Vernalophrys algivore gen. nov., sp. nov. (Rhizaria: Cercozoa: Vampyrellida), a New Algal Predator Isolated from Outdoor Mass Culture of Scenedesmus dimorphus

Microbial contamination is the main cause of loss of biomass yield in microalgal cultures, especially under outdoor environmental conditions. Little is known about the identities of microbial contaminants in outdoor mass algal cultures. In this study, a new genus and species of vampyrellid amoeba, Vernalophrys algivore, is described from cultures of Scenedesmus dimorphus in open raceway ponds and outdoor flat-panel photobioreactors. This vampyrellid amoeba was a significant grazer of Scenedesmus and was frequently associated with a very rapid decline in algal numbers. We report on the morphology, subcellular structure, feeding behavior, molecular phylogeny, and life cycle. The new amoeba resembles Leptophrys in the shape of trophozoites and pseudopodia and in the mechanism of feeding (mainly by engulfment). It possesses two distinctive regions in helix E10_1 (nucleotides 117 to 119, CAA) and E23_1 (nucleotides 522 and 523, AG) of the 18S rRNA gene. It did not form a monophyletic group with Leptophrys in molecular phylogenetic trees. We establish a new genus, Vernalophrys, with the type species Vernalophrys algivore. The occurrence, impact of the amoeba on mass culture of S. dimorphus, and means to reduce vampyrellid amoeba contamination in Scenedesmus cultures are addressed. The information obtained from this study will be useful for developing an early warning system and control measures for preventing or treating this contaminant in microalgal mass cultures.     

Saccamoeba lacustris,sp. nov. (Amoebozoa: Lobosea: Hartmannellidae), a new lobose amoeba,parasitized by the novel chlamydia ‘Candidatus Metachlamydia lacustris’ (Chlamydiae: Parachlamydiaceae)

An amoeba isolated from an aquatic biotope, identified morphologically as Saccamoeba limax, was found harbouring mutualistic rod-shaped gram-negative bacteria. During their cultivation on agar plates, a coinfection also by lysis-inducing chlamydia-like organisms was found in some subpopulations of that amoeba. .Here we provide a molecular-based identification of both the amoeba host and the two bacterial endosymbionts. Analysis of the 18S rRNA gene revealed that this strain is the sister-group to Glaeseria, for which we proposed the name Saccamoeba lacustris. The rod-shaped endosymbiont was identified as a member of Variovorax paradoxus group (Comamonadaceae, Beta-Proteobacteria). No growth on bacteriological agars was recorded, hence this symbiont might be strictly intracellular. The chlamydia-like parasite was unable to infect Acanthamoeba and other amoebae in coculture, showing high host specificity. Phylogenetic analysis based on the 16S rDNA indicated that it is a new member of the family Parachlamydiaceae (order Chlamydiales), for which we proposed the name ‘Candidatus Metachlamydia lacustris’.     

Amoebae as Potential Environmental Hosts for Mycobacterium ulcerans and Other Mycobacteria, but Doubtful Actors in Buruli Ulcer Epidemiology

Background

The reservoir and mode of transmission of Mycobacterium ulcerans, the causative agent of Buruli ulcer, remain unknown. Ecological, genetic and epidemiological information nonetheless suggests that M. ulcerans may reside in aquatic protozoa.

Methodology/Principal Findings

We experimentally infected Acanthamoeba polyphaga with M. ulcerans and found that the bacilli were phagocytised, not digested and remained viable for the duration of the experiment. Furthermore, we collected 13 water, 90 biofilm and 45 detritus samples in both Buruli ulcer endemic and non-endemic communities in Ghana, from which we cultivated amoeboid protozoa and mycobacteria. M. ulcerans was not isolated, but other mycobacteria were as frequently isolated from intracellular as from extracellular sources, suggesting that they commonly infect amoebae in nature. We screened the samples as well as the amoeba cultures for the M. ulcerans markers IS2404, IS2606 and KR-B. IS2404 was detected in 2% of the environmental samples and in 4% of the amoeba cultures. The IS2404 positive amoeba cultures included up to 5 different protozoan species, and originated both from Buruli ulcer endemic and non-endemic communities.

Conclusions/Significance

This is the first report of experimental infection of amoebae with M. ulcerans and of the detection of the marker IS2404 in amoeba cultures isolated from the environment. We conclude that amoeba are potential natural hosts for M. ulcerans, yet remain sceptical about their implication in the transmission of M. ulcerans to humans and their importance in the epidemiology of Buruli ulcer.     

