Entamoeba Chitinase is Required for Mature Round Cyst Formation.

Entamoeba histolytica, a protozoan parasite, causes amoebiasis in humans. Amoebiasis transmission is solely mediated by chitin-walled cysts, which are produced in the large intestine of humans from proliferative trophozoites by a cell differentiation process called encystation. Resistance to environmental stresses, an essential characteristic for transmission, is attributed to the cyst wall, which is constructed from chitin and several protein components, including chitinase. Chitinase may play a key role in cyst wall formation; however, this has not been confirmed. Here, to elucidate the physiological role of chitinase during Entamoeba encystation, we identified a new chitinase inhibitor, 2,6-dichloro-4-[2-(1-piperazinyl)-4-pyridinyl]-N-(1,3,5-trimethyl-1H-pyrazol-4-yl)-benzenesulfonamide, by recombinant-Entamoeba chitinase-based screening of 400 Pathogen Box chemicals. This compound dose dependently inhibited native chitinase associated with Entamoeba invadens encystation, a model for E. histolytica encystation, with an 50% inhibitory concentration (IC50) of ∼0.6 µM, which is comparable to the IC50s (0.2 to 2.5 µM) for recombinant E. histolytica and E. invadens chitinases. Furthermore, the addition of this compound to E. invadens encystation-inducing cultures increased the generation of cyst walls with an abnormal shape, the most characteristic of which was a "pot-like structure." A similar structure also appeared in standard culture, but at a far lower frequency. These results indicate that chitinase inhibition increases the number of abnormal encysting cells, thereby significantly reducing the efficiency of cyst formation. Transmission electron microscopy showed that compound-treated encysting cells formed an abnormally loose cyst wall and an unusual gap between the cyst wall and cell membrane. Hence, Entamoeba chitinase is required for the formation of mature round cysts. IMPORTANCE Amoebiasis is caused by Entamoeba histolytica infection and is transmitted by dormant Entamoeba cells or cysts. Cysts need to be tolerant to severe environmental stresses faced outside and inside a human host. To confer this resistance, Entamoeba parasites synthesize a wall structure around the cell during cyst formation. This cyst wall consists of chitin and several protein components, including chitinase. The physiological roles of these components are not fully understood. Here, to elucidate the role of chitinase during cyst formation, we identified a new chitinase inhibitor by screening a library of 400 compounds. Using this inhibitor, we showed that chitinase inhibition causes the formation of abnormal cyst walls, the most characteristic of which is a "pot-like structure." This results in decreased production of mature cysts. Chitinase is therefore required for Entamoeba to produce mature cysts for transmission to a new host.

The dynamics of ultrastructural changes during Entamoeba invadens encystation.

Entamoeba histolytica infection causes amoebiasis, which is a global public health problem. The major route of infection is oral ingestion of E. histolytica cysts, cysts being the sole form responsible for host-to-host transmission. Cysts are produced by cell differentiation from proliferative trophozoites in a process termed 'encystation'. Therefore, encystation is an important process from a medical as well as a biological perspective. Previous electron microscopy studies have shown the ultrastructure of precysts and mature cysts; however, the dynamics of ultrastructural changes during encystation were ambiguous. Here, we analysed a series of Entamoeba invadens encysting cells by transmission electron microscopy. Entamoeba invadens is a model for encystation and the cells were prepared by short interval time course sampling from in vitro encystation-inducing cultures. We related sampled cells to stage conversion, which was monitored in the overall population by flow cytometry. The present approach revealed the dynamics of ultrastructure changes during E. invadens encystation. Importantly, the results indicate a functional linkage of processes that are crucial in encystation, such as glycogen accumulation and cyst wall formation. Hence, this study provides a reference for studying sequential molecular events during Entamoeba encystation.

Growth-promoting effects of the hydrogen-sulfide compounds produced by Desulfovibrio desulfuricans subsp. desulfuricans co-cultured with Escherichia coli (DH5α) on the growth of Entamoeba and Endolimax species isolates from swine.

