Entamoeba histolytica and Giardia lamblia: Growth responses to reducing agents

The growth responses of Entamoeba histolytica strains HM-1:IMSS and HK-9 to a variety of reducing agents were tested for one subculture in TYI-S-33 medium, prepared with no cysteine or ascorbic acid. Amoebae did not grow in this medium. Addition of l-ascorbic acid, d- or l-cysteine, or l-cystine each permitted the maximum growth observed. Dithiothreitol supported 68% maximum growth of HK-9 amoebae, but only 12% of HM-1. In contrast, growth of both strains was greatly diminished (0–13% growth) with 11 other compounds tested including glutathione, thiomalic acid, thioglycolic acid, and methionine. The growth responses of Giardia lamblia were similarly tested in TYI-S-33, as well as in TP-S-1 media. If l-cysteine was omitted from either medium, trophozoites did not grow, and eventually lysed. In TYI-S-33 medium, the requirement for l-cysteine was specific, whereas in TP-S-1 medium, other sulfhydryl compounds were partially effective and lower concentrations of l-cysteine satisfied the requirement. Ascorbic acid or l-cystine alone was totally ineffective; however, in combination, 30 to 60% of maximum growth was achieved. Once added to either medium, cysteine was rapidly oxidized. Amino acid analysis of the growth media revealed that the broth components of TP-S-1 medium contained 2.8 mM and TYI-S-33 broth 2.1 mM endogenous levels of cysteine (or half-cystine), with an additional 3 mM contributed by 10% serum.     

Entamoeba histolytica: Nucleic acid precursors affecting axenic growth

Treatment of Panmede and Trypticase, in water, with activated carbon and subsequently combining the filtered solution with glucose, cysteine, salts, serum and vitamins results in a medium depleted in the nucleic acid precursors required to sustain a high level of growth of axenic Entamoeba histolytica. Additions to the depleted medium which stimulated sustained growth, were the following, in the order of increasing efficacy: adenosine, adenine, AMP, AMP + GMP, AMP + GMP + UMP + CMP, or yeast ribonucleic acid. The last three additions restored growth to the level of cultures in TP-S-1 medium. No stimulation of sustained growth was found with GMP, IMP, UMP, or CMP, singly.     

Entamoeba histolytica and Giardia lamblia: effects of cysteine and oxygen tension on trophozoite attachment to glass and survival in culture media

Attachment of Entamoeba histolytica and of Giardia lamblia trophozoites to glass was monitored during the culture cycle. Attachment of each parasite was greatest during the exponential phase of axenic growth. The effects of l-cysteine upon the kinetics of attachment of trophozoites to glass were determined quantitatively. Attachment in complex growth media required cysteine, even under N₂, atmosphere. With cysteine, the rates of attachment were greatest for the first 2 hr, then continued more slowly. The numbers of attached trophozoites decreased immediately upon exposure to medium without cysteine. The role of cysteine in protecting trophozoites of both species from the lethal effects of oxygen was assessed using clonal growth in agar or agarose medium to determine viability following exposure to varying oxygen tensions in liquid medium. Cysteine was required for viability of trophozoites. Without cysteine, decreasing the oxygen tension prolonged survival. Under increased oxygen tension, cysteine delayed the onset of exponential killing. Although it has no thiol reducing group, l-cystine similarly protected E. histolytica.     

Entamoeba histolytica: Oxygen resistance and virulence

Entamoeba histolytica virulence has been attributed to several amoebic molecules such as adhesins, amoebapores and cysteine proteinases, but supporting evidence is either partial or indirect. In this work we compared several in vitro and in vivo features of both virulent E. histolytica (vEh) and non-virulent E. histolytica (nvEh) axenic HM-1 IMSS strains, such as complement resistance, proteinase activity, haemolytic, phagocytic and cytotoxic capacities, survival in mice caecum, and susceptibility to O₂. The only difference observed was a higher in vitro susceptibility of nvEh to O₂. The molecular mechanism of that difference was analyzed in both groups of amoebae after high O₂ exposure. vEh O₂ resistance correlated with: (i) higher O₂ reduction ( and H₂O₂ production); (ii) increased H₂O₂ resistance and thiol peroxidase activity, and (iii) reversible pyruvate: ferredoxin oxidoreductase (PFOR) inhibition. Despite the high level of carbonylated proteins in nvEh after O₂ exposure, membrane oxidation by reactive oxygen species was not observed. These results suggest that the virulent phenotype of E. histolytica is related to the greater ability to reduce O₂ and H₂O₂ as well as PFOR reactivation, whereas nvEh undergoes irreversible PFOR inhibition resulting in metabolic failure and amoebic death.     

