Lipid requirements for axenic cultivation ofEntamoeba histolytica

Fatty acids, cholesterol and glucose present in axenic medium are utilized by growingEntamoeba histolytica but the amoeba is unable to synthesize cholesterol from [U^-14 C^- ] glucose although the label is incorporated into the fatty acids and non-saponifiable fractions of the organism. Exogenously-added sonicated dispersions of cholesterol, Β-sitosterol, lanosterol, lecithin and lauric, palmitic, linoleic and stearic acids are ingested by the amoebae with subsequent loss in amoeboid movement. After a few hours the movement is regained. Cholesterol, lecithin and the fatty acids stimulate amoebic multiplication but are unable to replace serum in the medium either singly or in combination. CDRI Communication No. 2516.     

Effect of essential oil ofHyssopus officinalis on the lipid composition ofAspergillus fumigatus

Addition of the essential oil ofHyssopus officinalis to the culture medium ofAspergillus fumigatus induced alterations in both growth and lipid composition of this mould. Total lipids and sterols were reduced, whereas total phospholipids were increased. There were alterations in the proportions of fatty acids, neutral lipid and phospholipid fractions.     

The Induction of a Phospholipid Requirement and Morphological Abnormalities in Tetrahymena pyriformis by Growth at Supraoptimal Temperatures*†

SYNOPSIS. Supplementation of a chemically-defined medium that supported excellent axenic growth of Tetrahymena pyriformis, mating type II, variety 1, at 35 C, with nucleic acid derivatives allowed full growth at 37, and further addition of phospholipids permitted equivalent growth at 39 and 1/3 of that at 40. No other nutrients tested were active at 40. Population growth could be sustained continuously by serial sub-culture every 48 hr at 40 only if the medium contained synthetic phospholipids or phospholipids isolated from natural sources. The requirement was specific for phosphatidyl choline and phosphatidyl ethanolamine. Phospholipids which contained only saturated fatty acids, and ones which contained unsaturated acids were active. Phospholipids and neutral lipids isolated from 35-grown T. pyriformis were also effective. Phospholipid precursors, sterols, neutral lipids and fatty acids were not. A 48–72 hr exposure to 40 in either the unsupplemented medium or the nucleic acid derivatives-phospholipid-supplemented medium caused extreme variation in size and shape, fractured and displaced kineties, and abnormalities of karyokinesis. The frequency and degree of teratology was greater in the unsupplemented medium, but not markedly so. A possible metabolic basis for the temperature-induced phospholipid requirement and the morphological abnormalities is discussed.     

The utilization of sterols and other lipids by insect cells grown in vitro

Cells derived from Antheraea eucalypti were grown in vitro in a medium containing triglycerides, diglycerides, free fatty acids, and sterols as the main ‘neutral’ lipids. The sterol content of the medium was derived chiefly from the haemolymph component. The ‘neutral’ lipids in the cells were triglycerides, free fatty acids, and sterols. During growth over 6 days there was a quantitative balance between cholesterol and β-sitosterol gained by the cells and those sterols removed from the medium when allowance was made for losses from sterile medium. Cells metabolized more triglycerides and free fatty acids than they incorporated.     

Influence of lignin and oxygen on the growth and the lipid formation of the fungus Lentinus tigrinus

During cultivation of the filamentous fungus Lentinus tigrinus on a medium containing lignin, a high oxygen content stimulated the growth of the fungus and contributed to the yield of lipids. A high content of phosphatidic acid and a reduction in the level of phosphatidylethanolamine and phosphatidylserine were first detected in the composition of phospholipids. Changes in the composition of neutral lipids, such as variation in the ratio of esterified and free sterols, have occurred; thus, the amount of sterol esters reduced simultaneously with a decrease in the content of free fatty acids. Based on the obtained results, the possible role of phosphatidic acid as a second messenger in the process of the consumption of lignin by the fungus Lentinus tigrinus is discussed.     

Lipid Auxotrophy and the Effect on Lipid Composition of Entamoeba invadens*†

SYNOPSIS. A medium for the axenic cultivation of Entamoeba invadens has been developed. Serum, an essential constituent of conventional media, has been replaced by a mixture of albumin, unsaturated fatty acids, Tween, and cholesterol to control the lipid composition of the medium. Entamoeba invadens requires both cholesterol and unsaturated fatty acids for growth. The fatty acid composition of the phospholipids of the ameba reflects that of the medium to a great extent, especially with regard to the unsaturated fatty acids. The amount of membrane bounded cholesterol depends on the cholesterol concentration in the medium.     

