Effects of miltefosine on the proliferation, ultrastructure, and phospholipid composition of Angomonas deanei, a trypanosomatid protozoan that harbors a symbiotic bacterium.

Some trypanosomatids, such as Angomonas deanei formerly named as Crithidia deanei, present an obligatory intracellular bacterium, which maintains a mutualistic relationship with the host. Phosphatidylcholine (PC) is the major phospholipid in eukaryotes and an essential component of cell membranes playing structural, biochemical, and physiological roles. However, in prokaryotes, PC is present only in those species closely associated with eukaryotes, either in symbiotic or pathogenic interactions. In trypanosomatids, the endosymbiont envelope is composed by a reduced cell wall and by two membrane units that lack sterols and present cardiolipin (CL) and PC as the major phospholipids. In this study, we tested the effects of miltefosine in A. deanei proliferation, as well as, on the ultrastrucuture and phospholipid composition considering that this drug inhibits the CTP-phosphocholine cytidyltransferase (CCT), a key enzyme in the PC biosynthesis. Besides the low effect of miltefosine in cellular proliferation, treated protozoa presented ultrastructural alterations such as plasma membrane shedding and blebbing, mitochondrial swelling, and convolutions of the endosymbiont envelope. The use of (32) Pi as a tracer revealed that the production of PC, CL, and phosphatidylethanolamine decreased while phosphatidylinositol production remained stable. Mitochondrion and symbiont fractions obtained from protozoa treated with miltefosine also presented a decrease in phospholipid production, reinforcing the idea that an intensive metabolic exchange occurs between the host trypanosomatid and structures of symbiotic origin.

Characterization of a porin channel in the endosymbiont of the trypanosomatid protozoan Crithidia deanei.

Crithidia deanei is a trypanosomatid protozoan that harbours a symbiotic bacterium. The partners maintain a mutualistic relationship, thus constituting an excellent model for studying metabolic exchanges between the host and the symbiont, the origin of organelles and cellular evolution. According to molecular analysis, symbionts of different trypanosomatid species share high identity and descend from a common ancestor, a ß-proteobacterium of the genus Bordetella. The endosymbiont is surrounded by two membranes, like Gram-negative bacteria, but its envelope presents special features, since phosphatidylcholine is a major membrane component and the peptidoglycan layer is highly reduced, as described in other obligate intracellular bacteria. Like the process that generated mitochondria and plastids, the endosymbiosis in trypanosomatids depends on pathways that facilitate the intensive metabolic exchanges between the bacterium and the host protozoan. A search of the annotated symbiont genome database identified one sequence with identity to porin-encoding genes of the genus Bordetella. Considering that the symbiont outer membrane has a great accessibility to cytoplasm host factors, it was important to characterize this single porin-like protein using biochemical, molecular, computational and ultrastructural approaches. Antiserum against the recombinant porin-like molecule revealed that it is mainly located in the symbiont envelope. Secondary structure analysis and comparative modelling predicted the protein 3D structure as an 18-domain ß-barrel, which is consistent with porin channels. Electrophysiological measurements showed that the porin displays a slight preference for cations over anions. Taken together, the data presented herein suggest that the C. deanei endosymbiont porin is phylogenetically and structurally similar to those described in Gram-negative bacteria, representing a diffusion channel that might contribute to the exchange of nutrients and metabolic precursors between the symbiont and its host cell.

The bacterium endosymbiont of Crithidia deanei undergoes coordinated division with the host cell nucleus.

In trypanosomatids, cell division involves morphological changes and requires coordinated replication and segregation of the nucleus, kinetoplast and flagellum. In endosymbiont-containing trypanosomatids, like Crithidia deanei, this process is more complex, as each daughter cell contains only a single symbiotic bacterium, indicating that the prokaryote must replicate synchronically with the host protozoan. In this study, we used light and electron microscopy combined with three-dimensional reconstruction approaches to observe the endosymbiont shape and division during C. deanei cell cycle. We found that the bacterium replicates before the basal body and kinetoplast segregations and that the nucleus is the last organelle to divide, before cytokinesis. In addition, the endosymbiont is usually found close to the host cell nucleus, presenting different shapes during the protozoan cell cycle. Considering that the endosymbiosis in trypanosomatids is a mutualistic relationship, which resembles organelle acquisition during evolution, these findings establish an excellent model for the understanding of mechanisms related with the establishment of organelles in eukaryotic cells.