Entamoeba histolytica: Virulence enhancement of isoenzyme-stable parasites

Pathogenic Entamoeba histolytica isolated from patients with clinical amoebiasis can be differentiated from nonpathogenic E. histolytica obtained from asymptomatic carriers on the basis of the electrophoretic pattern of their isoenzymes. Virulence of different strains of axenically grown trophozoites of Entamoeba histolytica, as determined by various laboratory tests, such as damage to tissue culture monolayers, or their ability to cause an hepatic abscess in a hamster, are known to vary considerably. Reassociation of trophozoites of strain HK-9 with certain Escherichia coli strains for short periods of time markedly augmented their virulence, as tested by the above-mentioned methods. The bacterial association, however, did not cause any change in the electrophoretic pattern of amoebic isoenzymes (zymodeme).     

Giardia lamblia: Evaluation of roller bottle cultivation

A modified “outside-in” roller bottle with a high ratio of surface area to volume was used to cultivate Giardia lamblia. Yields were high, more so when bottles were rotated at 6 rph (9.3 ± 4.0 × 10⁸ trophozoites/bottle) than at 12 rph (4.2 ± 1.9 × 10⁸ trophozoites/bottle). The method was more efficient than stationary tube culture with respect to utilization of culture medium; trophozoite concentration after roller bottle culture (1.7 ± 0.8 × 10⁶ trophozoites/ml) was significantly higher (by a factor of 2.8) than concentrations obtained from stationary tube culture (0.6 ± 0.4 × 10⁶ trophozoites/ml, P < 0.002). Increased yields from roller bottle culture were not accounted for by a reduction in mean trophozoite generation time (roller culture, 10.7 ± 1.2 hr; stationary tube culture, 10.3 ± 0.6 hr) but may be related to prolongation of the period of log phase growth or increased trophozoite survival. Trophozoite yields expressed per unit surface area were significantly higher from roller bottle culture (7.2 ± 3.1 × 10⁵ trophozoites/cm²) than from stationary tubes (1.9 ± 1.0 × 10⁵ trophozoites/cm², P < 0.002). Attempts to cultivate G. lamblia in spin culture using polystyrene beads (Biosilon) as a microcarrier were unsuccessful, trophozoite growth being inhibited rather than promoted. Roller bottle culture of G. lamblia, however, is efficient, economical, and less laborious than stationary tube culture, particularly when more than 10⁸ trophozoites are required.     

Eimeria nieschulzi and Trichinella spiralis: Analysis of concurrent infection in the rat

The effects of concurrent primary infection of the rat with Eimeria nieschulzi and Trichinella spiralis on the number of oocysts of E. nieschulzi shed by the host and on the number, distribution, and fecundity of adult T. spiralis were analyzed. When rats were initially infected with E. nieschulzi followed 9 days later by infection with T. spiralis there occurred a significant decrease in the total numbers of adult worms in the small intestine, a significant shift in the position of these worms along the length of the small gut, a decrease in the fecundity of adult female worms, and a decrease in muscle parasitism when compared with rats infected with T. spiralis alone. When rats were initially infected with T. spiralis, followed 9 days later by infection with E. nieschulzi, there occurred a significant decrease in the numbers of oocysts shed over 24 hr on Days 7, 9, and 11 postinfection below that seen with rats infected only with Eimeria. These changes are discussed in terms of the enteropathophysiologic lesions and enteric inflammation known to occur during infections with these two parasites.     

