The action of the systemic fungicides tridemorph and fenpropimorph on sterol biosynthesis by the soil amoeba Acanthamoeba polyphaga

Tridemorph and fenpropimorph, two systemic fungicides known by their inhibitory effects on sterol biosynthesis in fungi and plants, were administered in vivo to the amoeba Acanthamoeba polyphaga. The compounds did not kill the cells, but modified completely their sterol pattern. Fungicide-exposed cells accumulated cyclopropylsterols indicating a partial blockage of the cyclopropane isomerase as in higher plants and delta 8-sterols indicating an inhibition of the delta 8----delta 7 isomerase as in fungi. Three new sterols, 4 alpha-methylergosta-9(11),24(28)-dienol, ergosta-6,8,22-trienol and poriferasta-6,8,22-trienol were isolated and identified, the former from control cells, the two latter from fungicide-treated cells. These results are in accordance with our previous results on the presence of cycloartenol as sterol precursor and confirm our hypothesis on a phylogenetic relationship of Acanthamoeba polyphaga with photosynthetic phyla.     

Sterol biosynthesis via cycloartenol and other biochemical features related to photosynthetic phyla in the amoeba Naegleria lovaniensis and Naegleria gruberi

The sterols and sterol precursors of two amoebae of the genus Naegleria, Naegleria lovaniensis and Naegleria gruberi were investigated. Cycloartenol, the sterol precursor in photosynthetic organisms, is present in both amoebae. In N. lovaniesis, it is accompanied by lanosterol and parkeol, as well as by the 24,25-dihydro derivatives of these triterpenes. One of the most striking features of these amoebae is the accumulation of 4 alpha-methylsterols which are present in similar amounts as those of 4,4-desmethylsterols (3-5 mg/g, dry weight). 4 alpha-Methylergosta-7,22-dienol was identified as a new compound. Ergosterol was the major 4,4-desmethylsterol, accompanied by small amounts of C27 and other C28 sterols. Treatment of N. lovaniensis with fenpropimorph modified the sterol pattern of this amoeba and inhibited its growth. This fungicide, known to inhibit steps of sterol biosynthesis in fungi and plants, induced the disappearance of 4 alpha-methyl-delta 7-sterols and the appearance of the unusual delta 6,8,22-ergostatrienol as in A. polyphaga. These results might be explained by a partial inhibition of the delta 8----delta 7 isomerase, the small amounts of delta 7-sterols formed being converted into ergosterol which is still present in fenpropimorph-exposed cells. De novo sterol biosynthesis in N. lovaniensis was shown by incorporation of [1-14C]acetate into sterols and sterol precursors, especially cycloartenol. Lanosterol and parkeol were not significantly labelled. Furthermore, [3-3H]squalene epoxide was efficiently cyclized by a cell-free system of this amoeba into cycloartenol, and again no significant radioactivity was detected in lanosterol and parkeol. This shows that cycloartenol, the sterol precursor in plants and algae, is also the sterol precursor in Naegleria species, and that these amoebae, like A. polyphaga, are related by some biosynthetic pathways to photosynthetic phyla. Lanosterol, the sterol precursor in non-photosynthetic phyla (animal and fungi) and parkeol are more likely dead-ends of this biosynthetic pathway. The peculiar phylogenetic position of these protozoa was further emphasized by the action of indole acetic acid and other auxine-like compounds on their growth. Indeed amoebic growth was enhanced in the presence of these higher plant growth hormones. The differences in the sterol composition of the protozoa we have hitherto examined is related to their sensitivity toward polyene macrolide antibiotics.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparative in vitro activity of chlorine and bromine on the cysts of free-living amoebae

A continuous flow type apparatus is used for investigation of the chlorine and bromide activity in vitro on the cysts of two free-living amoeba strains : Naegleria gruberi and Acanthamoeba polyphaga. The Naegleria cysts are sensitive more to chlorine than to bromine ; those of Acanthamoeba are perfectly resistant to these disinfectants in the limit of the available concentrations in swimming-pools. The sensibility to these disinfectants stays the same at 25 degrees C and 35 degrees C.     