The Bacterium Endosymbiont of Crithidia deanei Undergoes Coordinated Division with the Host Cell Nucleus

In trypanosomatids, cell division involves morphological changes and requires coordinated replication and segregation of the nucleus, kinetoplast and flagellum. In endosymbiont-containing trypanosomatids, like Crithidia deanei, this process ismore complex, as each daughter cell contains only a single symbiotic bacterium, indicating that the prokaryote must replicate synchronically with the host protozoan. In this study, we used light and electron microscopy combined with three dimensional reconstruction approaches to observe the endosymbiont shape and division during C. deanei cell cycle. We found that the bacterium replicates before the basal body and kinetoplast segregations and that the nucleus is the last organelle to divide, before cytokinesis. In addition, the endosymbiont is usually found close to the host cell nucleus, presenting different shapes during the protozoan cell cycle. Considering that the endosymbiosis in trypanosomatids is a mutualistic relationship, which resembles organelle acquisition during evolution, these findings establish an excellent model for the understanding of mechanisms related with the establishment of organelles in eukaryotic cells.

Roles of the endosymbiont and leishmanolysin-like molecules expressed by Crithidia deanei in the interaction with mammalian fibroblasts.

Crithidia deanei is an insect trypanosomatid that harbors a bacterial endosymbiont in its cytoplasm. In this work, we have demonstrated the influence of the endosymbiont on the interaction of C. deanei with mammalian fibroblasts, also implicating the surface leishmanolysin-like molecules of C. deanei in this process. The wild strain of C. deanei expressed a higher amount (2-fold) of leishmanolysin-like molecules in the parasite surface than the aposymbiotic strain. The treatment of parasites with anti-leishmanolysin antibodies or the fibroblasts with purified leishmanolysin-like molecules from C. deanei significantly reduced the association index. The aposymbiotic strain of C. deanei presented interaction rates about 2- and 3-fold lower with fibroblasts than the endosymbiont-bearing counterpart after 1 and 2h, respectively. However, the association indexes were similar after 3 and 4h of interaction. Additionally, we observed a 2-fold increase in the association index after 24-96 h of parasite-fibroblast interaction when compared to the interaction process performed for 4h, irrespective to the presence of the endosymbiont, suggesting that fibroblasts support multiplication and survival of C. deanei. Both parasite strains were able to induce fibroblast lysis. Interestingly, the wild strain led to a 2-fold increase in fibroblasts death in comparison to the aposymbiotic strain after 48-96 h. We also showed that both wild and aposymbiotic biotinylated live parasites recognized the same receptor in the fibroblast cells.

The kinetoplast ultrastructural organization of endosymbiont-bearing trypanosomatids as revealed by deep-etching, cytochemical and immunocytochemical analysis.

The endosymbiont-bearing trypanosomatids present a typical kDNA arrangement, which is not well characterized. In the majority of trypanosomatids, the kinetoplast forms a bar-like structure containing tightly packed kDNA fibers. On the contrary, in trypanosomatids that harbor an endosymbiotic bacterium, the kDNA fibers are disposed in a looser arrangement that fills the kinetoplast matrix. In order to shed light on the kinetoplast structural organization in these protozoa, we used cytochemical and immunocytological approaches. Our results showed that in endosymbiont-containing species, DNA and basic proteins are distributed not only in the kDNA network, but also in the kinetoflagellar zone (KFZ), which corresponds to the region between the kDNA and the inner mitochondrial membrane nearest the flagellum. The presence of DNA in the KFZ is in accordance with the actual model of kDNA replication, whereas the detection of basic proteins in this region may be related to the basic character of the intramitochondrial filaments found in this area, which are part of the complex that connects the kDNA to the basal body. The kinetoplast structural organization of Bodo sp. was also analyzed, since this protozoan lacks the highly ordered kDNA-packaging characteristic of trypanosomatid and represents an evolutionary ancestral of the Trypanosomatidae family.

