Search

Promastigote forms of Phytomonas serpens, Leptomonas samueli, and Leishmania tarentolae express cytoskeletal giant proteins with apparent molecular masses of 3,500 kDa (Ps 3500), 2,500 kDa (Ls 2500), and 1,200 kDa (Lt 1200). respectively. Polyclonal antibodies to it 1200 and to Ps 3500 specifically recognize similar polypeptides of the same genera of parasite. in addition to reacting with giant polypeptides of the Leptomonas species, anti-is 2500 also cross reacts with Ps 3500, and with a 500-kDa polypeptide of Leishmania. Confocal immunofluorescence and immunogold electron microscopy showed major differences in topological distribution of these three proteins, though they partially share a common localization at the anterior end of the cell body skeleton. Furthermore, Ps 3500. Ls 2500, and it 1200 are in vivo phosphorylated at serine and threonine residues, whereas, in vitro phosphorylation of cytoskeletal fractions reveal that only Ps 3500 and Ls 2500 are phosphorylated. Heat treatment (100 degrees C) of high salt cytoskeletal extracts demonstrates that Ps 3500 and Ls 2500 remain stable in solution, whereas it 1200 is denatured. Kinase assays with immunocomplexes of heat-treated giant proteins show that only Ps 3500 and Ls 2500 are phosphorylated. These results demonstrate the existence of a novel class of megadalton phosphoproteins in promastigote forms of trypanosomatids that appear to be genera specific with distinct cytoskeletal functions. in addition, there is also evidence that Ps 3500 and is 2500, in contrast to it 1200, seem to be autophosphorylating serine and threonine protein kinases, suggesting that they might play regulatory roles in the cytoskeletal organization. (C) 2000 Wiley-Liss, Inc. Univ São Paulo, Dept Parasitol, Inst Ciencias Biomed, BR-05508900 São Paulo, Brazil Universidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, Escola Paulista Med, São Paulo, Brazil Universidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, Escola Paulista Med, São Paulo, Brazil Web of Science

In order to review the taxonomy of the genus Herpetomonas through phylogenetic and morphological analyses we barcoded 527 insect trypanosomatids by sequencing the V7V8 region of the small subunit ribosomal RNA (SSU rRNA) gene. Fifty two flagellates, 90% of them from Diptera, revealed to be related to known species of Herpetomonas. Sequences of entire glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) and SSU rRNA genes were employed for phylogenetic inferences including representatives of all genera of Trypanosomatidae. In the resulting phylogenetic trees, the selected flagellates clustered into a monophyletic assemblage that we are considering as the redefined genus Herpetomonas. Internal transcribed spacer 1 (ITS1) rDNA sequences and putative secondary structures of this region were compared for evaluation of inter- and intraspecific variability. The flagellates were classified in six already known species and five new species. In addition, two Leptomonas spp. were moved to Herpetomonas, now comprising 13 valid species, while four species were excluded from the genus. Light and electron microscopy revealed the extreme polymorphism of Herpetomonas, hindering genus and species identification by morphological characteristics. Our findings also showed that some species of Herpetomonas are generalist parasites of flies and appear to be as cosmopolitan as their hosts.

Apoptosis and/or programmed cell death have been described in examples ranging from fungi to man as gene-regulated processes with roles in cell and tissue physiopathology. These processes require the operation of an intercellular communicating network able to deliver alternative signals for cells with different fates and is thus considered a prerogative of multicellular organisms. Promastigotes from Leishmania (Leishmania) amazonensis, when shifted from their optimal in vitro growth temperature (22 degrees C) to the temperature of the mammalian host (37 degrees C), die by a calcium-modulated mechanism. More parasites die in the presence of this ion than in its absence, as detected by a colorimetric assay based on the activity of mitochondrial and cytoplasmic dehydrogenases which measures cell death, independently of the process by which it occurs. A heat shock, unable to induce detectable parasite death (34 degrees C for 1 h), is able to significantly raise the concentration of intracellular free calcium in these cells. Heat-shocked parasites present ultrastructural and molecular features characteristic of cells dying by apoptosis. Morphological changes, observed only in the presence of calcium, are mainly nuclear. Cytoplasmic organelles are preserved. Heat shock is also able to induce DNA cleavage into an oligonucleosomal ladder detected in agarose gels by ethidium bromide staining and autoradiography of [alpha 32P]ddATP-labeled fragments. These results indicate that death by apoptosis is not exclusive of multicellular organisms.

