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PubMed1994

Molecular evidence that the myxozoan protists are metazoans.

The evolutionary origins of the protistan phylum, Myxozoa, have long been questioned. Although these obligate parasites are like protozoans in many features, several aspects of their ontogeny and morphology have implied a closer relationship to metazoan lineages. Phylogenetic analyses of 18S ribosomal RNA sequences from myxozoans and other eukaryotes, with the use of parsimony, distance, and maximum-likelihood methods, support the hypothesis that myxozoans are closely related to the bilateral animals. These results suggest that the Myxozoa, long considered an assemblage of protozoans, should be considered a metazoan phylum.

Animals↗
PubMed1997

Morphological and compositional shifts in an experimental bacterial community influenced by protists with contrasting feeding modes.

In a two-stage continuous-flow system, we studied the impacts of different protozoan feeding modes on the morphology and taxonomic structure of mixed bacterial consortia, which were utilizing organic carbon released by a pure culture of a Rhodomonas sp. grown on inorganic medium in the first stage of the system. Two of three second stages operated in parallel were inoculated by a bacterivorous flagellate, Bodo saltans, and an algivorous ciliate, Urotricha furcata, respectively. The third vessel served as a control. In two experiments, where algal and bacterial populations grew at rates and densities typical for eutrophic waters, we compared community changes of bacteria, algae, and protozoa under quasi-steady-state conditions and during the transient stage after the protozoan inoculation. In situ hybridization with fluorescent oligonucleotide probes and cultivation-based approaches were used to tentatively analyze the bacterial community composition. Initially the cell size distribution and community structure of all cultivation vessels showed similar patterns, with a dominance of 1- to 2.5-(mu)m-long rods from the beta subdivision of the phylum Proteobacteria ((beta)-Proteobacteria). Inoculation with the ciliate increased bacterial growth in this substrate-controlled variant, seemingly via a recycling of nutrients and substrate released by grazing on algae, but without any detectable effect on the composition of bacterial assemblage. In contrast, an inoculation with the bacterivore, B. saltans, resulted in a decreased proportion of the (beta)-Proteobacteria. One part of the assemblage (<4% of total bacterial numbers), moreover, produced large grazing-resistant threadlike cells. As B. saltans ingested only cells of <3 (mu)m, this strategy yielded a refuge for (symbl)70% of total bacterial biomass from being grazed. Another consequence of the heavy predation in this variant was a shift to the numerical dominance of the (alpha)-Proteobacteria. The enhanced physiological status of the heavily grazed-upon segment of bacterial community resulted in a much higher proportion of CFU (mean, 88% of total bacterial counts) than with other variants, where CFU accounted for (symbl)30%. However, significant cultivation-dependent shifts of the bacterial community were observed toward (gamma)-Proteobacteria and members of the Cytophaga/Flavobacterium group, which demonstrated the rather poor agreement between cultivation-based approaches and oligonucleotide probing.

Journal Article↗
PubMed2006

Direct and indirect effects of protist predation on population size structure of a bacterial strain with high phenotypic plasticity.

We studied the impact of grazing and substrate supply on the size structure of a freshwater bacterial strain (Flectobacillus sp.) which showed pronounced morphological plasticity. The cell length varied from 2 to >40 microm and encompassed rods, curved cells, and long filaments. Without grazers and with a sufficient substrate supply, bacteria grew mainly in the form of medium-sized rods (4 to 7 microm), with a smaller proportion (<10%) of filamentous forms. Grazing experiments with the bacterivorous flagellate Ochromonas sp. showed that freely suspended cells of <7 microm were highly vulnerable to grazers, whereas filamentous cells were resistant to grazing and became enriched during predation. A comparison of long-term growth in carbon-limited chemostats with and without grazers revealed that strikingly different bacterial populations developed: treatments with flagellates were composed of >80% filamentous cells. These attained a biomass comparable to that of populations in chemostats without grazers, which were composed of medium-sized rods and c-shaped cells. Carbon starvation resulted in a fast decrease in cell length and a shift towards small rods, which were highly vulnerable to grazing. Dialysis bag experiments in combination with continuous cultivation revealed that filament formation was significantly enhanced even without direct contact of bacteria with bacterivores and was thus probably stimulated by grazer excretory products.

Animals↗
PubMed2002

Methods for cultivation of luminal parasitic protists of clinical importance.

Cultivation of luminal protistan parasites has a long history. In this review we discuss the methods and media that are most widely used for the establishment and maintenance of the following organisms in culture: Entamoeba histolytica, Giardia intestinalis, Trichomonas vaginalis, Dientamoeba fragilis, Blastocystis hominis, and Balantidium coli. While cultivation is of limited importance in the diagnostic laboratory, it is essential to most research laboratories, and it is toward the latter that this review is primarily aimed.

Animals↗
PubMed2006

Transmembrane molecules for phylogenetic analyses of pathogenic protists: Leishmania-specific informative sites in hydrophilic loops of trans- endoplasmic reticulum N-acetylglucosamine-1-phosphate transferase.