Certain Desulfovibrio sp. (anaerobic sulfate-reducing bacteria) are indigenous to swine cecum and colon, which are also common habitats for parasitic amoebae such as Entamoeba polecki and Entamoeba suis. In this study, we evaluated the growth-promoting effects of D. desulfuricans co-cultured with Escherichia coli (DH5α) and its products [e.g., hydrogen sulfide (H2S) and certain iron-sulfide (FeS) compounds] using Robinson's medium, on the 4 amoeba isolates from swine-Entamoeba polecki subtype (ST)-1, E. polecki ST-3, Entamoeba suis, and Endolimax sp., and, consequently, a continuous culture system for these amoebae was established. However, this novel culture system was required to regulate the excess H2S dissolved in the medium by increasing air space as amoeba isolates thrive only in large air spaces (30-40%). The effects of air space and H2S and FeS compounds on the growth of E. polecki ST-1 (TDP-5) were determined. E. polecki ST-1 (TDP-5) thrived well in culture bottles with an air space of 30-40% (aerobic) (H2S: ~250-400 µmoles/L), but did not grow at all in an air space < 5% (microaerobic) ( H2S:~800 µmoles/L) and in anaerobic vessels (H2S: 20-30 µmoles/L). In both H2S-depleted and FeS compound-depleted conditions, the amoebae sp. could not thrive either. It was hypothesized that an appropriate concentration of H2S and FeS compounds might function as important physiologically active components of electron carriers such as FeS and ferredoxin.

Unique Features of Entamoeba Sulfur Metabolism; Compartmentalization, Physiological Roles of Terminal Products, Evolution and Pharmaceutical Exploitation.

Sulfur metabolism is essential for all living organisms. Recently, unique features of the Entamoeba metabolic pathway for sulfated biomolecules have been described. Entamoeba is a genus in the phylum Amoebozoa and includes the causative agent for amoebiasis, a global public health problem. This review gives an overview of the general features of the synthesis and degradation of sulfated biomolecules, and then highlights the characteristics that are unique to Entamoeba. Future biological and pharmaceutical perspectives are also discussed.

Reassessing the Role of Entamoeba gingivalis in Periodontitis.

The protozoan Entamoeba gingivalis resides in the oral cavity and is frequently observed in the periodontal pockets of humans and pets. This species of Entamoeba is closely related to the human pathogen Entamoeba histolytica, the agent of amoebiasis. Although E. gingivalis is highly enriched in people with periodontitis (a disease in which inflammation and bone loss correlate with changes in the microbial flora), the potential role of this protozoan in oral infectious diseases is not known. Periodontitis affects half the adult population in the world, eventually leads to edentulism, and has been linked to other pathologies, like diabetes and cardiovascular diseases. As aging is a risk factor for the disorder, it is considered an inevitable physiological process, even though it can be prevented and cured. However, the impact of periodontitis on the patient's health and quality of life, as well as its economic burden, are underestimated. Commonly accepted models explain the progression from health to gingivitis and then periodontitis by a gradual change in the identity and proportion of bacterial microorganisms in the gingival crevices. Though not pathognomonic, inflammation is always present in periodontitis. The recruitment of leukocytes to inflamed gums and their passage to the periodontal pocket lumen are speculated to fuel both tissue destruction and the development of the flora. The individual contribution to the disease of each bacterial species is difficult to establish and the eventual role of protozoa in the fate of this disease has been ignored. Following recent scientific findings, we discuss the relevance of these data and propose that the status of E. gingivalis be reconsidered as a potential pathogen contributing to periodontitis.

An NAD+-dependent novel transcription factor controls stage conversion in Entamoeba.

Developmental switching between life-cycle stages is a common feature among parasitic pathogens to facilitate disease transmission and pathogenesis. The protozoan parasite Entamoeba switches between invasive trophozoites and dormant cysts, but the encystation process remains poorly understood despite being central to amoebic biology. We identify a transcription factor, Encystation Regulatory Motif-Binding Protein (ERM-BP), that regulates encystation. Down-regulation of ERM-BP decreases encystation efficiency resulting in abnormal cysts with defective cyst walls. We demonstrate that direct binding of NAD+ to ERM-BP affects ERM-BP conformation and facilitates its binding to promoter DNA. Additionally, cellular NAD+ levels increase during encystation and exogenous NAD+ enhances encystation consistent with the role of carbon source depletion in triggering Entamoeba encystation. Furthermore, ERM-BP catalyzes conversion of nicotinamide to nicotinic acid, which might have second messenger effects on stage conversion. Our findings link the metabolic cofactors nicotinamide and NAD+ to transcriptional regulation via ERM-BP and provide the first mechanistic insights into Entamoeba encystation.

Cellular Events of Multinucleated Giant Cells Formation During the Encystation of Entamoeba invadens.