Summary Morphological events leading to cyst formation in a cyst-bearing Leptomonas sp. isolated from the hemipteran Oncopeltus varicolor were monitored by light and electron microscopy. In LIT cultures, cyst-bearing flagellates appear at the end of the lag phase and from then on the number of detached mature cysts increases steadily. The cysts develop at the point of the flagellum-cell body junction from the parental flagellate. Plasma-membranes of flagellate and cysts remain connected while they remain attached. Immature cysts present microtubules and a large tubular mitochondrion. Mature cysts are dense, ovalshaped, with few distinguishable organelles, such as a modified nucleus, a rudimentary flagellar pocket and a network of fibrils possibly of kinetoplast origin. During excystation, cysts gradually regain the missing organelles and morphological features of active promasti-gotes. Even cysts, with a very dense and distinct chromatin, retain the ability to undergo cell division in culture.

SUMMARYTwo domestic guinea-pigs (Cavia porcellus), bought in Pinheiros, São Paulo State, Brazil, were taken by their owners a farm in the rural district of Capão Bonito, close to the Atlantic Forest, São Paulo, where they both developed tumour-like and ulcerating lesions on the ears. The causative agent was identified as Leishmania (L.) enriettii, based on biological characters and isoenzyme profiles. Sources of the parasite in wild mammals, and the possible sandfly vector species discussed.

We comparatively examined the nutritional, molecular and optical and electron microscopical characteristics of reference species and new isolates of trypanosomatids harboring bacterial endosymbionts. Sequencing of the V7V8 region of the small subunit of the ribosomal RNA (SSU rRNA) gene distinguished six major genotypes among the 13 isolates examined. The entire sequences of the SSU rRNA and glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) genes were obtained for phylogenetic analyses. In the resulting phylogenetic trees, the symbiont-harboring species clustered as a major clade comprising two subclades that corresponded to the proposed genera Angomonas and Strigomonas. The genus Angomonas comprised 10 flagellates including former Crithidia deanei and C. desouzai plus a new species. The genus Strigomonas included former Crithidia oncopelti and Blastocrithidia culicis plus a new species. Sequences from the internal transcribed spacer of ribosomal DNA (ITS rDNA) and size polymorphism of kinetoplast DNA (kDNA) minicircles revealed considerable genetic heterogeneity within the genera Angomonas and Strigomonas. Phylogenetic analyses based on 16S rDNA and ITS rDNA sequences demonstrated that all of the endosymbionts belonged to the Betaproteobacteria and revealed three new species. The congruence of the phylogenetic trees of trypanosomatids and their symbionts support a co-divergent host-symbiont evolutionary history.

The course of experimental oral infection of Musca domestica with Herpetomonas samuelpessoai and two new isolates from houseflies was followed for up to 40 days using optical and electron microscopy. The fly's rectum was found to be the preferential site of colonization by flagellates, mainly at places near papillar insertions but not on the papillae themselves. Midgut infection was transient, and infection of the crop did not last longer than 1 week. Promastigotes were the predominant colonizing forms and could be seen either free in the gut lumen or attached via hemidesmosomes to the rectum wall. Adherent flagellates exhibited flagellar projections and surface membrane blebs. Many flagellates displayed longitudinal body foldings yielding star-like images in cross sections. Opisthomastigotes could be seen in small numbers as free swimmers, mainly in the rectum.

Summary Electron microscopy of thin sections and detergent extracted preparations of certain trypanosomatids of the genus Phytomonas revealed the unusual presence in some flagellates of an intracytoplasmic flagellum which appears as a naked axoneme encircling the nucleus before emerging at the anterior end of the cell. Another feature of these atypical flagellates is the absence of the flagellar pocket normally present in trypanosomatids at the emergence of the flagellum.

The Brindley's glands of Panstrongylus megistus were studied under the antomic, histologic and ultrastructural point of view. These glands located in the insect's methatorax are paired and have an opening near the third parir of the feet. Beside this aperture, ther are evaporation areas. Shape, sixe and aspect of the gland vary according to the feeding status. The glands are composed by a tubular part corresponding to the duct and a sack-like portion corrsponding to the secretory part. By electron microscopy we observed that the basal part of the epithelium has many interdigitations associated with mitochondria. On the apical surface where epicuticular foldings are located an electonlucent space is often seen. The glands are composed of the following elements: 1) superficial epithelial cells, located just below the apical surface foldings; 2) secretory cells; which are long and have an intracellular canalicule which changes according to the functional state of the cell; 3) a collecting duct to the secretory cells and covered with an epicuticle, reaching up to the gland's lumen; and 4) cells around the duct.

