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.

Stimulated transport of adenosine, guanosine and hypoxanthine in Crithidia luciliae: metabolic machinery in which the parasite has a distinct advantage over the host.

Nutritional insufficiency is a common environmental extreme to which parasitic protozoa are routinely exposed. In this study of purine salvage mechanisms we illustrate some successful adaptations of the parasite Crithidia luciliae to its environment, particularly in the case of purine stress. In purine-depleted conditions, the insect trypanosome C. luciliae has the ability to increase the rates of transport of adenosine, guanosine and hypoxanthine and the activity of the exoenzyme 3'nucleotidase (3'NTase) during the growth cycle. The dramatic increase in these activities appears after a 72-h period in culture. The increased activity of the purine transporters and 3'NTase could be suppressed by addition to the medium of a purine supplement such as adenosine or hypoxanthine (100 microM). Under conditions where the concentration of purines in the medium could be closely regulated, C. luciliae grown in purine-replete medium (> or = 75 microM purine) exhibited low rates of purine transport and activity of 3'NTase. In comparison, parasites transferred to medium with a low purine source (< or = 7.5 microM adenosine) had levels of adenosine, guanosine and hypoxanthine transport elevated 25-40-fold. The results link the simultaneous increase in activity of the nucleoside and base transporters, 3'NTase activity and a general increase in the purine salvage of C. luciliae to the concentration of purines available at any time to the parasite.

Crithidia luciliae: regulation of purine nucleoside transport by extracellular purine concentrations.

During the growth cycle of the protozoan parasite Crithidia luciliae, there was a dramatic concomitant increase in the rate of adenosine and guanosine transport and 3' nucleotidase (3'NTase) activity after 72-94 hr. The simultaneous increased activities of the nucleoside transporters and 3'NTase could be suppressed by addition to the medium of a purine supplement such as adenosine (100 microM). C. luciliae grown in purine-replete medium (> or = 75 microM adenosine) exhibited low rates of adenosine and guanosine transport whilst parasites transferred to a defined serum-free medium containing < or = 7.5 microM adenosine demonstrated elevated levels of both adenosine and guanosine transport up to 25- to 40-fold. The increased activity of the nucleoside transporters was inhibited by cycloheximide (10 microM). Under conditions of purine depletion 3'AMP and 3'GMP inhibited the adenosine and guanosine transporters, respectively. However, in the presence of a purine supplement (100 microM), neither 3'AMP nor 3'GMP was an effective inhibitor of nucleoside transport. Our results link the increased activity of the nucleoside transporters to the increased activity of the 3'NTase, indicating the activation of a purine salvage system not previously reported in other organisms.

A 31P nuclear magnetic resonance study of Crithidia luciliae.

31P NMR has been used to observe phosphorus-containing compounds in both perchloric acid and KOH extracts and in whole cell suspensions of Crithidia luciliae. Cells were grown in Bone and Steinert medium, or in a modified RPMI culture medium and harvested after about 72 h in mid- to late log phase. 31P NMR spectra of the perchloric acid extracts indicated that 3-phosphoglycerate, which is normally at low concentrations in most cells, was the dominant phosphorus-containing compound in the sugar phosphate region. 3-Phosphoglycerate is the end product of glycosomal glycolysis and our finding is consistent with previous observations of the failure to detect prior glycolytic intermediates. Other metabolites observed were ATP, ADP, NAD(P)+, phosphoenolpyruvate and low molecular weight polyphosphates (PPn, n less than 20). The presence of high-molecular-weight polyphosphates was established by spectra recorded on extracts obtained through subsequent treatment of the insoluble fraction with KOH. 31P NMR experiments on whole cells indicated that the average main internal pH of cells in late-log growth phase was approx. pH 7.2 +/- 0.1, using the orthophosphate resonance as an indicator. The cells responded to the addition of glucose (final concentration approx. 35 mM) with a decrease in pH, both internal (delta pH = -0.9 (55 min)-1) and external (delta pH = -1.3 (15 min)-1). Polyphosphates and ATP could not be observed in whole cell experiments, although perchloric acid extracts of identically treated cells showed no significant depletion of these compounds.

Inhibitors of ergosterol biosynthesis and growth of the trypanosomatid protozoan Crithidia fasciculata.

