Axenic Cultivation of Entamoeba dispar Brumpt 1925, Entamoeba insolita Geiman and Wichterman 1937 and Entamoeba ranarum Grassi 1879

ABSTRACT. Three species of Entamoeba have been grown in axenic culture for the first time. In two cases, novel methods for adapting the organisms to growth without bacteria were employed. While E. ranarum was axenized by the classic technique of Diamond, from a monoxenic culture with Trypanosoma cruzi as the associate, both E. dispar and E. insolita were first grown in axenic culture medium supplemented with lethally irradiated bacteria. From there, E. insolita was axenized directly, but E. dispar initially required the presence of fixed bacteria. After prolonged culture under this technically axenic but unwieldy culture system, E. dispar was eventually adapted to growth in the absence of added bacteria.     

Purine Metabolism in Trypanosomatids*

SYNOPSIS. Purine nucleotide biosynthesis was studied in culture forms of Trypanosoma cruzi strain Y, Crithidia deanei (a reduviid trypanosomatid with an endosymbiote) and an aposymbiotic strain of C. deanei (obtained by curing C. deanei with chloramphenicol). Trypanosoma cruzi was found to synthesize purine nucleotides only from the preformed bases adenine and guanine (“salvage” pathway), adenine being incorporated into both adenine and guanine nucleotides. Similar results were obtained with guanine, indicating that this flagellate has a system for the interconversion of purine nucleotides. Crithidia deanei was able to synthesize purine and pyrimidine nucleotides from glycine (“de novo” pathway) and purine nucleotides from adenine and guanine (“salvage” pathway). Adenine was incorporated into both adenine and guanine nucleotides, while guanine was incorporated into guanine nucleotides only, indicating the presence of a metabolic block at the level of GMP reducaase. The aposymbiotic C. deanei strain was unable to utilize glycine for the synthesis of purine nucleotides, although glycine was utilized for synthesizing pyrimidine nucleotides. These results suggest that the endosymbiote is implicated in the de novo purine nucleotide pathway of the C. deanei-endosymbiote complex. The incorporation of adenine and guanine by aposymbiotic C. deanei strain followed a pattern similar to that observed for C. deanei.     

Cryopreservation of Ochromonas in Liquid Nitrogen with Reproducibility of Thiamin Assay

The first successful cryopreservation of Ochromonas danica and Ochromonas malhamensis is reported. The freezing method was consistently reproducible for the former, but not for the latter. Ochromonas danica cultures established from frozen material still could be used as test organisms for assay of thiamin. This is the first report of a protozoon retaining its assay property after being frozen to -196 C.     

Methionine Regeneration and Aspartate Aminotransferase in Parasitic Protozoa

Aspartate aminotransferases have been cloned and expressed from Crithidia fasciculata, Trypanosoma brucei brucei, Giardia intestinalis, and Plasmodium falciparum and have been found to play a role in the final step of methionine regeneration from methylthioadenosine. All five enzymes contain sequence motifs consistent with membership in the Ia subfamily of aminotransferases; the crithidial and giardial enzymes and one trypanosomal enzyme were identified as cytoplasmic aspartate aminotransferases, and the second trypanosomal enzyme was identified as a mitochondrial aspartate aminotransferase. The plasmodial enzyme contained unique sequence substitutions and appears to be highly divergent from the existing members of the Ia subfamily. In addition, the P. falciparum enzyme is the first aminotransferase found to lack the invariant residue G197 (P. K. Mehta, T. I. Hale, and P. Christen, Eur. J. Biochem. 214:549-561, 1993), a feature shared by sequences discovered in P. vivax and P. berghei. All five enzymes were able to catalyze aspartate-ketoglutarate, tyrosine-ketoglutarate, and amino acid-ketomethiobutyrate aminotransfer reactions. In the latter, glutamate, phenylalanine, tyrosine, tryptophan, and histidine were all found to be effective amino donors. The crithidial and trypanosomal cytosolic aminotransferases were also able to catalyze alanine-ketoglutarate and glutamine-ketoglutarate aminotransfer reactions and, in common with the giardial aminotransferase, were able to catalyze the leucine-ketomethiobutyrate aminotransfer reaction. In all cases, the kinetic constants were broadly similar, with the exception of that of the plasmodial enzyme, which catalyzed the transamination of ketomethiobutyrate significantly more slowly than aspartate-ketoglutarate aminotransfer. This result obtained with the recombinant P. falciparum aminotransferase parallels the results seen for total ketomethiobutyrate transamination in malarial homogenates; activity in the latter was much lower than that in homogenates from other organisms. Total ketomethiobutyrate transamination in Trichomonas vaginalis and G. intestinalis homogenates was extensive and involved lysine-ketomethiobutyrate enzyme activity in addition to the aspartate aminotransferase activity. The methionine production in these two species could be inhibited by the amino-oxy compounds canaline and carboxymethoxylamine. Canaline was also found to be an uncompetitive inhibitor of the plasmodial aspartate aminotransferase, with a K(i) of 27 microm.     