Entamoeba histolytica, the causative agent of amoebiasis, does not form cysts in vitro, so reptilian pathogen Entamoeba invadens is used as an Entamoeba encystation model. During the in vitro encystation of E. invadens, a few multinucleated giant cells (MGC) were also appeared in the culture along with cysts. Like the cyst, these MGC's were also formed in the multicellular aggregates found in the encystation culture. Time-lapse live cell imaging revealed that MGC's were the result of repeated cellular fusion with fusion-competent trophozoites as a starting point. The early MGC were non-adherent, and they moved slowly and randomly in the media, but under confinement, MGC became highly motile and directionally persistent. The increased motility resulted in rapid cytoplasmic fissions, which indicated the possibility of continuous cell fusion and division taking place inside the compact multicellular aggregates. Following cell fusion, each nucleus obtained from the fusion-competent trophozoites gave rise to four nuclei with half genomic content. All the haploid nuclei in MGC later aggregated and fused to form a polyploid nucleus. These observations have important implications on Entamoeba biology as they point toward the possibility of E. invadens undergoing sexual or parasexual reproduction.

A Flow Cytometry Method for Dissecting the Cell Differentiation Process of Entamoeba Encystation.

Amoebiasis is caused by Entamoeba histolytica infection, a protozoan parasite belonging to the phylum Amoebozoa. This parasite undergoes a fundamental cell differentiation process from proliferative trophozoite to dormant cyst, termed "encystation." The cysts formed by encystation are solely responsible for the transmission of amoebiasis; therefore, Entamoeba encystation is an important subject from both biological and medical perspectives. Here, we have established a flow cytometry strategy for not only determining the percentage of formed cysts but also for monitoring changes in cell populations during encystation. This strategy together with fluorescence microscopy enables visualization of the cell differentiation process of Entamoeba encystation. We also standardized another flow cytometry protocol for counting live trophozoites. These two different flow cytometry techniques could be integrated into 96-well plate-based bioassays for monitoring the processes of cyst formation and trophozoite proliferation, which are crucial to maintain the Entamoeba life cycle. The combined two systems enabled us to screen a chemical library, the Pathogen Box of the Medicine for Malaria Venture, to obtain compounds that inhibit either the formation of cysts or the proliferation of trophozoites, or both. This is a prerequisite for the development of new drugs against amoebiasis, a global public health problem. Collectively, the two different 96-well plate-based Entamoeba bioassay and flow cytometry analysis systems (cyst formation and trophozoite proliferation) provide a methodology that can not only overcome the limitations of standard microscopic counting but also is effective in applied as well as basic Entamoeba biology.

Flow cytometric characterization of encystation in Entamoeba invadens.

Entamoeba histolytica causes dysentery and liver abscess mostly in countries that lack proper sanitation. Infection is acquired by ingestion of the cyst form in contaminated food or water. E. histolytica does not encyst in vitro; thus, E. invadens, a reptilian parasite that encysts in vitro, has been used as a surrogate. Cysts are small and possess chitin-rich walls. These are characteristics that may be exploited by flow cytometry. We stained encysting E. invadens cells with a fluorescent chitin stain, and analyzed fluorescence and forward scatter by flow cytometry. We demonstrate that flow cytometry can be used to track differentiation, reveal unique cell populations, and evaluate encystation inhibitors.

Presence of Parasites in Environmental Waters in Samsun and Its Districts.

OBJECTIVE: The aim of this study was to detect the presence of parasites in environmental waters in Samsun and its districts. METHODS: At the center of Samsun, 13 stations were determined. The research was performed between March 2012 and February 2013, and every month, water samples were collected on the dates stated. The samples were stained with Kinyoun acid-fast, modified trichrome, and trichrome dyes after examining with the direct bond. The preparations were evaluated in terms of parasitologic under a light microscope. RESULTS: Totally, 180 of 228 water samples analyzed were from streams; of these, 48 were drinking water samples. The following were found: 142 Giardia spp., 132 Cryptosporidium spp., 56 Cyclospora spp., 38 microsporidia, 47 Blastocystis spp., 38 Entamoeba coli cysts, 18 Dientamoeba, 9 Chilomastix, 9 Strongyloides spp., and 6 hookworms. CONCLUSION: The widespread use of animal husbandry and agriculture in the region and the use of stream surroundings as a grazing area increase the presence of some determined protozoa during a certain period. Parasitological studies in humans and animals in the region should be conducted, and control programs should be applied.

First molecular epidemiology of Entamoeba histolytica, E. dispar and E. moshkovskii infections in Yemen: different species-specific associated risk factors.