Productivity gradient affects the temporal dynamics of testate amoebae in a neotropical floodplain

Testate amoeba communities are influenced by temporal variation in the productivity levels in the environment, and may be used as an indicator group for these changing conditions. Here, we analysed the effect of temporal variation in the levels of productivity variables on testate amoeba community of the upper Paraná River floodplain. We evaluated the hypothesis that the frequency and abundance of the testate amoeba community change along an environmental gradient, with different taxa establishing at different points along the gradient in response to changes in the levels of productivity variables. We predicted that the number of species would increase and decrease at points associated with higher and lower levels of productivity variables, respectively. Testate amoeba species were sampled quarterly between 2000 and 2012 from six lakes in the upper Paraná River floodplain, Brazil. We recorded 110 species belonging to 11 families. Threshold Indicator Taxa Analysis identified positive and negative significant shift points in response to the concentration of chlorophyll-a, total nitrogen, and total phosphorus on the frequency and abundance of the testate amoeba community. Our results indicated that change intervals in the levels of productivity variables were associated with the establishment of different taxa. The main bioindicator species of productivity were Difflugia acuminata, D. amphoralis, D. helvetica multilobata, D. kempny, D. lobostoma multilobata, D. parva, D. schurmanni, D. ventricosa, and Lesquereusia ovalis. These species were linked to the increase and decrease in the levels of productivity, confirming the ecological importance of the role of these organisms as bioindicators in aquatic ecosystems.     

Peculiarities of Signal Transduction from Arginine-Vasopressin Receptors Located on the Apical Surface of Epithelial Cells of the Frog Urinary Bladder and on the Amoeba Outer Membrane

Mechanisms of action of arginine-vasopressin (AVP) on water transport across the wall of urinary bladder of the frog R. temporaria L. and the outer membrane of the amoeba A. proteus were considered. AVP and its functional analogs were added to the basolateral and apical surfaces of the frog urinary bladder membrane and to the amoeba outer surface. In amoeba the AVP effect was evaluated from action on frequency of contractions of contractile vacuole that represents its organ of water—electrolyte homeostasis. It was shown that the system of signal transduction from the apical AVP receptors differed considerably from that located on the basolateral membrane and had a marked similarity with the system of signal transduction from AVP-sensitive structures on the outer membrane of amoeba. The urinary bladder apical membrane as well as the amoeba outer membrane has a mixed type of sensitivity to AVP with unidirectional (not antagonistic like on the basolateral membrane) involvement of systems coupled to receptors of the V₁ and V₂ type. We suggest that the system of signal transduction from apical AVP receptors corresponds to the earlier stage of evolutionary development.     

Acanthamoeba mauritaniensis genotype T4D: An environmental isolate displays pathogenic behavior

Acanthamoeba spp. are free-living amoebae with a worldwide distribution. These amoebae can cause granulomatous amoebic encephalitis and amoebic keratitis in humans. Proteases are considered virulence factors in pathogenic Acanthamoeba. The objective of this study was to evaluate the behavior of Acanthamoeba mauritaniensis, a nonpathogenic amoeba. We analyzed the cytopathic effect of A. mauritaniensis on RCE1(5 T5) and MDCK cells and compared it to that of Acanthamoeba castellanii. A partial biochemical characterization of proteases was performed in total crude extracts (TCE) and conditioned medium (CM). Finally, we evaluated the effect of proteases on tight junction (TJ) proteins and the transepithelial electrical resistance of MDCK cells. The results showed that this amoeba can induce substantial damage to RCE1(5T5) and MDCK cells. Moreover, the zymograms and Azocoll assays of amoebic TCE and CM revealed different protease activities, with serine proteases being the most active. Furthermore, A. mauritaniensis induced the alteration and degradation of MDCK cell TJ proteins with serine proteases. After genotyping this amoeba, we determined that it is an isolate of Acanthamoeba genotype T4D. From these data, we suggest that A. mauritaniensis genotype T4D behaves similarly to the A. castellanii strain.     