We detected and cultivated isolates of Trypanosoma (Megatrypanum) theileri from cattle and water buffaloes in Sao Paulo state, southeastern Brazil, which were characterized by comparing morphological, growth and molecular features. Although isolates from cattle and water buffalo were morphologically indistinguishable, they differed in their growth characteristics. A dendrogram based on randomly amplified polymorphic DNA (RAPD) patterns indicated close-genetic relationships among all isolates from both species, which were all tightly clustered together and distant from Megatrypanum species from wild mammals. In addition, isolates within the T. theileri-cluster were clearly segregated into two host-associated groups. The sequence of a synapomorphic RAPD-derived DNA fragment (Tth625), which was shared by all T. theileri trypanosomes from cattle and buffalo but not detected in any of 13 other trypanosome species, was used as target for a conventional T. theileri-specific PCR assay. We also defined RAPD fragments (Tthc606 and Tthb606) that distinguished cattle from buffalo isolates. Thus, distinct growth features and genetic variability distinguished between isolates from cattle and water buffaloes of the same geographic origin, suggesting an association of these isolates with their host species. The trypanosomes from water buffalo reported here are the first T. theileri-like isolates from the Asiatic buffalo (Bubalus bubalis) to be continuously cultured and compared with cattle isolates using biological and molecular methods.

Leishmania (Leishmania) amazonensis LV79 (MPRO/BR/72/M1841) has been adapted to grow at 33 C as amastigote-like (AL) organisms in modified UM-54 medium initially adjusted to a pH of 4.8-5.0. Axenic cultures could be routinely restarted from parasites recovered from footpad lesions obtained by inoculation of BALB/c mice with preadapted culture stages. Morphological features, proteinase activities, and infectivity of AL organisms were examined during the in vitro growth cycle, and differences were found between log- and stationary-phase parasites. Stationary-phase AL organisms were morphologically similar to lesion amastigotes, did not react with a paraflagellar rod-specific monoclonal antibody in western blots, and contained proteinase activities resolving identically to the enzymes of lesion amastigotes in gelatin gels. Whereas typical megasomes could be identified in about a third of the stationary-phase AL population, the organelles were rarely seen in log-phase organisms. Azocaseinolytic activity progressively increased during the exponential growth phase and reached its highest values (approximately 65-70% of those determined in lesion amastigotes) at the stationary phase; the association of total proteinase activity with increased expression of cysteine proteinases was indicated by the strong inhibition of azocasein hydrolysis by E-64, the intensified banding of the 28-, 31-, and 35-kDa proteinases in gelatin gels, and the higher susceptibility of stationary-phase AL organisms to L-leucine methyl ester. Although overall axenic amastigotes were less infective to BALB/c mice than were lesion-derived parasites, stationary-phase AL organisms were more infective than were log-phase parasites. Medium pH increased during the exponential growth phase, but dropped in the stationary phase, when the observed morphological, biochemical, and biological changes became apparent.

A giant protein with an apparent molecular mass of 2,300-kDa was identified in the Triton X-100 soluble fraction of Crithidia luciliae thermophila. Polyclonal antibody raised against this protein reacted by immunoblot analysis with proteins of similar molecular mass in Crithidia fasciculata and Crithidia oncopelti. In addition, the antibody immunoprecipitates the protein either after in vivo phosphorylation with [32P]orthophosphoric acid or after metabolically labeling with [35S]methionine. Indirect immunofluorescence microscopy analysis performed either with fixed or with live parasites showed a single fluorescent spot at the level of the flagellar pocket region. Immunogold electron microscopy of thin sections of the parasite revealed that the antigen is localized at a restricted area of the spongiome, between the contractile vacuole and the flagellar pocket. Furthermore, Triton X-114 phase separation of whole cell membrane proteins, metabolically labeled with [35S]methionine, demonstrated that the giant protein remains in the aqueous phase. These results indicate that this phosphoprotein behaves as a peripheral membrane protein localized at the spongiome region, suggesting that it might be involved in the osmoregulatory process.