OBJECTIVES: To investigate the molecular epidemiology of Entamoeba histolytica, E. dispar and E. moshkovskii infections among rural communities in Yemen. METHODS: In a community-based study, faecal samples were collected from 605 participants and examined by wet mount, formalin-ether sedimentation, trichrome staining and nested multiplex PCR techniques. Demographic, socio-economic and environmental information was collected using a pre-tested questionnaire. RESULTS: Overall, 324 (53.6%) of the samples were positive for Entamoeba cysts and/or trophozoites by microscopic examination. Molecular analysis revealed that 20.2%, 15.7% and 18.2% of the samples were positive for E. histolytica, E. dispar and E. moshkovskii, respectively. Multivariate analysis showed different sets of species-specific risk factors among these communities. Educational level was identified as the significant risk factor for E. histolytica; age and gender were the significant risk factors for E. moshkovskii; and sources of drinking water and consumption of unwashed vegetables were the significant risk factors for E. dispar. Moreover, living in coastal/foothill areas and presence of other infected family members were risk factors for both E. histolytica and E. moshkovskii infections. CONCLUSION: The study reveals that Entamoeba spp. infection is highly prevalent among rural communities in Yemen, with E. histolytica, E. dispar and E. moshkovskii differentiated for the first time. Identifying and treating infected family members, providing health education pertinent to good personal and food hygiene practices and providing clean drinking water should be considered in developing a strategy to control intestinal parasitic infections in these communities, particularly in the coastal/foothill areas of the country.

The Potential of Lactobacillus casei and Entercoccus faecium Combination as a Preventive Probiotic Against Entamoeba.

Travellers' diarrhoea caused by enteric protozoa like Entamoeba histolytica is among the most common protozoan diseases in developing countries. In developing countries, amoebiasis is the second most prevalent protozoan disease. This protozoan parasite is often known to coexist as a part of the normal gut microbiota. It is estimated that around 50-60 % of population in developing countries might be harbouring Entamoeba in an asymptomatic manner. Due to physiological perturbation or upon immuno-compromise, it can become virulent and then cause diarrhoea, bloody stools and may invade other organs if left untreated. Nitroimidazole drugs, namely metronidazole and tinidazole, are widely used to treat protozoan infections. These drugs often show dose-dependent side effects. With emerging antibiotic resistance, novel therapeutics to prevent parasitic infections is required. This study aims to study effect of probiotics on prevention of Amoebiasis. In this study, we have investigated the effect of selected probiotics on the growth of Entamoeba. From the list of probiotics being currently used, five bacterial strains were selected for testing. These probiotic strains were co-cultured with Entamoeba, and their effect on Entamoeba proliferation was checked. Of the five probiotics chosen, individual treatments of Lactobacillus casei and Enterococcus faecium showed a significant reduction of up to 71 % in parasite survival only at higher CFUs. When the two probiotics were used in combination, the percentage of survival reduced gradually further to 80 % at a total CFU of 109 cells/ml of bacteria. The study lays the foundation for providing cost-effective prophylactic treatment for amoebiasis without the overuse of antibiotics.

Cell-matrix interactions of Entamoeba histolytica and E. dispar. A comparative study by electron-, atomic force- and confocal microscopy.

Invasion of tissues by Entamoeba histolytica is a multistep process that initiates with the adhesion of the parasite to target tissues. The recognition of the non-invasive Entamoeba dispar as a distinct, but closely related protozoan species raised the question as to whether the lack of its pathogenic potential could be related to a weaker adhesion due to limited cytoskeleton restructuring capacity. We here compared the adhesion process of both amebas to fibronectin through scanning, transmission, atomic force, and confocal microscopy. In addition, electrophoretic and western blot assays of actin were also compared. Adhesion of E. histolytica to fibronectin involves a dramatic reorganization of the actin network that results in a tighter contact to and the subsequent focal degradation of the fibronectin matrix. In contrast, E. dispar showed no regions of focal adhesion, the cytoskeleton was poorly reorganized and there was little fibronectin degradation. In addition, atomic force microscopy using topographic, error signal and phase modes revealed clear-cut differences at the site of contact of both amebas with the substrate. In spite of the morphological and genetic similarities between E. histolytica and E. dispar the present results demonstrate striking differences in their respective cell-to-matrix adhesion processes, which may be of relevance for understanding the invasive character of E. histolytica.

Entamoeba invadens: Identification of a SERCA protein and effect of SERCA inhibitors on encystation.