L-Proline uptake in Crithidia deanei is influenced by its endosymbiont bacterium.

Crithidia deanei, a monoxenic trypanosomatid, presents an endosymbiotic bacterium in its cytoplasm. Both the protozoan and the bacterium maintain intensive metabolic exchange, resulting in an interesting model to study the coevolution of metabolisms. The relevance of l-proline for the growth of C. deanei and its transport into these cells was studied. Both the endosymbiont-containing (wild) and the endosymbiont-free protozoa (aposymbiont or cured) strains, when grown in medium supplemented with l-proline, reached higher cell densities than those grown in unsupplemented media. We biochemically characterized the uptake of l-proline in both the wild (K(m)=0.153+/-0.022 mM, V(max)=0.239+/-0.011 nmol min(-1) per 4 x 10(7) cells) and the aposymbiont strains (K(m)=0.177+/-0.049 mM, V(max)=0.132+/-0.012 nmol min(-1) per 4 x 10(7) cells). These data suggest a single type of proline transporter whose activity is upregulated by the presence of the symbiotic bacterium. Proline transport was further characterized and was found to be insensitive to the extracellular concentration of Na+, but sensitive to K+ and pH. The abolition of proline uptake by respiratory chain inhibitors and valinomycin indicates that the proline transport in C. deanei is dependent on the plasma membrane K+ gradient.

Phosphatidylcholine synthesis in Crithidia deanei: the influence of the endosymbiont.

In this study, the role of phospholipid biosynthetic pathways was investigated in the establishment of the mutualistic relationship between the trypanosomatid protozoan Crithidia deanei and its symbiotic bacterium. Although the endosymbiont displays two unit membranes, it lacks a typical Gram-negative cell wall. As in other intracellular bacteria, phosphatidylcholine is a major component of the symbiont envelope. Here, it was shown that symbiont-bearing C. deanei incorporates more than two-fold (32)Pi into phospholipids as compared with the aposymbiotic strain. The major phospholipid synthesized by both strains was phosphatidylcholine, followed by phosphatidylethanolamine and phosphatidylinositol. Cellular fractioning indicated that (32)Pi-phosphatidylcholine is the major phospholipid component of the isolated symbionts, as well as of mitochondria. Although the data indicated that isolated symbionts synthesized phospholipids independently of the trypanosomatid host, a key finding was that the isolated bacteria synthesized mostly phosphatidylethanolamine, rather than phosphatidylcholine. These results indicate that phosphatidylcholine production by the symbiont depends on metabolic exchanges with the host protozoan. Insight about the mechanisms underlying lipid biosynthesis in symbiont-bearing C. deanei might help to understand how the prokaryote/trypanosomatid relation has evolved in the establishment of symbiosis.

An endosymbiont positively modulates ornithine decarboxylase in host trypanosomatids.

Some trypanosomatids, such as Crithidia deanei, are endosymbiont-containing species. Aposymbiotic strains are obtained after antibiotic treatment, revealing interesting aspects of this symbiotic association. Ornithine decarboxylase (ODC) promotes polyamine biosynthesis and contributes to cell proliferation. Here, we show that ODC activity is higher in endosymbiont-bearing trypanosomatids than in aposymbiotic cells, but isolated endosymbionts did not display this enzyme activity. Intriguingly, expressed levels of ODC were similar in both strains, suggesting that ODC is positively modulated in endosymbiont-bearing cells. When the aposymbiotic strain was grown in conditioned medium, obtained after cultivation of the endosymbiont-bearing strain, cellular proliferation as well as ODC activity and localization were similar to that observed in the endosymbiont-containing trypanosomatids. Furthermore, dialyzed-heated medium and trypsin treatment reduced ODC activity of the aposymbiont strain. Taken together, these data indicate that the endosymbiont can enhance the protozoan ODC activity by providing factors of protein nature, which increase the host polyamine metabolism.