Six nitrogen-, sulfur- and cyclopropane-containing derivatives of cholestanol were examined as inhibitors of growth and sterol biosynthesis in the trypanosomatid protozoan Crithidia fasciculata. The concentrations of inhibitors in the culture medium required for 50% inhibition of growth were 0.32 microM for 24-thia-5 alpha,20 xi-cholestan-3 beta-ol (2), 0.009 microM for 24-methyl-24-aza-5 alpha,20 xi-cholestan-3 beta-ol (3), 0.95 microM for (20,21),(24,-25)-bis-(methylene)-5 alpha,20 xi-cholestan-3 beta-ol (4), 0.13 microM for 22-aza-5 alpha,20 xi-cholestan-3 beta-ol (5), and 0.3 microM for 23-azacholestan-3-ol (7). 23-Thia-5 alpha-cholestan-3 beta-ol (6) had no effect on protozoan growth at concentrations as high as 20 microM. Ergosterol was the major sterol observed in untreated C. fasciculata, but significant amounts of ergost-7-en-3 beta-ol, ergosta-7,24(28)-dien-3 beta-ol, ergosta-5,7,22,24(28)-tetraen-e beta-ol, cholesta-8,24-dien-3 beta-ol, and, in an unusual finding, 14 alpha-methyl-cholesta-8,24-dien-3 beta-ol were also present. When C. fasciculata was cultured in the presence of compounds 2 and 3, ergosterol synthesis was suppressed, and the principal sterol observed was cholesta-5,7,24-trien-3 beta-ol, a sterol which is not observed in untreated cultures. The presence of this trienol strongly suggests that 2 and 3 specifically inhibit the S-adenosylmethionine:sterol C-24 methyltransferase but do not interfere with the normal enzymatic processing of the sterol nucleus. When C. fasciculata was cultured in the presence of compounds 5 and 7, the levels of ergosterol and ergost-7-en-3 beta-ol were suppressed, but the amounts of the presumed immediate precursors of these sterols, ergosta-5,7,22,24(28)-tetraen-3 beta-ol and ergosta-7,24-(28)-dien-3 beta-ol, respectively, were correspondingly increased. These findings suggest that 5 and 7 specifically inhibit the reduction of the delta 24(28) side chain double bond. When C. fasciculata was cultured in the presence of compound 4, ergosterol synthesis was suppressed, but the sterol distribution in these cells was complex and not easily interpreted. Compound 6 had no significant effect on sterol synthesis in C. fasciculata.

Levels of polyamines, glutathione and glutathione-spermidine conjugates during growth of the insect trypanosomatid Crithidia fasciculata.

Levels of the polyamines spermidine and putrescine and the major intracellular thiols glutathione (GSH), glutathionylspermidine (GSH-SPD) and dihydrotrypanothione [bis-(glutathionyl)spermidine); T[SH]2] were measured by high performance liquid chromatography throughout the growth cycle of the insect trypanosomatid Crithidia fasciculata. The amount of total spermidine, putrescine and glutathione (free and conjugated to spermidine) was found to be elevated during growth. Of the total spermidine, 30 to 50% was found conjugated to glutathione during the exponential growth phase, increasing to 60 to 70% at stationary phase. T[SH]2 was the principal intracellular thiol during exponential growth (12.1 to 17.4 nmol per 10(8) cells), whereas GSH-SPD was the major thiol in stationary phase (26.2 nmol per 10(8) cells). GSH levels changed little during the growth cycle and represented a constant proportion (10 to 12%) of the total intracellular glutathione. On dilution of stationary phase cells into fresh medium, a rapid decrease in GSH-SPD levels was observed to be associated with synthesis of T[SH]2. This process reached 90% completion by 15 min, with steady state achieved by 120 min. As the total spermidine and glutathione pools did not increase during this interval, it could be calculated that this rapid redistribution of metabolites resulted in the release of 13 nmol per 10(8) cells unconjugated spermidine without de novo synthesis. This mechanism for rapidly elevating the intracellular concentration of free spermidine may be advantageous to this organism in rapidly adapting to favourable growth conditions.

Efecto de quinonas y nitrofuranos sobre Trypanosoma mega y Crithidia fasciculata / Effect of quinones and nitrofurans on Trypanosoma mega and Crithidia fasciculata

La valoración in vitro de tripanocidas activos sobre Trypanosoma cruzi es el primer paso para el desarrollo de nuevos agentes terapéuticos contra la enfermedad de Chagas. Para verificar si una información equivalente puede lograrse con organismos no patógenos, se estudió la acción de varios tripanocidas de t. cruzi sobre T. mega y Crithidia fasciculada. Las drogas se ensayaron sobre el crecimiento de los protozoarios que se midió por la turbiedad de la suspensión celular en medio de cultivo líquido. Una serie de quinonas, incluyendo quinonas lipofílicas, lapachonas, quinina-iminas, benzoquinonas, un quinol (miconidina) y nitrofuranos (nifurtimox y análogos derivados del grupo (5-nitro-2-furfurilideno)-amino (grupo NF) inhibieron el crecimiento de los organismos, especialmente el de T. mega, con I50 menores de 5 micronM, para los compuestos mas activos. La sensibilidad de T. mega fue similar a la de T. cruzi y significativamente mayor que la de C. fasciculata. El cultivo de muestras de T. mega, preincubados con las mismas drogas,d emostró efectos irreversibles con los NF-derivados del pirazol, imidazol, indazol y benzimidazol pero no con el nifurtimox. En iguales condiciones, C. fasciculata fue afectada solamente por la ß-lapachona y una naftoquinona-imina. Estos resultados califican a T. mega como un modelo adecuado para el ensayo inicial de quimioterápicos anti-chagásicos, como lo son C. fasciculata y T. brucei para los tripanosomas africanos