Experimental infections of the phytophagous Hemiptera Dysdercus peruvianus with different trypanosomatids were studied for up to 55 days by light microscopy while the course of infection with Leptomonas seymouri and the Leptomonas isolate 49/553G.O. was analyzed by electron microscopy. Rates of infection of D. peruvianus varied according to the infecting flagellate. The lower part of the midgut was found to be the preferential site of colonization where most flagellates were found isolated or arranged in clumps or rosettes. Specialized junctional structures with host cells were never observed. Flagellates could also be seen inside midgut cells within a parasitophorous vacuole. Infection of haemocoele and salivary glands was also observed.

Macrófagos obtidos do peritoneo de camundongos após estímulo, com peptona, foram cultivados em lamínulas, infectados com tripomastigotas das cepas F e Y de T. cruzi, obtidos de cultivo de tecidos ou do sangue de camundongos infectados. Os parasitas, obtidos de cultivo de tecidos, tanto da cepa Y como os da cepa F, são interiorizados por macrófagos em proporção muito mais elevada do que os sanguícolas. Parasitas de cultivo de tecidos incubados com soro de camundongos normais, ou soro híperimune específico em diluição sub-aglutinante, comportam-se essencialmente como parasitas não opsonizados. Foram observadas diferenças a nível ultraestrutural na fase inicial de interação entre macrófagos e tripomastigotas das duas origens. Após 30 minutos, tripomastigotas de cultivo de tecidos localizam-se em agrupamentos na área de contato com os macrófagos. Enquanto os tripomastigotas sanguícolas estão na maioria das vezes no interior de vacúolos fagocíticos largos, após 3 horas de interação os tripomastigotas de cultura situam-se em um único vacúolo estreito. Tanto as formas de cultivo de tecidos quanto os tripomastigotas sanguícolas da cepa Y multiplicam-se em macrófagos; os tripomastigotas sanguícolas da cepa F são destruídos no interior da célula hospedeira, enquanto os tripomastigotas de cultivo de tecidos desta cepa são capazes de multiplicar-se. Mouse peritoneal elicited macrophages cultured on coverslips were infected with Trypanosoma cruzi trypomastigotes from both the F strain and Y strain obtained either from tissue culture or from the bloodstream of infected mice. Both the Y strain and F parasites obtained from tissue culture were interiorized by macrophages at a much higher rate than bloodstream trypomastigotes. Tissue culture parasites incubated with normal mouse serum, mouse plasma obtained at the 7th day after infection, or specific hyperimmune serum at subagglutinating concentration, behaved essentially as non-opsonized parasites. Ultrastructural differences were seen at the early interaction phase between macrophages and trypimastigotes from both sources. After 30 minutes, tissue culture trypomastigotes were located in clusters at the area of contact with macrophages. While bloodstream trypomastigotes, at 3 hours post-infection were most frequently enclosed in a loose phagocytic vacuole, tissue culture trypomastigotes were enclosed in single tight vacuoles. Both tissue culture and bloodstream trypomastigotes of the Y strain multiplied within macrophages; F strain bloodstream trypomastigotes did not develop within the host cells, while tissue culture trypomastigotes multiplied.

SummaryThe interaction between mouse peritoneal macrophages and ‘Y’ strain Trypanosoma cruzi bloodstream forms was studied at optical and electron microscopical levels. The method of marking lysosomes with Thorotrast, either before or after infection of cell monolayers with parasites, revealed that secondary lysosomes fused with phagosomes shortly after trypanosome interiorization. In spite of this, 24 h later most parasites were no longer in a vacuole but lay free within the host cell cytoplasm, multiplying actively. At this time, and up to shortly before 96 h when parasites escaped to the external milieu, most parasitized cells were not lethally injured, as revealed by the Trypan blue dye-exclusion test. Only when parasites were released into the external medium was this situation reversed and infected macrophages took up the dye.

The initial interaction between infective forms of Trypanosoma cruzi and host cells in vivo was studied at the ultrastructural level. In order to follow these events, T. cruzi bloodstream forms were inoculated into the cheek-pouch of hamsters—a peculiar region devoid of lymphatic vessels. This region was chosen as injection site because, unlike other regions, trypanosomes remained there and multiplied locally up to 15 days after inoculation. Parasites were detected initially outside cells or inside neutrophils. Only after the first week following inoculation were developing and multiplying trypanosomes seen inside macrophages or other resident cells. Parasites persisted until 15–20 days after inoculation, but by about the 28th day they were no longer seen.