Calcium has an important role on signaling of different cellular processes, including growth and differentiation. Signaling by calcium also has an essential function in pathogenesis and differentiation of the protozoan parasites Entamoeba histolytica and Entamoeba invadens. However, the proteins of these parasites that regulate the cytoplasmic concentration of this ion are poorly studied. In eukaryotic cells, the calcium-ATPase of the SERCA type plays an important role in calcium homeostasis by catalyzing the active efflux of calcium from cytoplasm to endoplasmic reticulum. Here, we reported the identification of SERCA of E. invadens (EiSERCA). This protein contains a putative sequence for endoplasmic reticulum retention and all domains involved in calcium transport identified in mammalian SERCA. By immunofluorescence assays, an antibody against SERCA of E. histolytica detected EiSERCA in a vesicular network in the cytoplasm of E. invadens trophozoites, co-localizing with calreticulin. Interestingly, EiSERCA was redistributed close to plasma membrane during encystation, suggesting that this pump could participate in regulate the calcium concentration during this process. In addition, thapsigargin and cyclopiazonic acid, both specific inhibitors of SERCA, affected the number and structure of cysts, supporting the hypothesis that calcium flux mediated by SERCA has an important role in the life cycle of Entamoeba.

Ribosomal RNA and protein transcripts persist in the cysts of Entamoeba invadens.

In most organisms rDNA transcription ceases under conditions of growth stress. However, we have earlier shown that pre-rRNA accumulates during encystation in Entamoeba invadens. We labeled newly-synthesized rRNA during encystation, with [methyl-(3)H] methionine in the presence of chitinase to enable uptake of isotope. Incorporation rate reduced after 24h, and then increased to reach levels comparable with normal cells. The label was rapidly chased to the ribosomal pellet in dividing cells, while at late stages of encystation the ratio of counts going to the pellet dropped 3-fold. The transcript levels of selected ribosomal protein genes also went down initially but went up again at later stages of encystation. This suggested that rRNA and ribosomal protein transcription may be coordinately regulated. Our data shows that encysting E. invadens cells accumulate transcripts of both the RNA and protein components of the ribosome, which may ensure rapid synthesis of new ribosomes when growth resumes.

Actin, RhoA, and Rab11 participation during encystment in Entamoeba invadens.

In the genus Entamoeba, actin reorganization is necessary for cyst differentiation; however, its role is still unknown. The aim of this work was to investigate the role of actin and encystation-related proteins during Entamoeba invadens encystation. Studied proteins were actin, RhoA, a small GTPase involved through its effectors in the rearrangement of the actin cytoskeleton; Rab11, a protein involved in the transport of encystation vesicles; and enolase, as an encystment vesicles marker. Results showed a high level of polymerized actin accompanied by increased levels of RhoA-GTP during cell rounding and loss of vacuoles. Cytochalasin D, an actin polymerization inhibitor, and Y27632, an inhibitor of RhoA activity, reduced encystment in 80%. These inhibitors also blocked cell rounding, disposal of vacuoles, and the proper formation of the cysts wall. At later times, F-actin and Rab11 colocalized with enolase, suggesting that Rab11 could participate in the transport of the cyst wall components through the F-actin cytoskeleton. These results suggest that actin cytoskeleton rearrangement is playing a decisive role in determining cell morphology changes and helping with the transport of cell wall components to the cell surface during encystment of E. invadens.

The ribosomal RNA transcription unit of Entamoeba invadens: accumulation of unprocessed pre-rRNA and a long non coding RNA during encystation.

The ribosomal RNA genes in Entamoeba spp. are located on extrachromosomal circular molecules. Unlike model organisms where rRNA transcription stops during growth stress, Entamoeba histolytica continues transcription; but unprocessed pre-rRNA accumulates during stress, along with a novel class of circular transcripts from the 5'-external transcribed spacer (ETS). To determine the fate of rRNA transcription during stage conversion between trophozoite to cyst we analyzed Entamoeba invadens, a model system for differentiation studies in Entamoeba. We characterized the complete rDNA transcription unit by mapping the ends of pre-rRNA and mature rRNAs. The 3' end of mature 28S rRNA was located 321 nt downstream of the end predicted by sequence homology with E. histolytica. The major processing sites were mapped in external and internal transcribed spacers. The promoter located within 146 nt upstream of 5' ETS was used to transcribe the pre-rRNA. On the other hand, a second promoter located at the 3' end of 28S rDNA was used to transcribe almost the entire intergenic spacer into a long non coding (nc) RNA (>10 kb). Interestingly we found that the levels of pre-rRNA and long ncRNA, measured by northern hybridization, decreased initially in cells shifted to encystation medium, after which they began to increase and reached high levels by 72 h when mature cysts were formed. Unlike E. histolytica, no circular transcripts were found in E. invadens. E. histolytica and E. invadens express fundamentally different ncRNAs from the rDNA locus, which may reflect their adaptation to different hosts (human and reptiles, respectively). This is the first description of rDNA organization and transcription in E. invadens, and provides the framework for further studies on regulation of rRNA synthesis during cyst formation.