The MAK16 Gene of Entamoeba histolytica and Its Identification in Isolates from Patients

To identify sequences of Entamoeba histolytica associated with the development of amebic liver abscess (ALA) in hamsters, subtractive hybridization of cDNA from E. histolytica HM-1:IMSS under 2 growth conditions was performed: 1) cultured in axenic medium and 2) isolated from experimental ALA in hamsters. For this procedure, 6 sequences were obtained. Of these sequences, the mak16 gene was selected for amplification in 29 cultures of E. histolytica isolated from the feces of 10 patients with intestinal symptoms and 19 asymptomatic patients. Only 5 of the 10 isolates obtained from symptomatic patients developed ALA and amplified the mak16 gene, whereas the 19 isolates from asymptomatic patients did not amplify the mak16 gene nor did they develop ALA. Based on the results of Fisher''s exact test (P<0.001), an association was inferred between the presence of the mak16 gene of E. histolytica and the ability to develop ALA in hamsters and with the patient''s symptoms (P=0.02). The amplification of the mak16 gene suggests that it is an important gene in E. histolytica because it was present in the isolates from hamsters that developed liver damage.     

The interplay between Entamoeba and enteropathogenic bacteria modulates epithelial cell damage

Background

Mixed intestinal infections with Entamoeba histolytica, Entamoeba dispar and bacteria with exacerbated manifestations of disease are common in regions where amoebiasis is endemic. However, amoeba–bacteria interactions remain largely unexamined.

Methodology

Trophozoites of E. histolytica and E. dispar were co-cultured with enteropathogenic bacteria strains Escherichia coli (EPEC), Shigella dysenteriae and a commensal Escherichia coli. Amoebae that phagocytosed bacteria were tested for a cytopathic effect on epithelial cell monolayers. Cysteine proteinase activity, adhesion and cell surface concentration of Gal/GalNAc lectin were analyzed in amoebae showing increased virulence. Structural and functional changes and induction of IL-8 expression were determined in epithelial cells before and after exposure to bacteria. Chemotaxis of amoebae and neutrophils to human IL-8 and conditioned culture media from epithelial cells exposed to bacteria was quantified.

Principal Findings

E. histolytica digested phagocytosed bacteria, although S. dysenteriae retained 70% viability after ingestion. Phagocytosis of pathogenic bacteria augmented the cytopathic effect of E. histolytica and increased expression of Gal/GalNAc lectin on the amoebic surface and increased cysteine proteinase activity. E. dispar remained avirulent. Adhesion of amoebae and damage to cells exposed to bacteria were increased. Additional increases were observed if amoebae had phagocytosed bacteria. Co-culture of epithelial cells with enteropathogenic bacteria disrupted monolayer permeability and induced expression of IL-8. Media from these co-cultures and human recombinant IL-8 were similarly chemotactic for neutrophils and E. histolytica.

Conclusions

Epithelial monolayers exposed to enteropathogenic bacteria become more susceptible to E. histolytica damage. At the same time, phagocytosis of pathogenic bacteria by amoebae further increased epithelial cell damage.

Significance

The in vitro system presented here provides evidence that the Entamoeba/enteropathogenic bacteria interplay modulates epithelial cell responses to the pathogens. In mixed intestinal infections, where such interactions are possible, they could influence the outcome of disease. The results offer insights to continue research on this phenomenon.     

Entamoeba histolytica and Entamoeba invadens: Effects of temperature and oxygen tension on growth and survival

The temperature ranges for axenic growth of Entamoeba histolytica strain HM-1 and Entamoeba invadens strain 165 clone II in TYI-S-33 medium were: 32 to 40 C for E. histolytica with an optimum of 35.5 to 37 C; whereas the range for E. invadens 165 II was 20 to 30 C, optimum 24 to 27 C. Growth of strain HM-1 at 40 C was dependent upon the cell density of the culture used as a source of the inoculum. Clonal growth in agar was used to assay survival of Entamoeba spp. trophozoites after exposure to deleterious physical conditions. The lowest temperature for thermal killing of E. histolytica HM-1 was 41.5 C and for E. invadens 165, 35.5 C. Both were killed rapidly at 42 C, but slowly at 0 C. Killing of E. histolytica HM-1 trophozoites by exposure to increased oxygen tensions was a function of temperature and time. At 24 C and 35.5 C, there was little loss of viability during the first 7 hr exposure, then killing was quite rapid. The cells were killed sooner if the medium was preexposed to air. In contrast, at 0 C there was less killing by oxygen. E. invadens 165 II appeared to be more oxygen sensitive at 24 than at 0 C. Other E. histolytica strains tested were similar to HM-1 in their responses to temperature and air.     