Influence of the endosymbiont of Blastocrithidia culicis and Crithidia deanei on the glycoconjugate expression and on Aedes aegypti interaction.

Blastocrithidia culicis and Crithidia deanei are trypanosomatid protozoa of insects that normally contain intracellular symbiotic bacteria. The protozoa can be rid of their endosymbionts by antibiotics, producing a cured cell line. Here, we analyzed the glycoconjugate profiles of endosymbiont-harboring and cured strains of B. culicis and C. deanei by Western blotting and flow cytometry analyses using lectins that recognize specifically sialic acid and mannose-like residues. The absence of the endosymbiont increased the intensity of the lectins binding on both trypanosomatids. In addition, wild and cured strain-specific glycoconjugate bands were identified. The role of the surface saccharide residues on the interaction with explanted guts from Aedes aegypti gut was assessed. The aposymbiotic strains of B. culicis and C. deanei presented interaction rates 3.3- and 2.3-fold lower with the insect gut, respectively, when compared with the endosymbiont-bearing strains. The interaction rate of sialidase-treated cells of the wild and cured strains of B. culicis and C. deanei was reduced in at least 90% in relation to the control. The interaction of B. culicis (wild strain) with explanted guts was inhibited in the presence of mucin (56%), fetuin (62%), sialyllactose (64%) and alpha-methyl-D-mannoside (80%), while in C. deanei (wild strain) the inhibition was 53%, 56%, 79% and 34%, respectively. Collectively, our results suggest a possible involvement of sialomolecules and mannose-rich glycoconjugates in the interaction between insect trypanosomatids and the invertebrate host.

The microtubule analog protein, FtsZ, in the endosymbiont of trypanosomatid protozoa.

Blastocrithidia culicis and Crithidia deanei are trypanosomatids that harbor an endosymbiotic bacterium in their cytoplasm. In prokaryotes, numerous proteins are essential for cell division, such as FtsZ, which is encoded by filament-forming temperature-sensitive (fts) genes. FtsZ is the prokaryotic homolog of eukaryotic tubulin and is present in bacteria and archaea, and has also been identified in mitochondria and chloroplasts. FtsZ plays a key role in the initiation of cytokinesis. It self-assembles into the Z ring, which establishes the division plane during septation. In this study, immunoblotting analysis using a FtsZ polyclonal antibody, revealed a 40-kDa band characteristic of FtsZ in endosymbiont fractions and in whole trypanosomatid homogenates, but not in whole cell extracts of aposymbiotic strains. Confocal microscopy and ultrastructural analysis revealed a specific and dispersed labeling over the endosymbiont. Bars and ring-like structures, which are suggestive of the presence of Z-rings, were never observed, even during the division of the symbiont. This peculiar distribution of FtsZ may represent an arrangement of cytoskeleton protein intermediate between prokaryotic and eukaryotic cells. The endosymbiont ftsz gene was completely sequenced after amplification of DNA from symbiont-bearing trypanosomatids or from pure endosymbiont fractions, using PCR and specific primers. The sequences obtained from the endosymbionts from C. deanei and B. culicis were very similar, and were most closely related to bacteria from the genus Pseudomonas.

A metalloproteinase extracellularly released by Crithidia deanei.

Actively motile cells from a cured strain of Crithidia deanei released proteins in phosphate buffer (pH 7.4). The molecular mass of the released polypeptides, which included some proteinases, ranged from 19 to 116 kDa. One of the major protein bands was purified to homogeneity by a combination of anion-exchange and gel filtration chromatographs. The apparent molecular mass of this protein was estimated to be 62 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The incorporation of gelatin into SDS-PAGE showed that the purified protein presented proteolytic activity in a position corresponding to a molecular mass of 60 kDa. The enzyme was optimally active at 37 degrees C and pH 6.0 and showed 25% of residual activity at 28 degrees C for 30 min. The proteinase was inhibited by 1,10-phenanthroline and EDTA, showing that it belonged to the metalloproteinase class. A polyclonal antibody to the leishmanial gp63 reacted strongly with the released C. deanei protease. After Triton X-114 extraction, an enzyme similar to the purified metalloproteinase was detected in aqueous and detergent-rich phases. The detection of an extracellular metalloproteinase produced by C. deanei and some other Crithidia species suggests a potential role of this released enzyme in substrate degradation that may be relevant to the survival of trypanosomatids in the host.

