The two phosphofructokinase gene products of Entamoeba histolytica.

Two phosphofructokinase genes have been described previously in Entamoeba histolytica. The product of the larger of the two genes codes for a 60-kDa protein that has been described previously as a pyrophosphate (PP(i))-dependent enzyme, and the product of the second, coding for a 48-kDa protein, has been previously reported to be a PP(i)-dependent enzyme with extremely low specific activity. Here it is found that the 48-kDa protein is not a PP(i)-dependent enzyme but a highly active ATP-requiring enzyme (k(cat) = 250 s(-)1) that binds the cosubstrate fructose 6-phosphate (Fru-6-P) with relatively low affinity. This enzyme exists in concentration- and ATP-dependent tetrameric active and dimeric inactive states. Activation is achieved in the presence of nucleoside triphosphates, ADP, and PP(i), but not by AMP, P(i), or the second substrate Fru-6-P. Activation by ATP is facilitated by conditions of molecular crowding. Divalent cations are not required, and no phosphoryl transfer occurs during activation. Kinetics of the activated enzyme show cooperativity with Fru-6-P (Fru-6-P(0.5) = 3.8 mm) and inhibition by high ATP and phosphoenolpyruvate. The enzyme is active without prior activation in extracts of E. histolytica. The level of mRNA, the amount of enzyme protein, and the enzyme activity of the 48-kDa enzyme are about one-tenth that of the 60-kDa enzyme in extracts of E. histolytica trophozoites.

Cloning and expression of the gene for the active PPi-dependent phosphofructokinase of Entamoeba histolytica.

Pyrophosphate-dependent phosphofructokinase (PPi-PFK) from Entamoeba histolytica (HM-1) was purified from trophozoites. Oligonucleotide probes based on partial amino acid sequence were used to clone and sequence the gene and the cDNA of the enzyme. The molecular mass of the subunit was greater than, and the derived sequence significantly different from, that of the product of the PPi-PFK gene previously cloned from E. histolytica [Huang, Albach, Chang, Tripathi and Kemp (1995) Biochim. Biophys. Acta 1260, 215-217; Bruchhaus, Jacobs, Denart and Tannich (1996) Biochem. J. 316, 57-63]. The sequence identity between the two proteins was 17%. The sequence bore greater identity with the more phylogenetically advanced plant PPi-PFKs than with bacterial PPi-PFKs. The cloned cDNA was expressed and the protein purified. The kinetic properties were identical with those of the enzyme isolated from the organism. Furthermore, the specific activity was more than three orders of magnitude higher than that described for the product of the previously cloned E. histolytica PFK gene [Bruchhaus et al. (1996)]. The pH-dependence and apparent substrate affinities of the cloned enzyme were identical with those of the PPi-PFK in trophozoite extracts, indicating that the product of the cloned gene accounts for most if not all of the PFK activity in E. histolytica trophozoites.

Cloning and sequencing a putative pyrophosphate-dependent phosphofructokinase gene from Entamoeba histolytica.

Pyrophosphate-dependent phosphofructokinase (PPi-PFK) gene from Entamoeba histolytica was cloned from its genomic library and sequenced. The open reading frame has 1149 bp and codes for a protein of 41.5 kDa. The deduced amino acid sequence of E. histolytica PPi-PFK has 25 to 28% identity to the PPi-PFKs from Propionibacterium freudenreichii, Naegleria fowleri and potato. The amino acid residues known to contribute to the active site of PPi-PFK from P. freudenreichii are conserved.

Nucleic acids of Entamoeba histolytica.

This review will concentrate on certain aspects of the nucleic acids of Entamoeba histolytica. Utilization and synthesis of purines and pyrimidines will initially be briefly discussed, e.g. salvage vs. de novo pathways, uptake studies and recognition of at least 4 transport loci. Data will be presented which show that the distribution and synthesis of RNA (to a lesser extent DNA) in the nucleus is basically the opposite one finds in other eukaryotes, viz. most RNA (ribosomal?) is synthesized (or accumulates) in the peripheral chromatin (functional equivalent of nucleolus?). The DNA is distributed and synthesized primarily throughout the nucleus. It is usually so dispersed that it will not stain with e.g. the standard Feulgen technique, unless the DNA condenses around the endosome (not a nucleolar equivalent) prior to nuclear division. Isolation of rRNA was difficult due, in part, to potent and difficult to inhibit RNase(s), some of which are apparently intimately bound to ribosomal subunits. The 25S (1.3 kDa), 17S (0.8 kDa) and 5S rRNA were recovered after isolation with a high salt SDS-DEP technique. This is the only procedure which enables us to obtain high yields of 25S rRNA; guanidine or guanidinium which permits isolation of intact functional mRNA results in isolation of small amounts of 25S RNA relative to 17S RNA. The 25S RNA is "nicked" (apparently during nuclear processing) and dissociates readily into 17S (0.7 kDa) and 16S (0.6 kDa) species, and a more rigidly bound 5.8S species. A small amount of "unnicked" 25S RNA was recovered with guanidine.(ABSTRACT TRUNCATED AT 250 WORDS)

Phagocytosis of Campylobacter jejuni and its intracellular survival in mononuclear phagocytes.