Tritrichomonas foetus: Localization of filipin-sterol complexes in cell membranes

The polyene antibiotic, filipin, was used as a probe for the detection of sterols in the freeze-fractured plasma membrane and the flagellar membranes of the pathogenic protozoa, Tritrichomonas foetus. A homogeneous distribution of filipin-sterol complexes was seen throughout the plasma membrane, and the membrane of the three anterior and the one recurrent flagella. No or very few filipin-sterol complexes were observed in some specialized regions such as the base of the flagella (necklace), the portion of the recurrent flagellum, and that part of the cell body to which the flagellum was attached. The density of filipin-sterol complexes varied from one cell to the other. In some cells, about 205 complexes/μm² were seen. A larger number of filipin-sterol complexes were observed on both faces of the membrane of cytoplasmic structures, probably corresponding to vacuoles. No complexes were seen in the nuclear membrane and in the membrane of the endoplasmic reticulum. Very few or no complexes were observed in the membrane of the hydrogenosomes. Treatment of living cells with filipin induced aggregation of filipin-sterol complexes at some points of the plasma membrane.     

Effects of temperature on survival, development, growth and feeding of larvae of Yellowtail clownfish Amphiprion clarkii (Pisces: Perciformes)

To understand the physiological and ecological responses of marine fishes to the change of water temperature, newly-hatched larvae of Yellowtail clownfish Amphiprion clarkii were reared in captivity at water temperatures of 23, 26 and 29 °C till they completed the metamorphosis to juvenile phase, and larval survival, development, growth and feeding were evaluated during the experimental period. The results showed that water temperature influenced the physiological performance of larvae of A. clarkii significantly. The survival and growth rates of larvae of A. clarkii increased significantly with the increase of water temperature from 23 to 29 °C (P < 0.05). Water temperature also influenced larval development of A. clarkii significantly and larvae reared at 23 °C took longer time for post-larval development and metamorphosis compared to 26 and 29 °C (P < 0.05). Total length and body weight for post-larval development and metamorphosis decreased with the increase of water temperature from 23 to 29 °C (P < 0.05). Q₁₀ in developmental rate was higher than in daily growth rate at the same rearing temperature, indicating that at water temperature had greater influence on larval development than on growth. Water temperature also influenced larval feeding of A. clarkii significantly with feed ration (FR) and feed conversion efficiency (FCE) increased with the increase of water temperature from 23 to 29 °C (P < 0.05). There was a positive correlation between FR and specific growth rate (SGR) (P < 0.05) but not between FCE and SGR (P > 0.05), indicating that FR influenced growth rate significantly in larvae of A. clarkii. This study demonstrated that the physiological responses of larvae of A. clarkii to the change of water temperature and confirmed that water temperature influenced larval survival, development, growth and feeding significantly. This study suggests that the decline of larval survival and growth rates, extension of pelagic larval duration and reduction of larval feeding at lower temperature have ecological impacts on larval dispersal and metamorphosis, juvenile settlement and population replenishment in A. clarkii in the wild.     

Trypanosoma congolense: Thrombocyte survival in infected steers

Charolais steers infected with Trypanosoma congolense developed a thrombocytopenia that was first demonstrated shortly before the onset of parasitemia. The thrombocyte count progressively decreased from a level of 6 × 10⁵/mm³ on the 3rd day postinfection to l × 10⁵/mm³, its most depressed level, on the 11th day postinfection. The mean of the thrombocyte level of five infected bovines at the time of thrombocyte survival analysis was 195.6 ± 83.5 × 10³(± 2 SE) as compared to 998 ± 245.9 × 10³ in the control group. In parallel with depressed thrombocyte levels, the mean of the apparent half-lives of ⁵¹Cr-labeled thrombocytes was 1.3 ± 0.5 days as compared to 3.7 ± 0.5 days in control animals. A similar survival was noted in the apparent half-lives of ⁵¹Cr-labeled autologous and heterologous thrombocytes transfused to normal recipients.     