Down-Regulation of Cellulose Synthase Inhibits the Formation of Endocysts in Acanthamoeba

Acanthamoeba cysts are resistant to unfavorable physiological conditions and various disinfectants. Acanthamoeba cysts have 2 walls containing various sugar moieties, and in particular, one third of the inner wall is composed of cellulose. In this study, it has been shown that down-regulation of cellulose synthase by small interfering RNA (siRNA) significantly inhibits the formation of mature Acanthamoeba castellanii cysts. Calcofluor white staining and transmission electron microscopy revealed that siRNA transfected amoeba failed to form an inner wall during encystation and thus are likely to be more vulnerable. In addition, the expression of xylose isomerase, which is involved in cyst wall formation, was not altered in cellulose synthase down-regulated amoeba, indicating that cellulose synthase is a crucial factor for inner wall formation by Acanthamoeba during encystation.     

Acanthamoeba polyphaga-enhanced growth of Mycobacterium smegmatis

Background

Mycobacterium smegmatis is a rapidly-growing mycobacterium causing rare opportunistic infections in human patients. It is present in soil and water environments where free-living amoeba also reside, but data regarding M. smegmatis-amoeba relationships have been contradictory from mycobacteria destruction to mycobacteria survival.

Methodology/Principal Findings

Using optic and electron microscopy and culture-based microbial enumeration we investigated the ability of M. smegmatis mc² 155, M. smegmatis ATCC 19420^T and M. smegmatis ATCC 27204 organisms to survive into Acanthamoeba polyphaga trophozoites and cysts. We observed that M. smegmatis mycobacteria penetrated and survived in A. polyphaga trophozoites over five-day co-culture resulting in amoeba lysis and the release of viable M. smegmatis mycobacteria without amoebal cyst formation. We further observed that amoeba-co-culture, and lysed amoeba and supernatant and pellet, significantly increased five-day growth of the three tested M. smegmatis strains, including a four-fold increase in intra-amoebal growth.

Conclusions/Significance

Amoebal co-culture increases the growth of M. smegmatis resulting in amoeba killing by replicating M. smegmatis mycobacteria. This amoeba-M. smegmatis co-culture system illustrates an unusual paradigm in the mycobacteria-amoeba interactions as mycobacteria have been mainly regarded as amoeba-resistant organisms. Using these model organisms, this co-culture system could be used as a simple and rapid model to probe mycobacterial factors implicated in the intracellular growth of mycobacteria.     

Electron microscopic study of a pathogenic Acanthamoeba castellani strain: The presence of bacterial endosymbionts

Along general lines, the fine structure of the Acanthamoeba castellani Sn cell corresponds to the aspects described by other authors in the free forms. Of particular interest was the presence in the amoeba cytoplasm of some free formations, with a cell envelop and internal bacteria-like structure. The presence of these bacterial bodies free in the amoeba cytoplasm and without a surrounding host cell membrane as well as the aspects suggestive of cell division, led the authors to consider them as integrated in the Acanthamoeba Sn economy, correlating them with bacterial endosymbionts. Similar endosymbionts have not yet been described in Acanthamoeba and might be implicated in the pathogenic potential of the Sn strain.     

Acanthamoeba polyphaga is a possible host for Vibrio cholerae in aquatic environments

Acanthamoeba is a genus of free-living amoebae found to be able to host many bacterial species living in the environment. Acanthamoebae and Vibrio cholerae are found in the aquatic environments of cholera endemic areas. Previously it has been shown that V. cholerae O1 and O139 can survive and grow in Acanthamoeba castellanii. The aim of this study was to examine the ability of Acanthamoeba polyphaga to host V. cholerae O1 and O139. The interaction between A. polyphaga and V. cholerae strains was studied by means of viable amoeba cell counts and viable count of the bacteria in the absence and presence of amoebae. The viable count of intracellularly growing bacteria was estimated by utilizing gentamicin assay. Electron microscopy was used to determine the localization of V. cholerae inside A. polyphaga. The results showed that A. polyphaga enhanced growth and survival of V. cholerae, which grew and survived inside the amoeba cells for 2 weeks. The electron microscopy showed that A. polyphaga hosted intracellular V. cholerae localized in the vacuoles of amoeba cell. Neither the presence of V. cholerae together with A. polyphaga nor the intracellular localization of the bacteria inhibited growth and survival of A. polyphaga. The outcome of the interaction between these microorganisms may support strongly the role of A. polyphaga as host for V. cholerae O1 and O139.     