In vitro phagocytosis and intracellular survival of Campylobacter jejuni strain 2964 in mononuclear phagocytes were studied. The following three types of mononuclear phagocytes were used: a J774G8 peritoneal macrophage line derived from BALB/c mice, resident BALB/c peritoneal macrophages, and human peripheral blood monocytes. When C. jejuni and mononuclear phagocytes were combined at a ratio of 75:1, light microscopy, fluorescent microscopy, and electron microscopy all indicated that C. jejuni cells were readily phagocytized. The majority of C. jejuni cells were spirals immediately following ingestion and were rapidly converted to the coccal form within 4 to 8 h. Conversion from the spiral form to the coccal form was complete in the presence of phagocytes within 96 h. In control preparations without phagocytes, conversion began after 24 h and was complete after 48 h. The extent of phagocytosis over time was determined by observing Giemsa-stained preparations and counting the number of intracellular bacterial colony-forming units after removal of extracellular C. jejuni. Human monocytes ingested C. jejuni more rapidly and vigorously than murine macrophages. Intracellular survival of C. jejuni was examined by measuring the number of C. jejuni colony-forming units associated with phagocytes after phagocytosis for 2 h and removal of extracellular bacteria. C. jejuni survived intracellularly for up to 6 to 7 days.

Isolation and characterization of RNA of Entamoeba histolytica.

RNAs were isolated from Entamoeba histolytica with a high salt sodium dodecylsulfate-diethylpyrocarbonate technique. Majority species of 25 S, 17 S and 4 S RNAs were detected after sucrose gradient centrifugation. An additional 5 S RNA was detected by polyacrylamide gel electrophoresis. The molecular weights of these RNAs as determined by completely denaturing polyacrylamide gel electrophoresis were 1.31 X 10(6) (25 S), 0.803 X 10(6) (17 S), 4.0 X 10(4) (5 S) and 2.5 X 10(4) (4 S). The 25 S RNA was labile and dissociated under mild denaturing conditions (between 37 degrees C and 55 degrees C) into 17 S and 16 S RNAs with molecular weights of 0.700 X 10(6) and 0.614 X 10(6), respectively; under completely denaturing conditions an additional 5.8 S RNA with a molecular weight of 4.8 X 10(4) was detected. Evidence is presented which suggests that the lability of the 25 S RNA is the result of an in vivo cleavage rather than one which is generated during RNA isolation.

Purine nucleotide synthesis in Entamoeba histolytica: a preliminary study.

Entamoeba histolytica, axenically grown, were unable to incorporate 14C-glycine into extractable purines, even after 16 hours incubation in the presence of this compound. Similarly, amoebas did not incorporate 14C-sodium formate into purines. Such data indicate a total absence of a de novo purine biosynthetic pathway in this amoeba. When amoebas were incubated in the presence of 3H-adenine and 3H-adenosine for one hour, considerable radioactivity was associated with AMP, ADP and ATP; no appreciable radioactivity was recoverable in the various guanine residues. However, after 16 hours incubation, a small percentage of guanine derivatives were labelled. Amoebas incubated for similar time periods in medium containing 3H-guanosine yielded high levels of radioactive guanosine and guanine nucleotides after one hour, and 3H-guanosine was not converted to adenine derivatives; after 16 hours incubation some 3H-guanosine was converted to adenine derivatives.

Concepts of function of peripheral non-chromatin and endosome in Entamoeba histolytica.

The distribution of RNA (to a lesser extent DNA) in the nucleus of E. histolytica appeared to be the opposite of what one found in other eukaryotic cells (originally stated by Pan and Geiman in 1955). Autoradiographs of amebae labelled with 3H-thymidine revealed primarily randomly distributed DNA-3H (probably euchromatin) in interphase cells. This DNA was so low in amount in individual cells that it usually did not stain with the Feulgen procedure. A small amount of DNA-3H was also detected by autoradiography in the peripheral chromatin (= rDNA?; occasionally it was abundant) and endosome. Clumps of DNA-3H were also observed around the endosomal area in some amebae. This labelling pattern was not observed in 3H-uridine (for RNA) incorporation experiments. These data were consistent with the possibility that the endosome, which was sometimes Feulgen +, was a site of condensation of DNA prior to nuclear division. As detected by light and electron microscopy autoradiographic analysis of 3H-uridine incorporation, RNA was synthesized (or accumulated) primarily in the peripheral chromatin; lesser amounts were evenly distributed. Most of the peripherally located amebal nuclear RNA was probably rRNA and/or precursor rRNA. If this is the case, it is probable that the peripheral chromatin, although structurally unlike nucleoli, may be "functionally" comparable to nucleoli of eukaryotes.

Uptake of selected pyrimidines by axenically grown Entamoeba histolytica.

This study was conducted to determine the mode(s) of uptake of selected pyrimidine bases and nucleosides by axenically grown Entamoeba histolytica. Two-minute uptake studies with 3H-labeled compounds showed the following: 1) uridine and cytidine are taken up, in part, by a carrier mediated system; 2) uracil, cytosine, thymine and thymidine enter amebae via passive diffusion; both cytidine and uridine are taken up by passive diffusion when present at exogenous concentration greater than 500 muM; 3) specific carrier sites are used for transport of uridine-cytidine and uridine-adenosine; 4) cytidine and uridine uptake is markedly inhibited by iodoacetate and N-ethylmalemide; 5) hypoxanthine (10mM) stimulates uptake of uridine; 6) ribose fails to inhibit uptake of cytidine and uridine; 7) cytidine uptake exceeds that of uridine (i.e., apparent Vmax for: a) cytidine = 10.0 nmoles/2 min/10(6) amebae; b) uridine = 3.0 nmoles/2 min/10(6) amebae); 8) Kt for: cytidine = 0.30 mM; uridine = 0.21 mM; 9) Q 10's at 35.5 C vs 25.5 C for: a) cytidine = 3.9; b) uridine = 2.6.