SYNOPSIS. The ultrastructure of the crithidial phase of T. conorhini culture has been studied. The structure of the plasma membrane is not easy to make out; only at a few points in highly magnified picit is seen as a double osmiophilic membrane with an intermedilayer of low density. Sub-pellicular tubules are present also around the flagellar pocket. The nucleus usually has a single large nucleolus, but sometimes this may be double. The flagellum has the sual 2 central and 9 double peripheral fibers, the latter having lateral arms. The structure of the kinetoplast is similar to that of other rypanosomatids but the division of the organelle seems to be more complex than has been described: the central, DNA-bearing lamellae duplicate, forming a double transverse band; 1 of these bands probably migrates toward the side before invagination of the membrane completes the division. Mitochondria are long tubular structures with few cristae, disposed chiefly along the periphery of the cell. A communication between the kinetoplast and tubular mitochondria is very frequent. The endoplasmic reticulum is poorly developed, represented chiefly by smooth membranes surrounding vesicles, rough-surfaced membranes being scanty in most cells. Several inclusions may be found, some probably lipids, others being of unknown nature.

SYNOPSIS. In cultures of Trypanosoma conorhini the authors have detected “cyst-like bodies” (CLBs) that, in the initial phase, have the appearance of 2 or more overgrown crithidiae fused together; after disorganization of the internal structure of the parent flagellates and division of their organelles, new crithidiae are formed within the CLB. The wall of the CLB consists of an irregularly thick layer of cytoplasm surrounded by the periplast of the parent flagellates and a true cystic membrane is not evident. The daughter crithidiae eventually escape from the CLB leaving the periplast and other residues of the parent flagellates. It is suggested that this represents a method of reproduction entirely different from binary or multiple fission, that is probably common to other trypanosomes of the Section STERCORARIA, and that it may provide for genetic exchange even if it is not a true sexual process.

SYNOPSIS. A cytostome is described in culture forms and in intracellular stages of Trypanosoma cruzi and in culture forms of T. conorhini. The organelle opens into the flagellar pocket or close to it and runs deeply into the cell.

SYNOPSIS. An electron microscope study was made of the “cyst-like bodies” (CLBs) previously described in cultures of Trypanosoma conorhini. It is again suggested that CLBs are a process of reproduction which involves fusion of epimastigotes, repeated divisions of DNA-containing organelles and organization of daughter epimastigotes that, after being completely formed, may be found free within the large central “vacuole” of the CLB. This “vacuole,” it is now evident, results from the fused and muchdilated flagellar reservoirs of parent epimastigotes. Our interpretation of the CLBs and their possible genetic significance are discussed.

O protozoário ciliado ambiguum contém depósitos de hidroxiapatita, e foi estudado por F. G. R. Pautard em 1959 (Biochim. Biophys. Acta 35: 33, 1959). No presente trabalho um potencial piezoelétrico foi aplicado em organismos cultivados emlaboratórios a fim de estimular o crescimento destes depósitos a analisar o processo de mineralização. Estudou-se os agregados isolados (ossículos) através da microscopia eletrônica de transmissão e de varredura, combinadas com microanálise deraios-X, difração eletrônica de área selecionadas (DEAS) e outros métodos. Foram analisados dois tipos de amostras, provenientes de células normais e em jejum. Naquelas mantidas em jejum por aproximadamente 3 meses, observou-se uma mudançaprogressiva na forma do organismo de \"alongado\" para forma de \"esfera\". Neste estágio final, as células apresentavam-se carregadas de inclusões minerais. A síntese destes ossículos parece estar associada com a atividade da fosfatase alcalina,conforme demonstrado nos cortes ultra-finos utilizando o meio de incubação de Mayahara (Mayahara, H., et al. Histochemie 11: 88, 1967). Resultados obtidos no presente estudo, utilizando as técnicas acima mencionadas, confirmaram a natureza dahidroxiapatita destes ossículos. A aplicação do campo elétrico mostrou que ocorre uma mudança na forma dos cristalitos de agulha para placa. Os ossículos já formados mostram nos cortes ultrafinos, um arranjo radial dos cristalitos,. distribuídosem anéis concêntricos Identification and properties of hydroxyapatite from the protozoan spirostomum ambiguum, studied by optical-electronic methods