Rhizamoeba neglecta n. sp. (Amoebozoa,Tubulinea) from the bottom sediments of freshwater Lake Leshevoe (Valamo Island,North-Western Russia), with notes on the phylogeny of the order Leptomyxida

A new species of Leptomyxida, named Rhizamoeba neglecta was found during studies of the amoeba fauna of the inner Lake Leshevoe located at Valamo archipelago (The Lake Ladoga, North-Western Russia). Light-microscopical and ultrastructural studies indicated that it represents a new species of Leptomyxida. The partial 18S rDNA sequence of this amoeba is very similar to that of Leptomyxa reticulata.. These organisms, however, are very different in LM morphology and biology. Organisms assigned to the genus Rhizamoeba do not form a single clade in the 18S rDNA tree. This may indicate that the genus is an artificial grouping or that a number of studied strains were misidentified. The phylogeny and the systematics of leptomyxids require further investigation.     

From Grazing Resistance to Pathogenesis: The Coincidental Evolution of Virulence Factors

To many pathogenic bacteria, human hosts are an evolutionary dead end. This begs the question what evolutionary forces have shaped their virulence traits. Why are these bacteria so virulent? The coincidental evolution hypothesis suggests that such virulence factors result from adaptation to other ecological niches. In particular, virulence traits in bacteria might result from selective pressure exerted by protozoan predator. Thus, grazing resistance may be an evolutionarily exaptation for bacterial pathogenicity. This hypothesis was tested by subjecting a well characterized collection of 31 Escherichia coli strains (human commensal or extra-intestinal pathogenic) to grazing by the social haploid amoeba Dictyostelium discoideum. We then assessed how resistance to grazing correlates with some bacterial traits, such as the presence of virulence genes. Whatever the relative population size (bacteria/amoeba) for a non-pathogenic bacteria strain, D. discoideum was able to phagocytise, digest and grow. In contrast, a pathogenic bacterium strain killed D. discoideum above a certain bacteria/amoeba population size. A plating assay was then carried out using the E. coli collection faced to the grazing of D. discoideum. E. coli strains carrying virulence genes such as iroN, irp2, fyuA involved in iron uptake, belonging to the B2 phylogenetic group and being virulent in a mouse model of septicaemia were resistant to the grazing from D. discoideum. Experimental proof of the key role of the irp gene in the grazing resistance was evidenced with a mutant strain lacking this gene. Such determinant of virulence may well be originally selected and (or) further maintained for their role in natural habitat: resistance to digestion by free-living protozoa, rather than for virulence per se.     

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.     

The genus Sappinia: History, phylogeny and medical relevance

The genus Sappinia with the single species Sappinia pedata was established for an amoeba with two nuclei and pedicellate “cysts” by Dangeard in 1896. In 1912, Alexeieff transferred an also double nucleated, but apparently sexually reproducing amoeba to this genus as Sappinia diploidea, that had been described as Amoeba diploidea by Hartmann and Nägler in 1908. As the original isolates were lost, Michel and colleagues established a neotype for S. diploidea in 2006 and Brown and colleagues established a neotype for S. pedata in 2007. Molecular analyses have corroborated the differentiation between S. pedata and S. diploidea, however, the genus splits into more than two well separated clusters. Altogether, the genus Sappinia is now classified as a member of the Thecamoebidae and, moreover, as potentially pathogenic. In 2001, Gelman and colleagues reported a case of severe encephalitis in a non-immunocompromised young man caused by Sappinia.     

Searching for the sweet spot of amoebic gill disease of farmed Atlantic salmon: the potential role of glycan-lectin interactions in the adhesion of Neoparamoeba perurans

One of the first critical steps in the pathogenesis of amoebic gill disease (AGD) of farmed salmon is the adhesion of the causative amoeba to the host. The current study aimed to investigate the potential involvement of glycan-binding proteins expressed on the extracellular surface of Neoparamoeba perurans in gill tissue recognition and binding. The glycan-binding properties of the surface membrane of N. perurans and the carbohydrate binding profile of Atlantic salmon gill-derived epithelial cells were identified through the use of glycan and lectin microarrays, respectively. The occurrence of specific carbohydrate-mediated binding was then further assessed by in vitro attachment assays using microtitre plates pre-coated with the main glycan candidates. Adhesion assays were also performed in the presence of exogenous saccharides with the aim of blocking glycan-specific binding activity. Comparative analysis of the results from both lectin and glycan arrays showed significant overlap, as some glycans to which binding by the amoeba was seen were reflected as being present on the gill epithelial cells. The two main candidates proposed to be involved in amoeba attachment to the gills are mannobiose and N-acetylgalactosamine (GalNAc). Adhesion of amoebae significantly increased by 33.5 and 23% when cells were added to α1,3-Mannobiose-BSA and GalNAc-BSA coated plates. The observed increased in attachment was significantly reduced when the amoebae were incubated with exogenous glycans, further demonstrating the presence of mannobiose- and GalNAc-binding sites on the surfaces of the cells. We believe this study provides the first evidence for the presence of a highly specific carbohydrate recognition and binding system in N. perurans. These preliminary findings could be of extreme importance given that AGD is an external parasitic infestation and much of the current research on the development of alternative treatment strategies relies on either instant amoeba detachment or blocking parasite attachment.     

