Cell Surface Carbohydrate Differences in Wild and Mutant Strains of Crithidia fasciculata1

Wild type Crithidia fasciculata and three drug-resistant mutant strains that have shown “flagellar adherence” were studied as to their ability to agglutinate with lectins specific for receptor molecules containing N-acetyl glucosamine, N-acetyl galactosamine, galactose, mannose-like residues, fucose, and sialic acid. Escherichia coli with mannose-sensitive fimbriae was also used as an agglutination probe. The presence of D-GalNAc, D-Gal, and mannose-like residues was detected in the wild strain. Generally, in the mutants, residues of these sugar units were present in greater concentrations when compared to the wild type strain. β-Galactosidase treatment showed that β-D-Galp units are exposed on the cell membrane. All types of cell agglutination including flagellum-flagellum (F-F), flagellum-soma (F-S), and soma-soma (S-S) were observed when lectins were used; however, with E. coli only the F-F type of cell agglutination was observed with the wild type strain and the TFR^R1 mutant. All types of agglutination were observed with the other two mutants.     

Protein Synthesis in Isolated Symbionts from the Flagellate Protozoon Crithidia deanei1

Here we describe a method that allows the isolation of intact trypanosomatid symbionts in amounts sufficient for biochemical analysis. The isolated symbionts retain their characteristic morphological features and are reasonably free of subcellular debris. They actively incorporate [³H]leucine and [³⁵S]methionine into proteins. Chloramphenicol and rifampicin at 50 μg/ml almost completely inhibit the incorporation of protein precursors. The inhibition of protein synthesis by the antibiotics provides direct evidence for the existence of a prokaryotic protein-synthesizing system in this unusual intracellular structure. The pattern of protein synthesis of the isolated symbionts is complex. Several symbiont polypeptides are absent or poorly represented in the flagellate.     

Ecto-phosphatase activity on the cell surface of Crithidia deanei

In the present work we have partially characterized an ecto-phosphatase activity in Crithidia deanei, using viable parasites. This enzyme hydrolyzed p-nitrophenylphosphate at a rate of 3.55 +/- 0.47 nmol Pi/h x 10(8) cells. The dependence on p-NPP concentration shows a normal Michaelis-Menten kinetics for this phosphatase activity and the value of the apparent Km for p-NPP was 5.35 +/- 0.89 mM. This phosphatase activity was inhibited by the product of the reaction, the inorganic phosphate. Experiments using classical inhibitors of acid phosphatases, such as ZnCl2 and sodium fluoride, as well as inhibitors of phosphotyrosine phosphatase, such as sodium orthovanadate and ammonium molybdate, showed a decrease in this phosphatase activity, with different patterns of inhibition.     

Heme synthesis in Crithidia deanei: influence of the endosymbiote

The activity of the following enzymes involved in the biosynthesis of porphyrins was determined in endosymbiote-free and endosymbiote-containing Crithidia deanei grown in a chemically defined medium: succinyl Coenzyme A synthetase (Suc.CoA-S), 5-aminolevulinate synthetase (ALA-S), 4,5-dioxovaleric acid transaminase (DOVA-T), 5-aminolevulinate dehydratase (ALA-D), porphobilinogenase (PBGase), deaminase and heme synthetase (Heme-S). The amount of 5-aminolevulinic acid (ALA) and porphobilinogen, porphyrins and heme was also determined. ALA and PBG were detected in C. deanei. The levels of free porphyrins was low. Heme concentration was nil. The activity of ALA-D, deaminase and PBGase was not detected in C. deanei. The activity of Suc.CoA-S and ALA-S were twice higher in symbiote-containing than in aposymbiotic C. deanei. Aposymbiotic cells had a higher activity of DOVA-T than symbiote-containing cells. The level of Heme-S, measured using protoporphyrin as substrate, was twice as high in symbiote-containing than in symbiote-free cells.     

Extra Nutritional Requirements of Artificially Aposymbiotic Crithidia deanei*

SYNOPSIS. Chloramphenicol cured Crithidia deanei of its endosymbiote. The derived aposymbiotic strain had additional growth requirements: purin (as adenine), heme, arginine, histidine, isoleucine, leucine, phenylalanine, threonine, tryptophan, valine, pyridoxine, riboflavin, and pantothenate and liver infusion (replaceable by high nicotinamide).     