Amoebic PI3K and PKC Is Required for Jurkat T Cell Death Induced by Entamoeba histolytica

The enteric protozoan parasite Entamoeba histolytica is the causative agent of human amebiasis. During infection, adherence of E. histolytica through Gal/GalNAc lectin on the surface of the amoeba can induce caspase-3-dependent or -independent host cell death. Phosphorylinositol 3-kinase (PI3K) and protein kinase C (PKC) in E. histolytica play an important function in the adhesion, killing, or phagocytosis of target cells. In this study, we examined the role of amoebic PI3K and PKC in amoeba-induced apoptotic cell death in Jurkat T cells. When Jurkat T cells were incubated with E. histolytica trophozoites, phosphatidylserine (PS) externalization and DNA fragmentation in Jurkat cells were markedly increased compared to those of cells incubated with medium alone. However, when amoebae were pretreated with a PI3K inhibitor, wortmannin before being incubated with E. histolytica, E. histolytica-induced PS externalization and DNA fragmentation in Jurkat cells were significantly reduced compared to results for amoebae pretreated with DMSO. In addition, pretreatment of amoebae with a PKC inhibitor, staurosporine strongly inhibited Jurkat T cell death. However, E. histolytica-induced cleavage of caspase-3, -6, and -7 were not inhibited by pretreatment of amoebae with wortmannin or staurosporin. In addition, we found that amoebic PI3K and PKC have an important role on amoeba adhesion to host compartment. These results suggest that amebic PI3K and PKC activation may play an important role in caspase-independent cell death in Entamoeba-induced apoptosis.     

The Neutral Lipids of Paramecium tetraurelia: Changes with Culture Age and the Detection of Steryl Esters in Ciliary Membranes1

Neutral lipids, particularly triglycerides, accounted for the major decrease in the total lipid content in Paramecium cells that occurs with culture age. Sterols, triglycerides, and steryl esters were the major classes of neutral lipids in cells and isolated cilia. Free as well as high concentrations of esterified sterols were detected in purified ciliary membrane preparations. Stigmasterol and 7-dehydrostigmasterol were the major components of both free and esterified sterols of cells and cilia; however, when cholesterol was present in the growth medium, it was desaturated to 7-dehydrocholesterol and incorporated into cellular and ciliary lipids. Free fatty acids from cells and triglycerides from cells and cilia were low in polyunsaturated fatty acids and reflected the composition of fatty acids in the culture medium. An exception was the reduced concentration of stearate in triglycerides from whole cells. Greater than 50% of triglyceride fatty acids from cilia were saturated. The fatty acid compositions of cellular triglycerides and ciliary steryl esters did not change with culture age, but those of cellular steryl esters and ciliary triglycerides did change. In comparison with phospholipids, these neutral lipid fatty acid compositional changes were smaller. The sensitivity of these stigmasterol-containing cells to polyene antibiotics indicated that they were killed by nystatin > filipin > amphotericin B. The unexpected finding of high concentrations of steryl esters in ciliary membrane preparations is discussed.     

The role of exogenous lipids in lycopene synthesis in the mucoraceous fungus Blakeslea trispora

The addition of plant oils to the growth medium stimulated growth and lipid synthesis in the fungus Blakeslea trispora. However, only oils with high content of linoleic and especially linolenic acid enhanced lycopene formation. The increase in lycopene formation was accompanied by accumulation in the neutral lipid fraction of the fatty acids prevailing in plant oils. In contrast, the influence of exogenous lipids on the fatty acid composition of bulk fungal phospholipids was insignificant. Nonetheless, a marked increase in the content of membrane lipids and of their phosphatidylethanolamine content was revealed. Presumably, the main mechanism of stimulation of lycopene formation by the oils involves an increase in the solubility of lycopene in the triacylglycerols of the lipid bodies, which is due to an increase in the desaturation degree of their fatty acids. The predominance of linoleic and especially of linolenic fatty acid in plant oils is regarded as a criterion for selecting the oil species for the purpose of intensifying lycopene synthesis.     