Trophic coupling of a testate amoeba and <Emphasis Type="Italic">Microcystis</Emphasis> species in a hypertrophic pond

Seasonal changes in abundance of the testate amoeba Penardochlamys sp. and its food vacuole contents were investigated in relation to blooms of the cyanobacteria Microcystis spp. in a hypertrophic pond from April 1999 to March 2000. The behavior of the amoeba feeding on M. aeruginosa and M. wesenbergii was also observed in the laboratory. The amoeba was detectable from late May to November 1999 during the blooms of Microcystis spp. Cell densities of the amoeba fluctuated between 1.4 and 350 cells ml^–1 with some sporadic peaks, which did not coincide with rapid decreases in the abundance of Microcystis spp. Food vacuoles contained only Microcystis cells; other prey items were not found, suggesting that this amoeba utilized only the cyanobacteria as food. The amoeba was frequently found attached to Microcystis colonies, but was not associated with other suspended particles. Observation of the amoeba feeding revealed the feeding mechanism and that the amoeba was able to graze on both species of Microcystis. These results suggest that the trophic coupling of these organisms is substantial, although grazing by the amoeba is not sufficient to regulate the dynamics of Microcystis populations in this hypertrophic pond.     

Draft Genome Sequence of Methanobacterium formicicum DSM 3637, an Archaebacterium Isolated from the Methane Producer Amoeba Pelomyxa palustris

Here is reported the draft genome sequence of Methanobacterium formicicum DSM 3637, which was isolated from the methane-producing amoeba Pelomyxa palustris. This bacterium was determined to be an endosymbiont living in the cytoplasm of P. palustris and the source of methane; however, the global characteristics of its genome suggest a free-living lifestyle rather than an endosymbiotic one.     

Intracellular parasitism,the driving force of evolution of Legionella pneumophila and the genus Legionella

Legionella pneumophila is an intracellular pathogen that causes a severe pneumonia called Legionnaires' disease that is often fatal when not promptly diagnosed and treated. Legionella parasitize aquatic protozoa with which it co-evolved over an evolutionary long time. The close relationship between hosts and pathogens, their co-evolution, led to molecular interactions such as the exchange of genetic material through horizontal gene transfer (HGT). Genome sequencing of L. pneumophila and of the entire genus Legionella that comprises over 60 species revealed that Legionellae have co-opted genes and thus cellular functions from their eukaryotic hosts to a surprisingly high extent. Acquisition and loss of these eukaryotic-like genes and domains is an on-going process underlining the highly dynamic nature of the Legionella genomes. Although the large amount and diversity of HGT in Legionella seems to be unique in the prokaryotic world the analyses of more and more genomes from environmental organisms and symbionts of amoeba revealed that such genetic exchanges occur among all amoeba associated bacteria and also among the different microorganisms that infect amoeba. This dynamic reshuffling and gene-acquisition has led to the emergence of Legionella as human pathogen and may lead to the emergence of new human pathogens from the environment.     

Lactate dehydrogenase in amoeba,Acanthamoeba sp. in different phases and conditions of culture

• 1.1. The activity and kinetic changes of amoeba LDH in different phases and conditions of culture were investigated.
• 2.2. LDH of the amoeba is specific against d(−)LDH irrespective of the hypoxic conditions created.
• 3.3. In hypoxic conditions it was not possible to visualize the presence of another LDH isozyme of muscle type by kinetic or electrophoretic analysis.
• 4.4. However, the changes in the K_m value and the L:H ratio as well as the decrease of electrophoretic mobility of LDH band indicate the change in kinetic properties of the enzyme from an obviously heart type in oxygenated culture in the direction of a muscle type LDH in strongly hypoxic culture conditions.
• 5.5. The influence of factors producing either environmental or metabolic hypoxia on possible repression or induction of LDH in amoeba is discussed.