Identification and partial characterization of plasma membrane polypeptides of Crithidia guilhermei,crithidia deanei and Crithidia oncopelti

• 1.1. Components of the cell surface of Crithidia guilhermei, Crithidia deanei and Crithidia oncopelti were radioiodinated by the iodogen technique. The distribution of proteins in the detergent-poor (DPP) and detergent-enriched phase (DRP) were studied using a phase separation technique in Triton X-114 and one- and two-dimensional polyacrylamide gel electrophoresis in sodium dodecyl sulphate (1D and 2D SDS-PAGE).
• 2.2. Significant differences were noted in the proteins present in the DRP when the three species were compared.
• 3.3. Two major bands with mol. wt 28,000 and 56,000 and motility in the pH gradient of 7.4 and 6.3, respectively, were observed in C. guilhermei, but not discernible in C. deanei and C. oncopelti.
• 4.4. One polypeptide with mol. wt 50,000 and p1 4.9 was identified in the DRP of C. deanei.
• 5.5. A broad band with mol. wt 68,000–140,000 and pI 4.7–5.5 was clearly observed in the DRP of C. deanei and one or two polypeptides only present in the DPP were observed in the three Crithidia species analyzed.
• 6.6. Our observations show that C. guilhermei has characteristic surface polypeptides not found in C. deanei and C. oncopelti.
• 7.7. Our results, in association with those reported by others, show that the phase separation using Triton X-114 offers a simple approach to the separation and further analysis of a select group of proteins from the bulk of the cellular proteins.

     

Detection of Penicillin-binding Proteins in the Endosymbiont of the Trypanosomatid Crithidia deanei

ABSTRACT. Growth by serial transfers of the trypanosomatid Crithidia deanei in culture medium containing 1 mg/ml of the β-lactam antibiotics ampicillin or cephalexin resulted in shape distortion of its endosymbiont. The endosymbiont first appeared as filamentous structures with restricted areas of membrane damage. An increase of electron lucid areas was also observed in the endosymbiont matrix. The continuous treatment with β-lactam antibiotics, resulted in endosymbiont membranes fragmentation; and later on the space previously occupied by the symbiont was identified as an electron lucid area in the host cytoplasm. The putative targets of β-lactam antibiotic were two membrane-bound penicillin-binding proteins (PBPs) detected in the Sarkosyl-soluble fraction of purified symbionts labeled with [³H]-benzylpenicillin. The apparent molecular weight of the proteins were 90 kDa (PBP1) and 45 kDa (PBP2). PBP2 represented 85% of the total PBP content in the membrane fraction of the endosymbionts. Competition experiments using the tested antibiotics and [³H]-benzylpenicillin showed that ampicillin and cephalexin have half saturating concentrations considerably higher than [³H]-benzylpenicillin and indicated that PBP1 is the probable lethal target of the antibiotics tested. These results suggest that a physiologically active PBP is present in the cell envelope of C. deanei endosymbionts and may play important roles in the control of processes such as cell division and shape determination.     

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.     

Simple Nutrition of Crithidia deanei,a Reduviid Trypanosomatid with an Endosymbiont*

Crithidia deanei from the reduviid hemipteron, Zelus leucogrammus, unlike most lower trypanosomatids cultivated in defined medium, required only 2 amino acids, methionine and tyrosine; only 4 vitamins, folic acid, thiamine, biotin, and nicotinamide; and neither hemin nor a purine source. Electron microscopy reveals an endosymbiont, probably bacterial, which presumably provides the other basic trypanosomatid essential nutrients.     

Infection of mouse dermal fibroblasts by the monoxenous trypanosomatid protozoa Crithidia deanei and Herpetomonas roitmani

Traditionally, monoxenous trypanosomatid protozoa are not believed to infect vertebrate cells. Using light and electron microscopy, we show that the monoxenous trypanosomatids Crithidia deanei and Herpetomonas roitmani are able to infect dermal mouse fibroblasts in vitro. We present experimental evidence of phagocytosis of these trypanosomatids, and demonstrate their survival in vertebrate cells. This paper raises the question about the role of C. deanei and H. roitmani, and perhaps other monoxenous trypanosomatid species, in opportunistic infections of immunocompromised individuals and cutaneos lesions in vertebrate hosts.     

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.     

Symbiote-Free Hemoflagellates,Blastocrithidia culicis and Crithidia oncopelti: their Liver Factor Requirement and Serologic Identity*

SYNOPSIS. Several aposymbiotic strains of Blastocrithidia culicis and Crithidia oncopelti were cultivated in Trager's chemically defined medium as well as in a blood broth, both supplemented with 0.25% (v/v) liver extract concentrate. For all such strains, the liver extract was found to serve as an essential growth factor in the defined medium and as growth promoting additive in the blood broth. The active molecules were found to be water-soluble, heat stable, dialyzable, and probably nonlipid fractions. Antisera were developed in rabbit against all the available aposymbiotic strains. An almost total cross-reactivity at very high titers was observed in reciprocal agglutination test using strains with and without the bacterial symbiotes. These results indicate that the loss of the symbiotes does not affect the antigenic identity of B. culicis and C. oncopelti.     

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.     

