulosus hydatid cysts

Echinococcus granulosus is a parasitic cestode causing hydatidosis in intermediate hosts (human and herbivorous). Most symptoms of the disease occur by the pressure exerted on viscera by cysts that are formed upon ingestion of the parasite eggs excreted by definitive hosts (canines). Protoscoleces, the developmental form of the parasite infective to definitive hosts, are formed in the germinal nucleated layer of fertile hydatid cysts. For unknown reasons, some cysts are unable to produce protoscoleces (infertile hydatid cysts). In this study, analysis of DNA fragmentation using TUNEL and agarose gel electrophoresis showed higher levels of apoptosis in infertile cysts as compared to fertile cysts. Additionally, caspase 3 was detected both in fertile and infertile cysts; the activity of this enzyme was found to be higher in infertile cysts. We conclude that apoptosis may be involved in hydatid cyst infertility. This is the first report on the presence of programmed cell death in E. granulosus. J. Cell. Biochem. 100: 1200–1209, 2007.

IN VITRO SEGMENTATION INDUCTION OF MESOCESTOIDES CORTI (CESTODA) TETRATHYRIDIA

Mesocestoides corti is a suitable model for studying cestode development because of its ability to reproduce asexually and segment in vitro. The cultured parasite is also capable of sexual differentiation and, probably, reproduction. To establish conditions that increase the efficiency of in vitro M. corti larvae (tetrathyridia) segmentation, we tested the effects of an inducing agent and some physical parameters in cultures. We found that a 5% CO2–95% N2 gas phase, an incubation temperature of 39 C (instead of 37 C), and a 24-hr pretreatment with trypsin (105 BAEE/ml, BAEE = Nα-benzoil-l-arginine ethyl ester unit of trypsin activity) in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 20% fetal bovine serum (FBS) are able to increase individually or synergistically the segmentation rate of tetrathyridia. A segmentation rate of up to 100% was achieved on day 4 of culture, when all these conditions were used simultaneously, in comparison with an average rate of 40% obtained not before day 11 in cultures without any inducing treatment. Fetal bovine serum is essential for segmentation, and a concentration of 20% was established as the standard for induction.

Natural Sesquiterpene Lactones are Active Against Leishmania mexicana

In this paper, the effects of 3 natural sesquiterpene lactones, i.e., helenalin (Hln), mexicanin (Mxc), and dehydroleucodine (DhL), were evaluated using cultured Leishmania mexicana promastigotes. It was observed that the compounds inhibited the in vitro growth of the parasites at relatively low concentrations. The effect was rapid and irreversible with an estimated IC50 of 2–4 μM, while all the lactones were more effective than ketoconazole. Moreover, these compounds exhibited low cytotoxicity for mammalian cells. Hln induced strong vacuolization of the parasite cytoplasm, although pericellular microtubules were preserved. The 3 lactones induced DNA fragmentation as judged by the high labeling with the fluorescent TUNEL method, which was confirmed by electrophoresis on agarose gels. The ability of the parasites to invade Vero cells was also decreased by exposure to low concentrations of the compounds. We conclude that these compounds can affect the parasites life cycle, possibly through multiple mechanisms. Identification of the molecular targets of these natural products and their effects on amastigotes should be determined to evaluate the possible therapeutic use of the compounds against leishmaniasis.