Identification of C3 acceptors responsible for complement activation in Crithidia fasciculata

Crithidia fasciculata, an insect trypanosomatid is readily lysed by normal human serum at concentrations as low as 3%. Lysis occurs in the presence of Mg+2-EGTA and is antibody independent, indicating that the alternative pathway of complement activation is involved. Analysis of [131I]C3 deposition on C. fasciculata cells using C8-deficient serum, revealed that about 4 x 10(5) C3 molecules bound to each cell. Most of the C3 was bound to cells as C3b, part of it forming high molecular weight complexes, which could be dissociated by methylamine treatment at alkaline pH. To characterize the C3 acceptors on C. fasciculata, surface-iodinated cells were incubated with C8D or heat-inactivated serum, extracted and immunoprecipitated with anti-C3 or anti-arabinogalactan antisera. Analysis of the immunoprecipitated material on SDS gels showed high-molecular weight components, which disappeared after methylamine treatment, giving rise to a component of 200 kDa molecular size. This 200-kDa component corresponded to a purified arabinogalactan complex, which was immunoprecipitated from labeled cell extracts, without incubation with C8D, using anti-arabinogalactan antibodies. These results suggest that the arabinogalactan glycoconjugate is a C3 acceptor in C. fasciculata during complement activation. Purified arabinogalactan complexes were able to inactivate C3 in vitro. Solubilization in KOH to cleave the peptide moiety rendered it unable to inactivate C3. Apparently, the aggregated state of the purified arabinogalactan component at the cell surface is important for C3 deposition and activation.     

Toxicity of 4-Nitroquinoline 1-Oxide for Crithidia fasciculata

Growth inhibition of Crithidia fasciculata by 4-nitroquinoline 1-oxide (NQO) was observed in defined and complex media at 28 C. Aromatic amino acids, cysteine, and nicotinic acid, among several other substances, were ineffective in overcoming NQO toxicity. Dicoumarol and bovine albumin reversed NQO inhibition. While bovine albumin probably acted by the extra-cellular binding of NQO, dicoumarol inhibited the activity of DT-diaphorase, which reduces NQO to 4-hydroxyaminonitroquinoline 1-oxide (HAQO). The DT-diaphorase from C. fasciculata had the same characteristics as the enzyme from rat liver. The specific protection by dicoumarol against NQO inhibition suggests that HAQO is the active toxic substance for C. fasciculata.     

Toxicity of 4-nitroquinoline 1-oxide for Crithidia fasciculata.

Growth inhibition of Crithidia fasciculata by 4-nitroquinoline 1-oxide (NQO) was observed in defined and complex media at 28 C. Aromatic amino acids, cystein, and nicotinic acid, among several other substances, were ineffective in overcoming NQO toxicity. Dicoumarol and bovine albumin reversed NQO inhibition. While bovine albumin probably acted by the extra-cellular binding of NQO, dicoumarol inhibited the activity of DT-diaphorase, which reduces NQO to 4-hydroxyaminonitroquinoline 1-oxide (HAQO). The DT-diaphorase from C. fasciculata had the same characteristics as the enzyme from rat liver. The specific protection by dicoumarol against NQO inhibition suggests that HAQO is the active toxic substance for C. fasciculata.     

Sub-Pellicular Microtubules in Crithidia fasciculata

SYNOPSIS. Crithidia fasciculata sectioned transversally and studied with the electron microscope had gaps among an otherwise uniform row of sub-pellicular microtubules. Cultures were submitted to various treatments known to affect the structure of microtubules in other cell types. Only those very drastic treatments that killed the cells affected the sub-pellicular microtubules. Others, altho affecting the structure of the cell, left the sub-pellicular gaps and microtubules unchanged.     

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.     

Auxotrophic mutants of Crithidia fasciculata

In pursuance of genetic studies, after exposure to ethylmethanesulfonate, 11 auxotrophic mutants of Crithidia fasciculata were cloned. Three proved uracil dependent; 3 serine dependent; and the remainder have not had their auxotrophy defined.     

Biosynthesis of phosphatidylinositol in Crithidia fasciculata

Microsomal preparations from the protozoan (Crithidia fasciculata were shown to incorporate myo-[2-3H]inositol into phosphatidylinositol by both the CDPdiacylglycerol:myo-inositol phosphatidyltransferase reaction and by a myo-inositol exchange reaction. Non-ionic detergent and Mg2+ were necessary for the measurement of transferase activity. Untreated preparations could not be saturated with Mg2+, even at very high concentrations (50-75 mM). However, low concentrations of EGTA (75 micro M) both stimulated the activity 3-fold and reduced the Mg2+ required for saturation to 15-20 mM. EGTA also increased the apparent Km for CDPdiacylglycerol while increasing the sensitivity to substrate inhibition above 1 mM. The transferase activity was inhibited by relatively low concentrations of Ca2+ (50 micro M). This and the EGTA effect suggest a possible role for Ca2+ in the modulation of phosphatidylinositol synthesis. The myo-inositol exchange activity required Mn2+, was insensitive to Ca2+ inhibition and was only slightly stimulated by detergents and EGTA. This activity was preferentially inactivated by heating at 50 degrees C in the presence of Triton X-100. In a detergent solubilized preparation the exchange activity but not the transferase exhibited a non-specific requirement for phospholipid. The differences in properties of the two activities suggest the presence of a separate exchange enzyme.     