Crithidia deanei: influence of parasite gp63 homologue on the interaction of endosymbiont-harboring and aposymbiotic strains with Aedes aegypti midgut

The present study demonstrates that the endosymbiont of Crithidia deanei influences the expression of surface gp63 molecules. Ultrastructural immunocytochemical analysis shows the presence of the gp63-like protein in the protozoan flagellum and flagellar pocket, either attached to shed membranes or in a free form. This molecule is glycosylphosphatidylinositol (GPI) anchored to the plasma membrane as demonstrated by phospholipase C (PLC) treatment and cross-reacting determinant detection by immunoblotting. The gp63 molecule mediates the adhesive process of the protozoan to Aedes aegypti explanted guts, since the binding was reduced by pre-incubating the C. deanei parasites (wild and aposymbiotic strains) with anti-gp63 antibodies, PLC or PLC followed by anti-gp63 antibodies incubation. In addition, the number of wild C. deanei bound to A. aegypti explanted guts was twice as that of aposymbiotic parasites. Flow cytometry assays revealed that the reactivity of the wild strain with anti-gp63 antibodies was approximately twice as that of the aposymbiotic strain. We may conclude that higher expression of surface gp63 by the wild strain of C. deanei may positively influence this interaction, posing a prominent advantage for the endosymbiont-containing trypanosomatids.     

Surface charge and hydrophobicity of wild and mutant Crithidia fasciculata.

Surface charge of wild-type Crithidia fasciculata and three drug-resistant mutants (TR3, TFRR1, and FUR11) was studied by direct zeta-potential determination and ultrastructural cytochemistry. Surface tension was also investigated by measurements of the advancing contact angle formed by the protozoa monolayers with drops of liquids of different polarities. The individual zeta potential varies markedly among the C. fasciculata cells. The wild and FUR11 mutant strains displayed lower negative surface charge (-12.5 and -9.5 mV, respectively) as compared with the TR3 (-14.8 mV) and TFRR1 (-14.7 mV) mutant strains. Binding of cationized ferritin (CF) was observed at the cell surface of wild and mutant strains of C. fasciculata. Neuraminidase treatment reduced the negative surface charge in the TFRR1 and TR3 mutants in about 37 and 29%, respectively, whereas no significant change was observed with the wild and FUR11 mutant strains. These findings suggest that sialic acid residues are the major anionogenic groups on the surface of C. fasciculata. The density of sialic acid residues per cell in wild and mutant strains of C. fasciculata falls in a range of 1.4 x 10(4) to 3.6 x 10(4). Marked differences of hydrophobicity were also observed. For example, the TFRR1, FUR11, and TR3 drug-resistant mutant strains showed higher contact angle values (55.4, 54.2, and 49.3, respectively) than the wild-type (35.6), as assessed by alpha-bromonaphtalene.     

Flagellar Adherence of Crithidia fasciculata Cells in Culture1

In early log phase cultures of several of the drug-resistant mutants of Crithidia fasciculata that we have previously obtained, a high percentage of cells attach in pairs at the base of the flagellum. This process, which we have termed “flagellar adherence,” lasts for several hours in some cases and occasionally involves changes in cell morphology. The attachment occurs optimally in gently agitated cultures. Flagellar adherent pairs can be disassociated by vigorous agitation; the pairs reappear in the culture within one to three h after disassociation. These paired forms can be clearly distinguished from the normal cell division forms. Clones of flagellar adherent-competent mutant strains are uniformly able to form these pairs in culture. A low percentage of flagellar adherent forms can be induced in wild type cells by glucose starvation.     

Identification and Characterization of Variants of Crithidia luciliae with Altered Morphological and Surface Properties

ABSTRACT. Variants of a cloned laboratory stock of the trypanosomatid parasite Crithidia luciliae have been distinguished from "parental type" organisms. These variants accumulated spontaneously over time as the protozoan was maintained by continuous passage in a chemically defined medium. Cloned lines of these variants have been isolated by plating on nutrient agar and partially characterized on the basis of their growth characteristics in culture, their colony and cellular morphology as well as their surface protein expression. One cloned line consisted of motile, flagellated forms which, unlike "parental type" organisms, did not adhere to the surface of culture flasks. Another cloned line was composed of non-adherent, nonmotile, amastigote-like forms which were further distinguished from "parental type" cells by virtue of their constitutive expression, in nutrient-replete medium, of high levels of a surface membrane associated 3'-nucleotidase/nuclease (3'-N'ase) activity. Both the motile, flagellated and amastigote-like variants, like the "parental type" organisms, exhibited elevated levels of the 3'-N'ase activity upon exposure to purine starvation conditions. The variants described are of potential importance in elucidating the mechanism of induction of the highly regulated 3'-N'ase activity as well as for understanding the cytoskeletal systems and the surface properties of these protozoa.