A sequence database was created for the Leishmania N-acetylglucosamine-1-phosphate transferase (nagt) gene from 193 independent isolates. PCR products of this single-copy gene were analyzed for restriction fragment length polymorphism based on seven nagt sequences initially available. We subsequently sequenced 77 samples and found 19 new variants (genotypes). Alignment of all 26 nagt sequences is gap free, except for a single codon addition or deletion. Phylogenetic analyses of the sequences allow grouping the isolates into three subgenera, each consisting of recognized species complexes, i.e., subgenus Leishmania (L. amazonensis-L. mexicana, L. donovani-L. infantum, L. tropica, L. major, and L. turanica-L. gerbilli), subgenus Viannia (L. braziliensis, L. panamensis), and one unclassified (L. enriettii) species. This hierarchy of grouping is also supported by sequence analyses of selected samples for additional single-copy genes present on different chromosomes. Intraspecies divergence of nagt varies considerably with different species complexes. Interestingly, species complexes with less subspecies divergence are more widely distributed than those that are more divergent. The relevance of this to Leishmania evolutionary adaptation is discussed. Heterozygosity of subspecies variants contributes to intraspecies diversity, which is prominent in L. tropica but not in L. donovani-L. infantum. This disparity is thought to result from the genetic recombination of the respective species at different times as a rare event during their predominantly clonal evolution. Phylogenetically useful sites of nagt are restricted largely to several extended hydrophilic loops predicted from hypothetical models of Leishmania NAGT as an endoplasmic reticulum transmembrane protein. In silico analyses of nagt from fungi and other protozoa further illustrate the potential value of this and, perhaps, other similar transmembrane molecules for phylogenetic analyses of single-cell eukaryotes.

Amino Acid Sequence↗
PubMed2003

Cysteine proteases and cell differentiation: excystment of the ciliated protist Sterkiella histriomuscorum.

The process of excystment of Sterkiella histriomuscorum (Ciliophora, Oxytrichidae) leads in a few hours, through a massive influx of water and the resorption of the cyst wall, from an undifferentiated resting cyst to a highly differentiated and dividing vegetative cell. While studying the nature of the genes involved in this process, we isolated three different cysteine proteases genes, namely, a cathepsin B gene, a cathepsin L-like gene, and a calpain-like gene. Excystation was selectively inhibited at a precise differentiating stage by cysteine proteases inhibitors, suggesting that these proteins are specifically required during the excystment process. Reverse transcription-PCR experiments showed that both genes display differential expression between the cyst and the vegetative cells. A phylogenetic analysis showed for the first time that the cathepsin B tree is paraphyletic and that the diverging S. histriomuscorum cathepsin B is closely related to its Giardia homologues, which take part in the cyst wall breakdown process. The deduced cathepsin L-like protein sequence displays the structural signatures and phylogenetic relationships of cathepsin H, a protein that is known only in plants and animals and that is involved in the degradation of extracellular matrix components in cancer diseases. The deduced calpain-like protein sequence does not display the calcium-binding domain of conventional calpains; it belongs to a diverging phylogenetic cluster that includes Aspergillus palB, a protein which is involved in a signal transduction pathway that is sensitive to ambient pH.

Amino Acid Sequence↗
PubMed1999

Regulation and adaptation of glucose metabolism of the parasitic protist Leishmania donovani at the enzyme and mRNA levels.

Adaptation of the glucose metabolism of Leishmania donovani promastigotes (insect stage) was investigated by simultaneously measuring metabolic rates, enzyme activities, message levels, and cellular parameters under various conditions. Chemostats were used to adapt cells to different growth rates with growth rate-limiting or excess glucose concentrations. L. donovani catabolized glucose to CO(2), succinate, acetate, and pyruvate in ratios that depended on growth rate and glucose availability. Rates of glucose consumption were a linear function of growth rate and were twice as high in excess glucose-grown cells as in glucose-limited organisms. The major end product was CO(2), but organic end products were also formed in ratios that varied strongly with growth conditions. The specific activities of the 14 metabolic enzymes measured varied by factors of 3 to 17. Two groups of enzymes adapted specific activities in parallel, but there was no correlation between the groups. The activities of only one group correlated with specific rates of glucose metabolism. Total RNA content per cellular protein varied by a factor of 6 and showed a linear relationship with the rate of glucose consumption. There was no correlation between steady-state message levels and activities of the corresponding enzymes, suggesting regulation at the posttranscriptional level. A comparison of the adaptation of energy metabolism in L. donovani and other species suggests that the energy metabolism of L. donovani is inefficient but is well suited to the environmental challenges that it encounters during residence in the sandfly, its insect vector.

Adaptation, Physiological↗
PubMed2000

Improvement of silver impregnation technique (protargol) to obtain morphological features of protists ciliates, flagellates and opalinates.

The research on ciliates, flagelates and opalinates have been widespread by the utilization of techniques employing silver impregnation (protargol), modified by several authors. However, these are time consuming and its results are variable. The present work is a variant of the technique described by Tuffrau (1964, 1967) showing some adaptations made in our laboratory. The organisms can be preserved by different fixatives (alcoholic Bouin, Stieve's fluid, 2.5% glutaraldehyde and others) and then rinsed in destilled water followed by a fast clarification by 3% sodium hypochloride. If the organism is very sensitive to hypochloride, 4% sodium lauryl sulfate may be used and then washed 3 times in distilled water. The protista can be adhered to the glass slides with Mayer's glycerinated-albumin (1 glycerin vol. to 1 or 2 albumin vol.), diluted in water at a proportion of 1:10 Cv/v., or with 1% polylysine followed by fast washes with distilled water. After the slide preparation, they were covered with a layer of 0,8% Silver proteinate. Right after that, the slide has to be placed in a glass tray lined with moist tissue and covered to prevent the proteinate to dry. The tray was placed in a incubator at 40 degrees - 50 degrees C for 30 minutes. The slides are rinsed for 1 minute. with warm (35 degrees C) distilled water. The development of the material should be done with 0.4% hydroquinone with a maximum incubation time of 1 minute. It should be developed gradually, controlling the silver impregnation intensity by observation under optical microscope. Next, rinse in distilled water for 1 minute, and then, fix in 2,5% Sodium thiosulfate. Rinse the slide for two minutes before dehydrating it in an alcoholic serial 50-100 degrees. Finally rinse the slides in xylene. Mount the slides with Entellan MerckTM or Canada balsam.

Animals↗