Analysis of the Antigenic Relationships Among Trichomonas,Histomonas, Dientamoeba and Entamoeba III. Immunoelectrophoresis Technics*

Antigens prepared from Trichomonas gallinae, Histomonas meleagridis, Dientamoeba fragilis, Entamoeba invodens and Entamoeba histolytica were separated by electrophoresis in agar gels and reacted with antisera prepared in rabbits against each of the 5 species. The most numerous and strongest precipitin lines were obtained from reactions between the homologous antigens and antisera. Direct and cross-absorption reaction methods were employed with each antiserum and the various antigens to ascertain quantitatively the immunologic relationships among the several organisms. Trichomonas shared many common antigens with Histomonas, fewer with Dientamoeba and none with either species of Entamoeba. Histomonas was more closely related antigenically to Dientamoeba than to Trichomonas. The histomonad had only a few weakly cross-reacting antigens in common with the 2 Entamoeba species. Dientamoeba shared the most common antigens with Histomonas, fewer with Trichomonas and the fewest with Entamoeba. Somewhat stronger cross-reactivity was obtained with anti-Dientamoeba serum and E. invadens than between this immune serum and E. histolytica. The 2 species of Entamoeba shared the largest number of common antigens with each other, and to a much lesser extent both species cross reacted with Dientamoeba. Anti-Entamoeba sera had only a few weak cross-reacting precipitins with Histomonas. No antigenic relationship was found between either species of Entamoeba and Trichomonas.     

Long-Term Storage of Infective Microsporidian Spores in Liquid Nitrogen

Thirty-one species of microsporidia, isolated from insects and stored in liquid nitrogen for up to 25 yr, were infectious when removed from liquid nitrogen. The natural hosts of all of these microsporidia were terrestrial insects, representing six different insect orders: Coleoptera, Diptera, Hemiptera, Hymenoptera, Lepidoptera, and Orthoptera. All microsporidia from terrestrial insects that were tested survived storage in liquid nitrogen, while Nosema algerae , a microsporidium from aquatic mosquito hosts did not survive freezing in liquid nitrogen. A Nosema species from the alfalfa weevil, Hypera postica , lost some infectivity in a water storage medium after 25 yr in liquid nitrogen. Liquid nitrogen storage of microsporidian spores in 50% and 100% glycerol media reduced loss of infectivity and is recommended for extended storage of microsporidia from terrestrial insect hosts.     

Storage of Conidia of Penicillium chrysogenum in Liquid Nitrogen

Conidiated slope cultures of derivative of Penicillium chrysogenum Wis 54-1255 were stored at -196 or +4 C for a period of 3.5 years. After this time, the viability fell to 68% in the former case and to 4% in the latter. At the end of the experiment, 65 single conidial isolates from each series were tested for penicillin yield. Among those from conidia stored at -196 C, the spread of penicillin yields did not differ markedly from that of 65 single conidial isolates made as controls prior to storage. However, 18% of those from conidia stored at +4 C formed a subpopulation with substantially lower penicillin titers than those of control isolates. Storage at -196 C may reduce or prevent a possible source of penicillin yield decay, namely, the selection of spontaneous mutants of low titer present in small numbers in the original culture and selected, as viability decreased, by virtue of their increased longevity relative to that of the parental culture.     

Effect of Entomopathogenic Nematode Concentration on Survival during Cryopreservation in Liquid Nitrogen

Entomopathogenic nematodes are used for biological control of insect pests. A method for improved cryopreservation of infective juvenile stage nematodes has been developed using Steinernema carpocapsae and Heterorhabditis bacteriophora. Optimum survival for both species was achieved with 12,000 infective juveniles/ml in glycerol and 7,500/ml in Ringer''s solution. For S. carpocapsae, maximum survival also was observed with 60,000 infective juveniles/ml in glycerol and 25,000/ml in Ringer''s solution. These concentrations resulted in 100% post-cryopreservation survival of S. carpocapsae and 100% retention of original virulence to Galleria mellonella larvae. This is the first report of achieving 100% survival of an entomopathogenic nematode after preservation in liquid nitrogen. Maximum survival of H. bacteriophora following cryopreservation was 87%.     

Liquid Nitrogen Preservation of Standard Inoculum: Gas-Phase Storage

Liquid nitrogen storage was the most satisfactory of several methods tested for supplying standard Streptomyces viridoflavus inoculum for laboratory and pilot plant experimentation. Shake-flask cultures were subdivided into sterile cotton-plugged ampoules and stored in the gas space of a liquid nitrogen refrigerator. There were no detectable changes in viability or in candidin-producing capacity over a 12-month test period. The procedure also proved satisfactory with all other organisms tested.     