Homologous recombination occurs in Entamoeba and is enhanced during growth stress and stage conversion.

Homologous recombination (HR) has not been demonstrated in the parasitic protists Entamoeba histolytica or Entamoeba invadens, as no convenient method is available to measure it. However, HR must exist to ensure genome integrity, and possible genetic exchange, especially during stage conversion from trophozoite to cyst. Here we show the up regulation of mitotic and meiotic HR genes in Entamoeba during serum starvation, and encystation. To directly demonstrate HR we use a simple PCR-based method involving inverted repeats, which gives a reliable read out, as the recombination junctions can be determined by sequencing the amplicons. Using this read out, we demonstrate enhanced HR under growth stress in E. histolytica, and during encystation in E. invadens. We also demonstrate recombination between chromosomal inverted repeats. This is the first experimental demonstration of HR in Entamoeba and will help future investigations into this process, and to explore the possibility of meiosis in Entamoeba.

Transcriptome analysis of encystation in Entamoeba invadens.

Encystation is an essential differentiation process for the completion of the life cycle of a group of intestinal protozoa including Entamoeba histolytica, the causative agent of intestinal and extraintestinal amebiasis. However, regulation of gene expression during encystation is poorly understood. To comprehensively understand the process at the molecular level, the transcriptomic profiles of E. invadens, which is a related reptilian species that causes an invasive disease similar to that of E. histolytica, was investigated during encystation. Using a custom-generated Affymetrix platform microarray, we performed time course (0.5, 2, 8, 24, 48, and 120 h) gene expression analysis of encysting E. invadens. ANOVA analysis revealed that a total of 1,528 genes showed ≥3 fold up-regulation at one or more time points, relative to the trophozoite stage. Of these modulated genes, 8% (116 genes) were up-regulated at the early time points (0.5, 2 and 8h), while 63% (962 genes) were up-regulated at the later time points (24, 48, and 120 h). Twenty nine percent (450 genes) are either up-regulated at 2 to 5 time points or constitutively up-regulated in both early and late stages. Among the up-regulated genes are the genes encoding transporters, cytoskeletal proteins, proteins involved in vesicular trafficking (small GTPases), Myb transcription factors, cysteine proteases, components of the proteasome, and enzymes for chitin biosynthesis. This study represents the first kinetic analysis of gene expression during differentiation from the invasive trophozoite to the dormant, infective cyst stage in Entamoeba. Functional analysis on individual genes and their encoded products that are modulated during encystation may lead to the discovery of targets for the development of new chemotherapeutics that interfere with stage conversion of the parasite.

Growth of Entamoeba invadens in sediments with metabolically repressed bacteria leads to multicellularity and redefinition of the amoebic cell system.

Extracellular signaling and mechanisms of cell differentiation in Entamoeba are misunderstood. The main reason is the popular use of axenic media, which do not correspond to the natural habitats of Entamoeba. The axenic environment lacks the exogenous activators and repressors provided by natural habitats. Absent bacterial commensals understanding of the development of the amoebic cell system remains deficient. The present Aa(Sm) culture method using mixed sediments of antibiotically repressed Aerobacter aerogens and amoebae was developed to model in vitro extracellular signaling that induce multicellularity in cultures of E. invadens. Repressed oxygen consuming sediment bacteria supply E. invadens the hypoxic environment needed for differentiation and development. The amoebae themselves alter the environment by consuming the bacteria by phagocytosis thus reversing hypoxia. Exogenous activators are in this manner down regulated and suppressed. This feedback effect controls amoebic development and differentiation. Co-existing cell types and cell fractions with different life spans and cell cycle length could be identified. Aa(Sm) long term cultures contain continuous and non-continuous self renewing cell lines producing quiescent and terminally differentiated daughter cells (precysts) by asymmetric division. This culturing method helps to understand the intimate relationship between hypoxic environments and the multicellular behaviour of E. invadens and the interrelations existing between the distinct cell types.