Immunocytochemical detection of DNA and RNA in endosymbiont-bearing trypanosomatids.

Research about the kinetoplast of trypanosomatids has yielded valuable information about the organization of extranuclear structure. However, the ultrastructural localization of nucleic acids within these protozoa remains uncertain. We have applied cytochemical and immunocytochemical approaches to precisely identify DNA and RNA in lower endosymbiont-bearing trypanosomatids. Using the Terminal deoxynucleotidyl Transferase (TdT) immunogold technique, we showed that nuclear DNA is seen associated with the nuclear envelope during the trypanosomatid cell cycle. By combining the TdT technique with the acetylation method, which improves the contrast between structures containing fibrils and granules, we have demonstrated that the nucleolus of endosymbiont-bearing trypanosomatids is composed of two constituents: a granular component and a DNA-positive fibrillar zone. Moreover, we revealed that DNA of endosymbiotic bacteria consisted of electron-dense filaments which are usually in close contact with the prokaryote envelope. Using a Lowicryl post-embedding immunogold labeling procedure with anti-RNA antibodies, we showed the presence of RNA not only over the cytoplasm, the interchromatin spaces and the nucleolus, but also over the kinetoplast and virus-like particles present in Crithidia desouzai.

Differential expression of proteolytic enzymes in endosymbiont-harboring Crithidia species.

Crithidia oncopelti, Crithidia deanei and Crithidia desouzai are flagellates of the Trypanosomatidae family that present bacterium-like endosymbionts in their cytoplasm. Gelatin-SDS-PAGE analysis was used to characterize cell-associated and extracellular proteinases in these organisms. Our survey indicates that the proteolytic profiles of C. deanei and C. desouzai are identical; that C. oncopelti displays a distinct zymogram; and that species naturally lacking endosymbionts have a more complex extracellular proteolytic activity, which illustrates the heterogeneity of this genus. This is the first report on the presence of cysteine proteinases in the culture supernatant of monoxenic trypanosomatids, and by the use of wild and aposymbiotic strains from C. deanei we also demonstrated that the prokaryote endosymbiont somehow alters quantitatively the expression of extracellular proteinases in this trypanosomatid.

Monophyly of endosymbiont containing trypanosomatids: phylogeny versus taxonomy.

To obtain additional information on the phylogenetic relationships within the family Trypanosomatidae (order Kinetoplastida), we have sequenced the small subunit ribosomal RNA genes from the endosymbiont containing species Herpetomonas roitmani TCC080, Herpetomonas sp. TCC263, Crithidia oncopelti ATCC 12982 and a partial large subunit rRNA gene from H. roitmani. The small subunit sequences in the two isolates of Herpetomonas are very similar but not identical, and so are their restriction digest profiles of kinetoplast DNA. The size of minicircles in both isolates is 4.2 kilobases. The inferred ribosomal RNA phylogenetic trees shows the genera Herpetomonas and Crithidia as polyphyletic. Endosymbiont-bearing herpetomonads cluster with the endosymbiont-bearing crithidias and a blastocrithidia to form a monophyletic clade, whereas the endosymbiont-free members of these genera are found elsewhere in the tree. These data support the hypothesis of a monophyletic origin of endosymbiosis in trypanosomatid evolution and also suggest that a taxonomic revision is needed in order to better describe the natural affinities in this family.

16S ribosomal DNA sequence identities of beta-proteobacterial endosymbionts in three Crithidia species.

The 16S ribosomal DNA sequences of endosymbionts from the trypanosomatid protozoa (Crithidia spp.) are most homologous to that of Bordetella spp. This finding extends the polyphyletic origin of endosymbionts for the first time to the beta Proteobacteria. Biased base transitions and compensatory mutations of the symbionts' sequences that may contribute to their identity in the three Crithidia spp. are noted.