Respiratory Pigments of Crithidia fasciculata

Mitochondria were isolated from the heme-requiring insect trypanosomatid, Crithidia fasciculata, which had respiratory activity, showed a P/O ratio with succinate of 0.5 to 1.0, and contained 40 to 50% of the heme a and heme c found in the intact cells. Cytochromes b, c(555), possibly c(1), cytochrome oxidase, a carbon monoxide-binding pigment, and flavoproteins were detectable in the spectra of both intact cells and mitochondria. Cytochrome c(555) is a basic protein that was extracted from cells and mitochondria with salt solutions. The molar ratio of heme c to heme a was approximately 2:1 in both cells and mitochondria. This organism could possibly serve as a model for studies of the respiratory activity of the pathogenic trypanosomes.     

Activation of active-site cysteine residues in the peroxiredoxin-type tryparedoxin peroxidase of Crithidia fasciculata.

Tryparedoxin peroxidase (TXNPx), recently identified as the hydroperoxide-detoxifying enzyme of trypanosomatidae [Nogoceke, E., Gommel, D. U., Kiess, M., Kalisz, H. M. & Flohé, L. (1997) Biol. Chem. 378, 827-836], is a member of the peroxiredoxin family and is characterized by two VCP motifs. Based on a consensus sequence of TXNPx and peroxiredoxin-type peroxidases, eight TXNPx variants were designed, heterologously expressed in Escherichia coli, checked for alpha-helix content by CD and kinetically analysed. The variant Q164E was fully active, C52S, W87D and R128E were inactive and C173S, W87H, W177E and W177H showed reduced activity. Wild-type TXNPx and Q164E exhibit ping-pong kinetics with infinite maximum velocities, whereas saturation kinetics were observed with C173S and W177E. The data comply with a mechanism in which C52, primarily activated by R128 and possibly by W87, is first oxidized by hydroperoxide to a sulfenic acid derivative. C173, supported by W177, then forms an intersubunit disulfide bridge with C52. If C173 is exchanged with a redox-inactive residue (Ser) or is insufficiently activated, the redox shuttle remains restricted to C52. The shift in the kinetic pattern and decrease in specific activity of C173S and W177E may result from a limited accessibility of the oxidized C52 to tryparedoxin, which in the oxidized wild-type TXNPx presumably attacks the C173 sulfur of the disulfide bridge. The proposed mechanism of action of TXNPx is consistent with that deduced for the homologous thioredoxin peroxidase of yeast [Chae, H. Z., Uhm, T. B. & Rhee, S. G. (1994) Proc. Natl Acad. Sci. USA 91, 7022-7026] and is supported by molecular modelling based on the structure of the human peroxiredoxin 'hORF6' [Choi, H.-J., Kang, S. W. Yang, C.-H., Rhee, S. G. & Ryu, S.-E. (1998) Nat. Struct. Biol. 5, 400-406].     

Entamoeba dispar: Cultivation with Sterilized Crithidia fasciculata1

Four isolates of Entamoeba dispar identified by their hexokinase and phosphoglucomutase isoenzyme profile and by their failure to react with Entamoeba histolytica-specific monoclonal antibody (4G6) could be grown in either Diamond's BI-S-33 medium, newly developed BCSI-S (Biosate cysteine starch iron-serum) medium, or casein-free YI-S medium in the presence of Crithidia fasciculata (ReF-1:PRR) sterilized by heating 56° C for 30 min and subsequent incubation with 1% hydrogen peroxide for 24 hours at 4° C. After the cultures were maintained for over 50 passages, the amebae were identified as E. dispar by isoenzyme analysis, polymerase chain reaction with E. histolytica- and E. dispar-specific primers, i.e. p11 plus p12 and p13 plus p14, respectively, and by negative reactivity with monoclonal antibody 4G6. The flagellates added to the culture were judged to be metabolically inactive based on the results of nuclear magnetic resonance spectroscopy, electron microscopy, and polarographic analysis. All of these findings suggest that E. dispar can grow in vitro with metabolically inactive C. fasciculata as a culture associate.     

