Trichinella spiralis thymidylate synthase: cDNA cloning and sequencing, and developmental pattern of mRNA expression.

The persistent expression of thymidylate synthase activity has previously been demonstrated not only in adult forms, but also in non-developing muscle larvae of Trichinella spiralis and T. pseudospiralis, pointing to an unusual pattern of cell cycle regulation, and prompting further studies on the developmental pattern of T. spiralis thymidylate synthase gene expression. The enzyme cDNA was cloned and sequenced, allowing the characterization of a single open reading frame of 307 amino acids coding for a putative protein of 35,582 Da molecular weight. The amino acid sequence of the parasite enzyme was analysed, the consensus phylogenetic tree built and its stability assessed. The aa sequence identity with thymidylate synthase was confirmed by the enzymatic activity of the recombinant protein expressed in E. coli. As compared with the enzyme purified from muscle larvae, it showed apparently similar Vmax value, but higher Km(app) values desscribing interactions with dUMP (28.8 microM vs. 3.9 microM) and (6RS,alphaS)-N(5,10)-methylenetetrahydrofolate (383 microM vs. 54.7 microM). With the coding region used as a probe, thymidylate synthase mRNA levels, relative to 18S rRNA, were found to be similar in muscle larvae, adult forms and newborn larvae, in agreement with muscle larvae cells being arrested in the cell cycle.

Trichinella spiralis and Trichinella pseudospiralis: developmental patterns of enzymes involved in thymidylate biosynthesis and pyrimidine salvage.

Thymidylate synthase, dihydrofolate reductase and dUTPase specific activities were found to remain at a high and constant level in crude extracts from adult worms of Trichinella spiralis, as well as from muscle larvae of both Trichinella spiralis (isolated 1-24 months after infection) and Trichinella pseudospiralis (isolated 5.5-13 months after infection). The results obtained with Trichinella pseudospiralis muscle larvae isolated with the use of pepsin did not differ from those obtained when pepsin was not used. No thymidine kinase activity could be detected in muscle larvae of either species and thymidine phosphorylase could be found only in T. pseudospiralis larvae isolated without the use of pepsin. Muscle larvae of both species contained orotidylate phosphoribosyl transferase activity, pointing to a possibility of 5-fluorouracil activation. Uridine phosphorylase, another enzyme involved in 5-fluorouracil anabolism, was also present in T. pseudospiralis muscle larvae. Results of comparative studies on inhibition of purified T. spiralis and rat thymidylate synthases by substrate (4-thio-5-fluoro-dUMP, 2-thio-5-fluoro-dCMP and N4-hydroxy-dCMP) and cofactor (ZD 9331) analogues indicated only dUMP analogues to show feeble selectivity towards the parasite enzyme. A hypothesis is discussed, assuming high expression of thymidylate synthase in muscle larvae to be connected with their cells being arrested in the cell cycle.

Trichinella spiralis: the infectivity of synchronous newborn larvae of different ages inoculated intraocularly.

Trichinella spiralis infection results in the transformation of muscle cells into a new, non-muscular cell called the nurse cell, and the nurse cell-muscle larva complex is finally created. To investigate whether T. spiralis infectivity is NBL age-dependent, five groups of synchronous newborn larvae (sNBL) were obtained at 1, 9, 24, 48, and 72 h of age and were inoculated into mice by intravenous injection into the retro-orbital venous plexus. When both "young" groups of sNBL (1 and 9 h old) were injected, the highest number of larvae were capable of infecting the muscle cells. The highest infectivity of 80.0 % was observed for 9-h-old sNBL. In older sNBL the infectivity gradually decreased; thus, for 72-h-old sNBL the lowest level - 0.1% - was detected. Therefore, an "age limit" for NBL infectivity in the present study was precisely determined.

Trichinella spiralis: proteinases in the larvae.

Under in vitro conditions, muscle larvae of Trichinella spiralis secreted minute amounts of a cysteine proteinase into the outer environment from the stichosome. The proteinase hydrolyzed azocoll at pH 5.0 but not a number of synthetic N-blocked and N-unsubstituted proteinase substrates at this pH. The reducing compound dithioerythritol enhanced the enzyme activity, but the thiol-blocking reagent sodium-p-hydroxymercuribenzoate (0.1 mM) was without effect. Phenylmethylsulfonyl fluoride (PMSF) (2 mM) and leupeptin (100 mM) produced partial and complete inhibition, respectively, whereas soybean trypsin inhibitor, pepstatin A, and 1,10-phenanthroline were non-inhibitory. Calcium (1 mM) produced a slight decrease in the activity that was reversed by 1 mM EGTA. Although multiple proteinase activities were detected histochemically in the somatic muscles, stichosome, midgut, and genital primordium of the muscle larvae, none of these enzymes appeared to be the one secreted. Several histochemically demonstrable proteinases were also found in the cells of 48- to 72-h-old juveniles of the parasite. One was localized in the esophageal lumen and at or around the anterior esophagus of the larvae, where developing stichocytes are believed to occur. The proteinase hydrolyzed N-acetyl-L-methionine-L-naphthyl ester and was sensitive to the metal cation-complexing compound EGTA as well as to PMSF, an inhibitor of serine proteinases.

Trichinella pseudospiralis: X-ray analysis of the infected muscle cells and the muscle larva compartments after exponential growth of the larva in situ.