Protozoa and pathogenic bacteria: lessons learned from Legionella pneumophila

Bacterivorous protozoa and bacteria have been in co-existence since the origin of life. This co-existence has led unequivocally to the evolution of many different co-interactions. Most bacteria are ingested and digested, but many escape ingestion for various reasons. Others are ingested but evade digestion, and a few, notoriously Legionella pneumophila , even have the capacity of multiplying within the protozoan host. The aims of this study were to elucidate the interactions of various multi-drug-resistant Staphylococcus aureus (MRSA) strains, Listeria monocytogenes sv4b, and Escherichia coli K12 with the amoeba, Acanthamoeba polyphaga . To evaluate the interactions, we set up co-cultures in Neffs' amoebic saline, at a multiplicity of invasion (MOI) of 1:100 of amoeba to bacteria, and a temperature of 37°C, although the effects of MOI and temperature were also assessed. Survival of bacteria and amoeba was checked at regular intervals, coupled with microscopy. It was discovered under our test conditions, that E. coli was ingested and digested by A. polyphaga , but in contrast, L. monocytogenes , had the capacity to flourish in the presence of A. polyphaga . We also report, for the first time, that all six MRSA isolates tested, survived and replicated in association with A. polyphaga , in comparison to conditions where amoebae were absent. Indeed, we also have evidence suggesting that increases in MRSA, in the presence of A. polyphaga , may be attributable to intracellular survival and replication. These findings have profound implications for the hospital environment, where Acanthamoeba sp., are also commonly isolated. In conclusion, this study illustrates the significance of protozoa as vehicles augmenting the survival of MRSA and L. monocytogenes in the environment.     

Survival of Legionella pneumophila within cysts of Acanthamoeba polyphaga following chlorine exposure

The association between Legionella pneumophila and the free-living amoeba Acanthamoeba polyphaga was studied. Intracellular growth of L. pneumophila within amoebic trophozoite was confirmed by kinetic growth experiments, light and electron microscopy. Cysts produced from infected trophozoites were found to protect the legionellas from at least 50 mg/l free chlorine. The ability of L. pneumophila to survive within the cysts of A. polyphaga is suggested as a possible mechanism by which the organism evades disinfection and spreads to colonize new environments.     

Survival of Legionella pneumophila within cysts of Acanthamoeba polyphaga following chlorine exposure

The association between Legionella pneumophila and the free-living amoeba Acanthamoeba polyphaga was studied. Intracellular growth of L. pneumophila within amoebic trophozoite was confirmed by kinetic growth experiments, light and electron microscopy. Cysts produced from infected trophozoites were found to protect the legionellas from at least 50 mg/l free chlorine. The ability of L. pneumophila to survive within the cysts of A. polyphaga is suggested as a possible mechanism by which the organism evades disinfection and spreads to colonize new environments.     

Cryptococcus neoformans: gastronomic delight of a soil ameba.

During 7 days of incubation in vitro the trophozoite stage of the free-living soil amoeba, Acanthamoeba polyphaga, phagocytized and killed 78-97% of the cells of three strains of Cryptococcus neoformans. With one strain, incubation time was increased to nine days and 99% of the yeast cells were killed. It was calculated that during 4-9 days of incubation a single trophozoite phagocytized and killed a daily average of 84 yeast cells. The lethal effect of A. polyphaga on C. neoformans may represent a biological control mechanism in nature. Some of the surviving cells of C. neoformans developed into colonies containing pseudohyphae; these pseudolhyphal forms may be a biological 'escape hatch'.     

Learamoeba waccamawensis, N. G., N. Sp. (Heterolobosea: Vahlkampfiidae), a New Temperature-Tolerant Cyst-Forming Soil Amoeba

Soil from a roadside ditch at Lake Waccamaw, North Carolina, was tested for cyst-forming free-living amoebae, and water from the same ditch was tested for fecal coliform bacteria. Soil samples incubated at room temperature (21-23° C) yielded Acanthamoeba polyphaga, Amoeba sp., Hyperamaeba sp., Mayorella penardi. Naegleria gruberi, Naegleria minor , and unidentified ciliates, testaceans and slime molds. Incubation at 37-39° C yielded Acanthamoeba jacobsi, Platyamoeba schaefferi , and a new amoeba to be described herein. Fecal contamination of the ditch was confirmed by a direct membrane filtration technique that yielded a mean of 1,410 + 134 bacteria/ ml. The new amoeba is assigned to the class Heterolobosea Page & Blanton, 1985 on the basis of promitotic nuclear division, and a flagellated stage with a cytostome as seen in the genus Tetramitus. Amoebae studied in hanging drop preparations were flattened and irregular as described for the genus Stachyamoeba in the family Gruberellidae but changed to a lobose cylindrical form as described for species of Vahlkampfia in the family Vahlkampfiidae. A new genus and species, Learamoeba waccamawensis , is proposed for the amoeba described herein.     