Effects of Hydroxyurea on Crithidia fasciculata

SYNOPSIS Hydroxyurea (HU) inhibits increase in cell number in cultures of Crithidia fasciculata. Complete inhibition is produced by 8 mM and higher concentrations. If HU is not removed, population growth resumes in 45–50 h: if HU is removed, partially synchronous growth occurs through 2 cycles. During HU inhibition, the rate of DNA synthesis is reduced to 1% of that in exponentially growing cultures; protein and RNA syntheses continue at slightly reduced rates. Mean cell size and protein and RNA contents per cell increase; rate of oxygen consumption per mg cell protein remains constant. The behavior of a culture upon addition of HU and upon its removal agrees with predictions based on the hypothesis that the only direct effect of HU is to block DNA synthesis. The synchrony produced by HU is judged satisfactory for investigations of kinetoplast and nuclear replication but not for biochemical characterization of other aspects of the cell cycle.     

The ribonucleic acids of Crithidia fasciculata

Crithidia fasciculata ribosomes were found to be 80S and to dissociate into 58 and 41S subunits; on 5 to 50% sucrose gradients, rRNA was separated into 25, 18, and 5S components. The molecular sizes of the heavier rRNA species, estimated by polyacrylamide gel electrophoresis were 1.24 and 0.84 M (X 10(6) daltons). The 25S RNA has a tendency to interact with the 18S RNA to give a complex that is difficult to separate by sucrose gradient centrifugation. The 25S RNA is also unstable and dissociates into 0.73 and 0.57 M components. The 18S RNA has molecular size (0.84 M) higher than the 0.7 M reported for most eukaryotes, but similar to that of Euglena and Amoeba. Ribosomal RNA hybridized 0.29% of the nuclear DNA. Mitochondrial RNA, extracted by a rapid procedure was resolved into 16 and 5S components in sucrose gradients.     

The purine phosphoribosyltransferases of Crithidia fasciculata

The purine phosphoribosyltransferases of Crithidia fasciculata were identified and some of their properties described. The organism possesses three separate enzymes for the production of AMP, IMP, and GMP. The evidence for this comes from the observed differences in elution patterns from gel filtration columns, differences in heat sensitivity, and especially the clear separation of hypoxanthine phosphoribosyltransferase from guanine phosphoribosyltransferase by affinity chromatography on GMP-agarose. APRTase is activated most efficiently by Zn++, whereas HPRTase and GPRTase are activated most effectively by Co++. In no case did the product mononucleotides produce strong inhibition of the transferase activities.     

Growth-phase dependent substrate adhesion in Crithidia fasciculata

Crithidia fasciculata is a trypanosomatid flagellate that parasitizes several species of mosquito. Within the alimentary tract of its host, C. fasciculata exists in two forms: one is a non-motile form, attached in clusters to the lining of the gut, the other a more elongated form swimming freely in the gut lumen. We have developed an in vitro culture system that reproduces the appearance of these two distinct morphological forms. Using two different cultivation methods, shaking and stationary incubations, we have demonstrated that adherence phenotypes are growth-phase dependent. Organisms in the logarithmic phase of growth possess the ability to adhere to substrates; this ability is lost when the organism enters a stationary growth phase. Parasite adherence was independent of cultivation method or substrate. Furthermore, adherent forms of Crithidia maintained their adhesive properties following their removal from substrates. Our data reveal a growth-phase-regulated process of cell attachment that may influence the transmission and dissemination of this parasitic flagellate.     

The Catabolism of Tryptophan to Indole-3-Ethanol by Crithidia fasciculata and Phytomonas davidi1

Crithidia fasciculata and Phytomonas davidi catabolize tryptophan (TRP) to indole-3-ethanol, which was identified by both thin layer and gas chromatography. The catabolic pathway involved in this metabolic conversion is suggested to be similar to that proposed for other members of the family Trypanosomatidae. Although this catabolism occurs at both 25° and 37°C, the catabolic rate is greater at 37°C, a non-permissive growth temperature. Conditions that inhibit protein synthesis would appear to favor the catabolism of tryptophan to indole-3-ethanol. The possible importance of this catabolic pathway to these organisms is discussed.     

The origin of unconjugated pteridines in Crithidia fasciculata

Summary By using folic acid-2-¹⁴C in the growth medium as the sole source of pteridine it was found that the kinetoplastid flagellate, Crithidia fasciculata, produced a total of 87 mg of labeled biopterin and 23 g of 2-amino-4-hydroxy-6-hydroxymethylpteridine from 2 mg of folic acid in 2 liters of medium. Eighty percent of the biopterin and 70% of the 6-hydroxymethylpteridine was isolated from the spent medium, the remainder from the cells. These results and the results whereby the total pteridine was supplied as a folic acid analog make it obvious that this flagellate does convert folate to biopterin and is incapable of de novo pteridine synthesis.Supported in part by Research grants AM 01005 and CA 02924 from the National Institutes of Health, United States Public Health Service, and by Deutsche Forschungsgemeinschaft.Dedicated to Professor C. B. Van Niel on the occasion of his 70th birthday.