Intracellular lectin-binding sites in symbiont-bearing Crithidia species.

Crithidia oncopelti, C. deanei, and C. desouzai are flagellates of the Trypanosomatidae family that present bacterium-like endosymbionts in their cytoplasm. Direct and indirect lectin-gold labeling techniques were used at the electron microscopic level in Lowicryl K4M-embedded cells to demonstrate the presence of intracellular lectin-binding sites. We used the lectins Ulex europaeus I, Griffonia simplicifolia II, Ricinus communis I, Arachis hypogaea, G. simplicifolia I, Wistaria floribunda, Limulus polyphemus, and Canavalia ensiformis, which recognize alpha-L-fucose, alpha- and beta-N-acetylglucosamine, beta-galactose and beta-N-acetylgalactosamine, beta-galactose, alpha-galactose, beta-N-acetylgalactosamine, sialic acid and alpha-D-mannose, and alpha-D-glucose residues, respectively. The nucleus was the cellular structure most frequently labeled by the lectins. The Golgi complex was seldom labeled, whereas the endoplasmic reticulum and the flagellar pocket presented a large number of binding sites. Symbionts had their two unit membranes weakly labeled by the different lectins but displayed no labeling of the space between the membranes.

Herpetomonas roitmani (Fiorini et al., 1989) n. comb.: a trypanosomatid with a bacterium-like endosymbiont in the cytoplasm.

The trypanosomatid previously described as Crithidia roitmani is characterized here at the ultrastructural and biochemical levels. The data indicates that the parasite belongs to the Herpetomonas genus, and we therefore suggest the flagellate to be denominated as Herpetomonas roitmani n. comb. Cladistic analysis of isoenzyme data generated by eight different enzymes showed that the parasite presented a distinct banding pattern and could be grouped with some Herpetomonas spp., but not with Crithidia spp., used as reference strains. Accordingly, when the parasites were grown for longer periods in Roitman's defined medium, expontaneous differentiation from promastigotes to opisthomastigotes (typical of the Herpetomonas genus) occurred. Transmission electron microscopy revealed the presence of bacterium-like endosymbionts in the cytoplasm of all evolutive forms of the parasite. All morphological alterations characteristic of endosymbiont-bearing trypanosomatids could be observed.

Pesquisa de anticorpos anti DNA nativo pelos métodos de radioimunoensaio e da Crithidia luciliae: estudo comparativo

Anticorpos contra DNA de dupla hélice säo achado característico em pacientes com lúpus eritematoso sistêmico (Les. Várias técnicas säo descritas para demonstrar anticorpos antiDNA nativo. No Brasil, essa pesquisa vem sendo habitualmente realizada utilizando-se como substrato a Crithidia luciliae, que tem cinetoplasto rico em DNA nativo. A pesquisa por radioimunoensaio de Farr näo tem sido feita em nosso meio. Foram examinados soros de 82 pacientes de Belo Horizonte com diagnóstico preestabelecido: 65 com LES, 14 com doença mista de tecido conjuntivo (DMTC),dois com síndrome de Sjögren primária (SS) e um com esclerose sistêmica progressiva (ESP); a idade de 32 meses a 70 anos. Neste estudo, empregaram-se as duas técnicas, analisanbdo comparativamente sua sensibilidade e especificidade. Os testes foram executados no Instituto de Pesquisas Reumatológicas de Aachen (alemanha). Os resultados obtidos foram analisados como concordantes (positivo ou negtivo nas duas técnicas) e discordantes (positivo em uma e negativo em outra técnica). Oitenta e quatro por cento dos resultados säo concordantes e os discordantes säo distribuidos em 8,5% positivos ao radioimunoensaio. Dos 33 resultados positivos por um e/ou outro método, 29 pacientes (88%) têm diagnóstico de LES, e os outros quatro pacientes (12%) têm DMTC. Esses dados demonstram alta especificidade para o LES das duas técnicas e sensibilidade estatisticamente iguais em doenças do tecido conjuntivo