Counting Legionella cells within single amoeba host cells

Here we present the first attempt to quantify Legionella pneumophila cell numbers within individual amoeba hosts that may be released into engineered water systems. The maximum numbers of culturable L. pneumophila cells grown within Acanthamoeba polyphaga and Naegleria fowleri were 1,348 (mean, 329) and 385 (mean, 44) CFU trophozoite(-1), respectively.     

Quantation by flow microfluorometry of total cellular DNA in Acanthamoeba.

The DNA content of five species of Acanthamoeba was determined by flow microfluorometry. Acanthamoeba castellanii (AC-30), acanthamoeba polyphaga (APG and P-23), acanthamoeba rhysodes, acanthamoeba culbertsoni (A-1), and acanthamoeba royreba were grown in a casitone based medium 24-48 HR. The trophozoites were harvested, and evaluated for DNA-bound fluorescence. All species tested has DNA values between 2.0-5.0 pg/cell. These results placed DNA/cell values of Acanthamoeba slightly lower than DNA/cell values of other eucaryotic cells and much lower than Amoeba proteus values. These results indicate that FMF may be a useful adjunct in distinguishing Acanthamoeba cells from either eucaryotic cells or some other amoeba. However, differences in DNA/cell between species of Acanthamoeba are small and would not be useful in identification of species.     

Protozoan Acanthamoeba polyphaga as a Potential Reservoir for Campylobacter jejuni

We showed by a laboratory experiment that four different Campylobacter jejuni strains are able to infect the protozoan Acanthamoeba polyphaga. C. jejuni cells survived for longer periods when cocultured with amoebae than when grown in culture alone. The infecting C. jejuni cells aggregated in amoebic vacuoles, in which they were seen to be actively moving. Furthermore, a resuscitation of bacterial cultures that were previously negative in culturability tests was observed after reinoculation into fresh amoeba cultures. After spontaneous rupture of the amoebae, C. jejuni could be detected by microscopy and culturability tests. Our results indicate that amoebae may serve as a nonvertebrate reservoir for C. jejuni in the environment.     

Glutathione transferase activity in some flagellates and amoebae, and purification of the soluble glutathione transferases from Acanthamoeba

Glutathione transferase (GST) enzymes are toxicologically important from many points of view. Nine protozoans were investigated here for their GST content. Six aerobic amoebae had very different specific GST activities, but an anaerobic amoeba and two anaerobic flagellates did not have any GST activity, suggesting that the peroxidase activity of GST is an evolutionarily important property for aerobic organisms. The soluble GST isoenzymes of Acanthamoeba culbertsoni and A. polyphaga were purified and partially characterized. The same two cationic and one anionic GST isoenzyme were found in both Acanthamoeba ssp., while A. culbertsoni had one more cationic isoenzyme. It is concluded that GST in aerobic amoebae can play an important role in detoxication.     

Viability of Listeria monocytogenes in co-culture with Acanthamoeba spp.

Listeria monocytogenes is a human pathogen, ubiquitous in the environment, and can grow and survive under a wide range of environmental conditions. It contaminates foods via raw materials or food-processing environments. However, the current knowledge of its ecology and, in particular, the mode of environmental survival and transmission of this intracellular pathogen remains limited. Research has shown that several intracellular pathogens are able to survive or replicate within free-living amoebae. To examine the viability of L. monocytogenes in interaction with Acanthamoeba spp., bacteria were co-cultured with three freshly isolated amoebae, namely Acanthamoeba polyphaga, Acanthamoeba castellanii and Acanthamoeba lenticulata . The survival of bacteria and amoebae was determined using culture techniques and microscopy. Under the experimental conditions used, all amoebae were able to eliminate bacteria irrespective of the hly gene. Bacteria did not survive or replicate within amoeba cells. However, extra-amoebic bacteria grew saprophytically on materials released from amoebae, which may play an important role in the survival of bacteria under extreme environmental conditions.     

Metabolic interconversion of free sterols and steryl esters in Saccharomyces cerevisiae.