Entamoeba histolytica: Identification and partial characterization of α-mannosidase activity

Despite their well recognized importance in pathogenesis of Entamoeba histolytica there are few studies dealing with the assembly and secretion of glycoproteins that participate in the adhesion to target cells and in the dissemination of the parasite in infected tissues. Some of these studies refer to the identification and, in some cases, the characterization of glycosyl transferases and glycosidases involved in the biosynthesis of these macromolecules as well as to compartments involved in the amoeba dolichol-linked glycosylation pathway. While an N-glycan trimming α-mannosidase has been demonstrated in E. histolytica, little is known on its cellular distribution and properties. Here we describe the presence and partial biochemical characterization of soluble and MMF-associated forms of α-mannosidase and the separation of at least three internal membrane structures enriched with this glycosidase. Results are discussed in terms of the possible identity of α-mannosidase activity and the potential precursor-product relationship between the two enzyme forms.     

Trypanosoma cruzi: Isolation and characterization of a proteinase

Lysates of Trypanosoma cruzi epimastigotes were able to hydrolyze casein (K_m = 2.5 mg/ml) as well as bovine and human hemoglobins (K_m = 12.2 mg/ml); there was optimum activity was around pH 7.0. The proteinase activity detected with these substrates was enhanced by sodium diaminotetraacetate (EDTA) and reducing agents (SO^2?₃, mercaptoethanol, cysteine) and was inhibited by sulfhydryl reagents, thus suggesting an SH-dependent enzyme. Purification (60×) of the proteinase was carried out as follows: (1) precipitation at ?20 C, pH 4.5, with 80% acetone, (2) gel filtration on Sephadex G-200, (3) affinity chromatography on Sepharose 4B covalently linked to p-aminophenyl mercuric acetate. Only a single component (with an estimated molecular weight of 60,000) was detected in purified preparations by polyacrylamide gel electrophoresis. However, in addition to the major component identified as a proteinase, crossed immunoelectrophoresis experiments indicated the presence of at least three other antigens that apparently were devoid of proteinase activity. Optimum pH activity of the purified preparations was around pH 6.0 for casein and pH 3.0 for hemoglobins, but these activities probably are due to the one enzyme since they were altered identically by the same agents.     

Babesia microti: Biochemistry and function of hamster erythrocytes infected from a human source

Babesia microti, a protozoan parasite of mammalian erythrocytes was obtained from the blood of an infected human and maintained in golden hamsters, in which a parasitemia of 70% was obtained regularly. The hamsters' response—a subacute, hemolytic anemia—was studied with regard to oxygen affinity and red cell organic phosphate content. In addition, the reduced glutathione status of infected erythrocytes was observed because of the possible importance of this metabolite to parasite growth and red cell integrity. Infected animals developed a severe anemia with reticulocytosis; there occurred a 4-mm decrease in whole blood oxygen affinity without any change in erythrocytes' 2,3-diphosphoglycerate levels. The glutathione content of the infected animals' erythrocytes increased twofold during the course of the infection. In uninfected animals, in which anemia and reticulocytosis had been produced by bleeding, all changes seen in infected animals were reproduced. It was concluded that the changes in the infected animals were due to the anemia and reticulocytosis alone, and that the parasite played no role in these changes apart from being a cause of anemia and reticulocytosis.     

Entamoeba histolytica: Molecular cloning and characterization of a novel neutral sphingomyelinase

A novel neutral sphingomyelinase (nSMase) was characterized in Entamoeba histolytica trophozoites. SMase, a sphingomyelin-specific form of phospholipase C, catalyzes the hydrolysis of sphingomyelin to ceramide and phosphorylcholine. Three amebic putative nSMase genes were found to be actively transcribed. Mg²⁺-independent nSMase activity in the soluble fraction of the trophozoites was stimulated by Mn²⁺ and partially inhibited by Zn²⁺. nSMase activity of the recombinant protein EhnSM1, increased 4.5-fold in the presence of 0.5 mM Mn²⁺, and abolished by 5 mM Zn²⁺. A dose-dependent inhibition of rEhnSM1 was observed with scyphostatin, a specific inhibitor of nSMases. The EhnSM1 and EhnSM3 were detected in the soluble fraction of the amebic lysate as 35-37 kDa proteins by western blot analysis. Immunofluorescence assay showed that the overexpressed HA-tagged EhnSM1 and EhnSM3 were localized to the cytosol. The biological role of these novel E. histolytica nSMases described in this work remains to be determined.     