The distribution of nutrients such as Na, Cl, K, P, S, and Ca was investigated by energy-dispersive X-ray microanalysis (EDXA) on bulk-frozen mouse muscles infected with Trichinella pseudospiralis. In an attempt to gain information as to whether muscle larvae would modify the element status within muscles of their host. Significant increases in phosphorus concentrations within nurse cells and internal cells of the larva, e.g., the somatic muscle cells and the stichocytes, were shown. This could reflect changes in the energy metabolism and/or in the turnover of nucleic acids of the nurse cell. High phosphorus concentrations within the internal larva cells corresponded to high levels of expression of thymidylate synthase shown elsewhere in both T. pseudospiralis and T. spiralis muscle larvae, most likely reflecting more intensive DNA replication. Furthermore, the Na+-concentration gradient between the nurse cell and neighboring host muscle cells indicated the plasma membrane as a diffusion barrier for Na+. Moreover, the cuticle seemed to maintain the Cl--concentration gradient between the nurse cell and the internal cells of the larva. Nevertheless, the mechanisms involved in the transport of both ions, the collagen cuticle appeared, strong permeability that serves the transcuticular transport maintaining the Cl--concentration gradient.

Trichinella spiralis: induction of the basophilic transformation of muscle cells by synchronous newborn larvae. II. Electron microscopy study.

The present ultrastructure observations apply to the same experimental groups that were investigated in the first part of the study. The results of electron microscopy investigations correspond to those obtained using light microscopy methods. The ultrastructure data demonstrated that 1-h-old sNBL (group I) penetrated into the sarcoplasm of the muscle cells and transformed it basophilically, finally creating the "nurse cell-muscle larva complex." These larvae also caused transformation of the same muscle cells without being present in the sarcoplasm. The larvae of group II (9-h-old sNBL) preserved transformation potential as well, but it was less intensive. Not all NBL settled in the muscle cells; some of them remained in the intercellular spaces. Group II larvae present in the muscle cells underwent early degeneration and necrosis more often than did group I larvae; the inflammatory cell reactions in the vicinity of the larvae were more intensive. The basophilic transformation of muscle cells that did not contain larvae in their sarcoplasm was not intensive and often stopped at the level of cell nuclei. The larvae of group III (6-day-old sNBL) neither settled in the muscle cells nor transformed the cell sarcoplasm.

Trichinella spiralis: induction of the basophilic transformation of muscle cells by synchronous newborn larvae.

Newborn larvae (NBL) possess the "transformation potential" (TP) to induce the basophilic transformation of the muscle cell into the nurse cell and to create the nurse cell-muscle larva complex. For investigation as to whether TP is age-dependent, three groups of synchronous NBL (sNBL) were obtained at 1 h, 9 h, and 6 days of age, respectively, and were injected intramuscularly into mice. The TP was demonstrated to be high for both 1- and 9-h-old sNBL, but for 6-day-old sNBL, none was found. The earliest transformation of muscle cells was observed at 48 h postinfection (p.i.) in the first two groups of sNBL. Moreover, for 1-h-old sNBL the numbers of basophilically transformed muscle cells without larvae were 3-fold those of sNBL observed in histology sections. The results indicate that (1) due to the high TP observed in the 1-h-old sNBL group, NBL are born with TP; (2) the TP is lost by aging; and (3) the induction of the basophilic transformation of muscle cells is possible without direct contact between the muscle cell and NBL being required, as contact with released factor(s) alone may be enough.

Trichinella spiralis thymidylate synthase: developmental pattern, isolation, molecular properties, and inhibition by substrate and cofactor analogues.

Thymidylate synthase specific activity was found to remain at a constant level in crude extracts from muscle larvae, isolated (1-15 months after infection) by pepsin-HCI digestion, as well as from adult worms of Trichinella spiralis. The enzyme was purified and its molecular (monomer mol. wt 35 kD) and kinetic (sequential mechanism with the K(m) values 3.1 and 19 microM for dUMP and N5,10-methylenetetrahydrofolate, respectively) properties determined. 5-Fluoro-dUMP was a competitive, slow-binding inhibitor of the parasite enzyme. N5,10-methylenetetrahydrofolate analogues 10-propargy1-5,8- dideazafolate (CB3717), ZD1694, BW1843U89, and AG337 were weaker inhibitors of the parasite than regenerating rat liver enzyme. Inhibition by 10-propargyl-5,8-dideazafolate was strengthened by an increasing number of glutamate residues. Thymidine kinase activity could not be detected in the muscle larvae crude extracts.

Trichinella spiralis: X-ray analysis of the nurse cell-muscle larva complex after exponential growth of the larva.

The energy-dispersive X-ray analysis (EDXA) method was employed to determine how the exponential growth of the Trichinella spiralis larva affect the concentration of some metabolically active elements such as phosphorus, sulfur, sodium, chlorine, potassium, and calcium in host myocytes as well as inside the nurse cell-muscle larva complex. Two observations are noteworthy. First, the capsule of the complex reveals the highest Na+ and Cl-concentrations. We would suggest that these high Na+ and Cl-concentrations could be a part of the capsule-localized transport mechanisms covering the metabolic demands of the larva. Second, a substantial increase in the P concentration in the cells inside the complex, i.e., the nurse cell, somatic muscle of the larva, and larval stichosome was found. We postulate that energy metabolism in the nurse cell is entirely dependent on the metabolic requirements of the larva and that they remain together as one functional unit.