The interconversion of free and esterified sterols was followed radioisotopically with [U-14C]acetate and [methyl-14C]methionine. In pulse-chase experiments, radioactivity first appeared mainly in unesterified sterols in exponential-phase cells. Within one generation time, the label equilibrated between the free and esterified sterol pools and subsequently accumulated in steryl esters in stationary-phase cells. When the sterol pools were prelabeled by growing cells aerobically to the stationary phase and the cells were diluted into unlabeled medium, the prelabeled steryl esters returned to the free sterol form under several conditions. (i) During aerobic growth, the prelabeled sterols decreased from 80% to 45% esters in the early exponential phase and then returned to 80% esters as the culture reached the stationary phase. (ii) Under anaerobic conditions, the percentage of prelabeled steryl esters declined continuously. When growth stopped, only 15% of the sterols remained esterified. (iii) In the presence of an inhibitor of sterol biosynthesis, which causes accumulation of a precursor to ergosterol, prelabeled sterols decreased to 40% steryl esters while the precursor was found preferentially in the esterified form. These results indicate that the bulk of the free sterol and steryl ester pools are freely interconvertible, with the steryl esters serving as a supply of free sterols. Furthermore, there is an active cellular control over what types of sterol are found in the free and esterified sterol pools.     

Novel bacterial endosymbionts of Acanthamoeba spp. related to the Paramecium caudatum symbiont Caedibacter caryophilus

Acanthamoebae are increasingly being recognized as hosts for obligate bacterial endosymbionts, most of which are presently uncharacterized. In this study, the phylogeny of three Gram-negative, rod-shaped endosymbionts and their Acanthamoeba host cells was analysed by the rRNA approach. Comparative analyses of 16S rDNA sequences retrieved from amoebic cell lysates revealed that the endosymbionts of Acanthamoeba polyphaga HN-3, Acanthamoeba sp. UWC9 and Acanthamoeba sp. UWE39 are related to the Paramecium caudatum endosymbionts Caedibacter caryophilus, Holospora elegans a n d Holospora obtusa . With overall 16S rRNA sequence similarities to their closest relative, C. caryophilus , of between 87% and 93%, these endosymbionts represent three distinct new species. In situ hybridization with fluorescently labelled endosymbiont-specific 16S rRNA-targeted probes demonstrated that the retrieved 16S rDNA sequences originated from the endosymbionts and confirmed their intracellular localization. We propose to classify provisionally the endosymbiont of Acanthamoeba polyphaga HN-3 as ' Candidatus Caedibacter acanthamoebae', the endosymbiont of Acanthamoeba sp. strain UWC9 as ' Candidatus Paracaedibacter acanthamoebae' and the endosymbiont of Acanthamoeba sp. strain UWE39 as ' Candidatus Paracaedibacter symbiosus'. The phylogeny of the Acanthamoeba host cells was analysed by comparative sequence analyses of their 18S rRNA. Although Acanthamoeba polyphaga HN-3 clearly groups together with most of the known Acanthamoeba isolates (18S rRNA sequence type 4), Acanthamoeba sp. UWC9 and UWE39 exhibit < 92% 18S rRNA sequence similarity to each other and to other Acanthamoeba isolates. Therefore, we propose two new sequence types (T13 and T14) within the genus Acanthamoeba containing, respectively, Acanthamoeba sp. UWC9 and Acanthamoeba sp. UWE39.     

Nutritional studies on Acanthamoeba culbertsoni and development of chemically defined medium

A chemically defined medium containing 11 amino acids, 3 vitamins, 6 inorganic salts and glucose, yielding maximum cell densities of 1.5-2.5 x 10(7) cells/ml, has been developed for Acanthamoeba culbertsoni with a mean generation time (MGT) of 10 h. A medium containing six amino acids viz. arginine, methionine, leucine, isoleucine, valine and glycine along with other components could also support good albeit slower growth (MGT 27 h) of the amoeba. Acetate did not serve as a suitable carbon/energy source for A. culbertsoni. This organism bears close resemblance in its nutritional requirements to other Acanthamoeba especially A. polyphaga.     

Intracellular sterol transport in eukaryotes,a connection to mitochondrial function?