Entamoeba histolytica thioredoxin reductase: Molecular and functional characterization of its atypical properties

Background

Entamoeba histolytica, an intestinal protozoan that is the causative agent of amoebiasis, is exposed to elevated amounts of highly toxic reactive oxygen and nitrogen species during tissue invasion. Thioredoxin reductase catalyzes the reversible transfer of reducing equivalents between NADPH and thioredoxin, a small protein that plays key metabolic functions in maintaining the intracellular redox balance.

Methods

The present work deals with in vitro steady state kinetic studies aimed to reach a better understanding of the kinetic and structural properties of thioredoxin reductase from E. histolytica (EhTRXR).

Results

Our results support that native EhTRXR is a homodimeric covalent protein that is able to catalyze the NAD(P)H-dependent reduction of amoebic thioredoxins and S‐nitrosothiols. In addition, the enzyme exhibited NAD(P)H dependent oxidase activity, which generates hydrogen peroxide from molecular oxygen. The enzyme can reduce compounds like methylene blue, quinones, ferricyanide or nitro-derivatives; all alternative substrates displaying a relative high capacity to inhibit disulfide reductase activity of EhTRXR.

Conclusions and general significance

Interestingly, EhTRXR exhibited kinetic and structural properties that differ from other low molecular weight TRXR. The TRX system could play an important role in the parasite defense against reactive species. The latter should be critical during the extra intestinal phase of the amoebic infection. So far we know, this is the first in depth characterization of EhTRXR activity and functionality.     

Members of the Entamoeba histolytica transmembrane kinase family play non-redundant roles in growth and phagocytosis

Entamoeba histolytica contains a large and novel family of transmembrane kinases (TMKs). The expression patterns of the E. histolytica TMKs in individual trophozoites and the roles of the TMKs for sensing and responding to extracellular cues were incompletely characterised. Here we provide evidence that single cells express multiple TMKs and that TMK39 and TMK54 likely serve non-redundant cellular functions. Laser-capture microdissection was used in conjunction with microarray analysis to demonstrate that single trophozoites express more than one TMK gene. Anti-peptide antibodies were raised against unique regions in the extracellular domains of TMK39, TMK54 and PaTMK, and TMK expression was analysed at the protein level. Flow cytometric assays revealed that populations of trophozoites homogeneously expressed TMK39, TMK54 and PaTMK, while confocal microscopy identified different patterns of cell surface expression for TMK39 and TMK54. The functions of TMK39 and TMK54 were probed by the inducible expression of dominant-negative mutants. While TMK39 co-localised with ingested beads and expression of truncated TMK39 interfered with trophozoite phagocytosis of apoptotic lymphocytes, expression of a truncated TMK54 inhibited growth of amoebae and altered the surface expression of the heavy subunit of the E. histolytica Gal/GalNAc lectin. Overall, our data indicates that multiple members of the novel E. histolytica TMK family are utilised for non-redundant functions by the parasite.     

Entamoeba histolytica: uptake of purine bases and nucleosides during axenic growth.

A comparison was made of the uptake mechanisms of selected purine bases and nucleosides by axenically grown Entamoeba histolytica. Adenine, adenosine, and guanosine were taken up, in part, by a “carrier”-mediated system. Guanine, hypoxanthine, and inosine entered amoebas via diffusion. Inhibitor studies support the presence of individual transport sites for adenine-adenosine and adenosine-guanosine. Additional sites for transport of adenine, adenosine, and guanosine are implied by “non-productive binding” involving guanine, hypoxanthine, and inosine. Uptake of adenine, adenosine, and guanosine was reduced by iodoacetate and N-ethylmaleimide. Ribose failed to inhibit uptake of purine nucleosides.     