Species-Specific Immunity Induced by Infection with Entamoeba histolytica and Entamoeba moshkovskii in Mice

Entamoeba histolytica, the parasitic amoeba responsible for amoebiasis, causes approximately 100,000 deaths every year. There is currently no vaccine against this parasite. We have previously shown that intracecal inoculation of E. histolytica trophozoites leads to chronic and non-healing cecitis in mice. Entamoeba moshkovskii, a closely related amoeba, also causes diarrhea and other intestinal disorders in this model. Here, we investigated the effect of infection followed by drug-cure of these species on the induction of immunity against homologous or heterologous species challenge. Mice were infected with E. histolytica or E. moshkovskii and treated with metronidazole 14 days later. Re-challenge with E. histolytica or E. moshkovskii was conducted seven or 28 days following confirmation of the clearance of amoebae, and the degree of protection compared to non-exposed control mice was evaluated. We show that primary infection with these amoebae induces a species-specific immune response which protects against challenge with the homologous, but not a heterologous species. These findings pave the way, therefore, for the identification of novel amoebae antigens that may become the targets of vaccines and provide a useful platform to investigate host protective immunity to Entamoeba infections.     

Entamoeba histolytica: concurrent irreversible loss of infectivity-pathogenicity and encystment potential after prolonged maintenance in axenic culture in vitro

The NIH-200 strain of Entamoeba histolytica became avirulent after more than 2 yr maintenance in axenic culture in vitro. In an attempt to restore virulence to the amoeba, it was transferred to Locke's egg rice-flour medium with various combinations of the following bacteria: Bacteroides sp., Clostridium perfringens, Escherichia coli, and Streptococcus faecalis. Similar cultures were established with a mixed bacterial flora (comprising many unknown species), with and without rice flour, and an attempt was made to induce encystation. Subsequent inoculation of amoebae from the various amoeba-bacteria cultures into the cecum of germfree and exgermfree guinea pigs harboring the same bacteria, as the culture-produced inoculum did not in any instance produce amoebic lesions or prolonged amoebic infections of the enteric lumen. All attempts to induce encystation were unsuccessful; the amoeba had lost its encystment potential, and this was believed to be intimately related to the irreversible loss of virulence.     

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: 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.     

Effect of NaCl and Polyamines on Plasma Membrane Lipids of Wheat Roots

Caryopses of a salt sensitive wheat cultivar (Triticum aestivum L. cv. Giza 163) were presoaked in 2.5 mM putrescine (Put), 5 mM spermidine (Spd) or 2.5 mM spermine (Spm) for 24 h and then subjected to 150 mM NaCl added to the growth medium for 15 d. Effects of NaCl and polyamines (PAs) on plasma membrane (PM) lipids, phospholipids, fatty acids, and free sterols were determined. NaCl treatment caused a decrease in total phospholipids, increase in saturated fatty acids and altered distribution of sterols and phospholipids. NaCl also induced increase in sterol/phospholipid ratio. PAs treatments (particularly Put and Spd) counterbalanced the NaCl deleterious effects on PM lipids.     

Growth Characteristics of Axenic Strains of Entamoeba histolytica Schaudinn, 1903*

SYNOPSIS. An account of the culture methods for axenic growth of Entamoeba histolytica is given. Three media, including Diamond's medium, have been reliable. At present 6 strains are maintained axenically; details of isolation and axenization are outlined. Growth characteristics and oxygen requirements of 2 strains are examined.     

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.     

Axenic cultivation of Entamoeba dispar in newly designed yeast extract-iron-gluconic acid-dihydroxyacetone-serum medium

Yeast extract-iron-gluconic acid-dihydroxyacetone-serum medium that allows axenic cultivation of Entamoeba dispar was designed based on casein-free yeast extract-iron-serum (YI-S) medium, and the usefulness of the medium was assessed. The main differences from YI-S medium are replacement of glucose by gluconic acid, addition of dihydroxyacetone and D-galacturonic acid monohydrate, and sterilization by filtration. This medium promoted the axenic growth of 5 strains of E. dispar (2 strains of nonhuman primate isolates and 3 strains of human isolates). In addition, to clarify the biological basis for the growth of E. dispar in this medium, analyses of relevant enzymes on the glycolytic pathway of the amoebae as well as of the protozoans that are the best culture supplement for amoebae are being performed.