Eukaryotic cells synthesize sterols in the endoplasmatic reticulum (ER) from where it needs to be efficiently transported to the plasma membrane, which harbors approximately 90% of the free sterol pool of the cell. Sterols that are being taken up from the environment, on the other hand, are transported back from the plasma membrane to the ER, where the free sterols are esterified to steryl esters. The molecular mechanisms that govern this bidirectional movement of sterols between the ER and the plasma membrane of eukaryotic cells are only poorly understood. Proper control of this transport is important for normal cell function and development as indicated by fatal human pathologies such as Niemann Pick type C disease and atherosclerosis, which are characterized by an over-accumulation of free sterols within endosomal membranes and the ER, respectively. Recently, a number of complementary approaches using Saccharomyces cerevisiae as a model organism lead to a more precise characterization of the pathways that control the subcellular transport of sterols and led to the identification of components that directly or indirectly affect sterol uptake at the plasma membrane and its transport back to the ER. A genetic approach that is based on the fact that yeast is a facultative anaerobic organism, which becomes auxotrophic for sterols in the absence of oxygen, resulted in the identification of 17 genes that are required for efficient uptake and/or transport of sterols. Unexpectedly, many of these genes are required for mitochondrial functions. A possible connection between mitochondrial biogenesis and sterol biosynthesis and uptake will be discussed in light of the fact that cholesterol transport into the inner membranes of mitochondria is a well established sterol transport route in vertebrates, where it is required to convert cholesterol into pregnenolone, the precursor of steroids.     

Mechanisms of sterol uptake and transport in yeast

Sterols are essential lipid components of eukaryotic membranes. Here we summarize recent advances in understanding how sterols are transported between different membranes. Baker's yeast is a particularly attractive organism to dissect this lipid transport pathway, because cells can synthesize their own major sterol, ergosterol, in the membrane of the endoplasmic reticulum from where it is then transported to the plasma membrane. However, Saccharomyces cerevisiae is also a facultative anaerobic organism, which becomes sterol auxotroph in the absence of oxygen. Under these conditions, cells take up sterol from the environment and transport the lipid back into the membrane of the endoplasmic reticulum, where the free sterol becomes esterified and is then stored in lipid droplets. Steryl ester formation is thus a reliable readout to assess the back-transport of exogenously provided sterols from the plasma membrane to the endoplasmic reticulum. Structure/function analysis has revealed that the bulk membrane function of the fungal ergosterol can be provided by structurally related sterols, including the mammalian cholesterol. Foreign sterols, however, are subject to a lipid quality control cycle in which the sterol is reversibly acetylated. Because acetylated sterols are efficiently excreted from cells, the substrate specificity of the deacetylating enzymes determines which sterols are retained. Membrane-bound acetylated sterols are excreted by the secretory pathway, more soluble acetylated sterol derivatives such as the steroid precursor pregnenolone, on the other hand, are excreted by a pathway that is independent of vesicle formation and fusion. Further analysis of this lipid quality control cycle is likely to reveal novel insight into the mechanisms that ensure sterol homeostasis in eukaryotic cells. Article from a special issue on Steroids and Microorganisms.     

Occurrence of squalene and sterols in Cellulomonas dehydrogenans (Arnaudi 1942) comb. nov. Hester 1971.

The neutral lipid fraction of the photochromogenic, coryneform bacterium Cellulomonas dehydrogenans (Arnaudi 1942) comb. nov. contains the sterol precursor squalene and at least two sterols, cholesterol and beta-sitosterol. The compounds were characterized by mass spectrometry and combination gas-liquid chromatography--mass spectrometry. De novo sterol biosynthetic ability was shown from incorporation of 14C from D-[U-14C]glucose into squalene and the sterol fraction. The squalene concentration approximated 0.002 to 0.005% of the total dry cell weight, and the sterols approximated 0.03 to 0.05%.     

Acanthamoeba pearcei N. Sp. (Protozoa: Amoebida) from Sewage Contaminated Sediments

ABSTRACT. Seabottom sediments from a discontinued Philadelphia-Camden 40-Mile ocean sewage disposal site were cultured for cyst-forming free-living amoebae. Barge delivered wastes were discharged at the site from 1973 until 1980 when the site was closed. One station at the southeast margin of the site was sampled at a depth of approximately 50 m, twice in 1978 and once in 1982, 1983 and 1984. Sediment from the 1978 collection yielded Acanthamoeba polyphaga, Vahlkampfia sp., and an unknown amoeba with stellate endocysts similar to those of A. astronyxis. Trophozoites and cysts of the isolate were typical of those described for the genus Acanthamoeba. Biochemical tests employing enzyme electrophoresis and morphological studies on live and stained specimens showed that the isolate was distinct from other well-described species within the family Acanthamoebidae Sawyer & Griffin, 1975.