Entamoeba histolytica: Effect on virulence, growth and gene expression in response to monoxenic culture with Escherichia coli 055

Monoxenic cultivation of pathogenic Entamoeba histolytica trophozoites with Escherichia coli serotype 055 which binds strongly to the Gal/GalNAc amoebic lectin, markedly improved the growth of E. histolytica and produced a significant decrease in cysteine proteinase activity and a lower cytopathic activity on monolayer cells after 3 months of monoxenic culture. However, after long term monoxenic culture (12 months) the proteolytic and cytopathic activities were recovered and the amoebic growth reached the maximum yield. Employing the GeneFishing^R technology and DNA macroarrays we detected differentially gene expression related to the amoebic interaction with bacteria. A number of differentially expressed genes encoding metabolic enzymes, ribosomal proteins, virulence factors and proteins related with cytoskeletal and vesicle trafficking were found. These results suggest that E. coli 055 has a nutritional role that strongly supports the amoebic growth, and is also able to modulate some biological activities related with amoebic virulence.     

Influence of oxygen and temperature on growth and metabolic performance of Paralichthys lethostigma (Pleuronectiformes: Paralichthyidae)

In this study, we apply Fry's classification of environmental factors to demonstrate the limiting effects of oxygen and its interaction with temperature on the growth of juvenile P. lethostigma. We also evaluated the properties of two metabolic indices, marginal metabolic scope (MMS) and limiting oxygen concentration (LOC), as indicators of metabolic scope. We found that oxygen limitation has its greatest impact near the optimum temperature for growth of the species. At 29 °C a reduction from 6.00 mg/L to 4.00 mg/L caused a 50% reduction in growth rate while at 27 °C the reduction had no significant effect on growth rate. The results are particularly relevant because these temperatures and oxygen concentrations are commonly observed in nursery areas during summer months. At all temperatures fish from the lowest oxygen treatment (1.75 mg/L) had negative growth rates. Comparisons between daily oscillating oxygen treatments and constant treatments failed to demonstrate significant effects. At temperatures past the optimum, growth rates between the 6.00 mg/L and 4.00 mg/L treatments were not statistically different. LOC was significantly affected by temperature, oxygen, and their interaction. Estimates were positively correlated with oxygen treatment (R² > 0.71) and negatively correlated with temperature at moderate and low oxygen concentrations (R² > − 0.84). MMS was significantly affected by temperature and oxygen and was significantly correlated with oxygen treatment (R² > − 0.91), but correlations with temperature were not as clear. In conclusion, oxygen and temperature interactions have significant effects on metabolic scope and growth rates of fish, well above the accepted hypoxia threshold of 2.00 mg/L and MMS has proved a useful estimator of the metabolic scope of the organism within an environment.     

Eimeria meleagrimitis, E. adenoeides, and E. dispersa: Severity of infection and changes in the intestinal mucosa of the Turkey

Glucose and methionine were malabsorbed in some intestinal regions of turkeys infected with Eimeria meleagrimitis, E. adenoeides, or E. dispersa. The decrease in absorption was not always related to the numbers of parasites in the cells or the extent of damage to the mucosa. With E. adenoeides, malabsorption was found in the jejunum even though parasites were not present. Conversely, with E. dispersa, no malabsorption was observed in the duodenum even though light microscopy showed numerous parasites. In many intestinal regions, damage to the mucosal surface visible with scanning electron microscopy (SEM) was slight or absent, although malabsorption was marked. No changes were noted with SEM in the structure and orientation of the brush border in these regions. Villar height was significantly reduced in the regions of heaviest infection when intestinal damage was visible. Conversely, the crypts of Lieberkühn were often two or three times as deep in infected poults as in uninfected poults. In general, no differences were found in the thickness of the circular and longitudinal muscle layers between the infected and uninfected poults. The dry weight of the intestinal tissue was less from infected poults than from uninoculated controls and was related to both region of the intestine and severity of the infection.