History of Staining the Staining of Blood and Parasitic Protozoa

By the term “blood stain” one ordinarily means a compound dye formed from the chemical union of an acid and a basic dye, and usually a compound of the eosin-methylene-blue group. It is well known today that the sodium salt of a color acid (e. g. eosin) and the chloride of a dye base (e. g. methylene blue) may be converted by simple metathesis into sodium chloride plus the compound dye (e. g. methylene blue eosinate), the latter being insoluble in water unless an excess is present of either the acid or the basic dye. In modern blood stains a compound dye of this type is dissolved in methyl alcohol and mixed with water on the slide at the moment of staining.     

Storage of Escherichia coli Strains Containing Plasmid DNA in Liquid Nitrogen

The stability of plasmids and lysogenic bacteriophage in 16 strains of Escherichia coli was studied following storage in liquid nitrogen for periods up to 2 years. A comparison was made with cultures which had undergone lyophilization. Plasmid loss was detected by plating on selective media containing antibiotics to which resistance was conferred by plasmid DNA. Loss of plasmid DNA was not evident following storage in liquid nitrogen or lyophilization.     

Cell-free Synthesis and Functional Characterization of Sphingolipid Synthases from Parasitic Trypanosomatid Protozoa

The Trypanosoma brucei genome has four highly similar genes encoding sphingolipid synthases (TbSLS1–4). TbSLSs are polytopic membrane proteins that are essential for viability of the pathogenic bloodstream stage of this human protozoan parasite and, consequently, can be considered as potential drug targets. TbSLS4 was shown previously to be a bifunctional sphingomyelin/ethanolamine phosphorylceramide synthase, whereas functions of the others were not characterized. Using a recently described liposome-supplemented cell-free synthesis system, which eliminates complications from background cellular activities, we now unambiguously define the enzymatic specificity of the entire gene family. TbSLS1 produces inositol phosphorylceramide, TbSLS2 produces ethanolamine phosphorylceramide, and TbSLS3 is bifunctional, like TbSLS4. These findings indicate that TbSLS1 is uniquely responsible for synthesis of inositol phosphorylceramide in insect stage parasites, in agreement with published expression array data (17). This approach also revealed that the Trypanosoma cruzi ortholog (TcSLS1) is a dedicated inositol phosphorylceramide synthase. The cell-free synthesis system allowed rapid optimization of the reaction conditions for these enzymes and site-specific mutagenesis to alter end product specificity. A single residue at position 252 (TbSLS1, Ser²⁵²; TbSLS3, Phe²⁵²) strongly influences enzymatic specificity. We also have used this system to demonstrate that aureobasidin A, a potent inhibitor of fungal inositol phosphorylceramide synthases, does not significantly affect any of the TbSLS activities, consistent with the phylogenetic distance of these two clades of sphingolipid synthases. These results represent the first application of cell-free synthesis for the rapid preparation and functional annotation of integral membrane proteins and thus illustrate its utility in studying otherwise intractable enzyme systems.     

Ornithine Decarboxylase and Trypanothione Reductase Genes in Leishmania braziliensis guyanensis

. Ornithine decarboxylase and trypanothione reductase are the key enzymes in polyamine and trypanothione metabolism in kinetoplastids. Using a heterologous Trypanosoma brucei brucei probe for ornithine decarboxylase and a mixed synthetic probe of 29 oligonucleotides for trypanothione reductase, we have detected the putative genes for these enzymes by Southern blot hybridization using genomic DNA of Leishmania braziliensis guyanensis MHOM/SR/80/CUMC I. The trypanothione reductase probe was constructed both from the conserved codon usage of the redox active site for other flavin oxidoreductases over a wide evolutionary scale, and the preferred codon usage for other genes in species of Leishmania .     

Cryopreservation of Trypanosoma cruzi Bloodstream Forms*

SYNOPSIS. Recovery rates of T. cruzi bloodstream forms subjected to several methods of cryopreservation in liquid nitrogen and at -73 C are reported. Inoculations of animals with cryopreserved and nonpreserved trypomastigotes revealed that prolonged storage at -196 C apparently did not change the biologic characteristics of different T. cruzi strains. The reproducibility and consistency of results suggest that “cryobanks'’or “reference centers'’may be established.     

Some Recent Advances in Our Understanding of Parasitic Protozoa*†

SYNOPSIS. Striking progress in our understanding of parasitic protozoa has been achieved thru the employment of advanced electron-microscopic, biochemical, immunologic, and cultivation methods. Some recent information gathered by means of these methods on trichomonad and trypanosomatid flagellates as well as on eimeriidian and plasmodiid haemosporiidian Sporozoa is discussed. It is emphasized that the parasitic protozoa when studied by the presently available sophisticated methods in the context of being parasites, not merely cells maintained on refined media, can aid us greatly in illuminating the highly complex functional aspects of host-parasite interactions.