Enteropathogenic Escherichia coli modulates the virulence and pathogenicity of Entamoeba dispar.

Amoebiasis is a disease caused by Entamoeba histolytica, affecting the large intestine of humans and occasionally leading to extra-intestinal lesions. Entamoeba dispar is another amoeba species considered commensal, although it has been identified in patients presenting with dysenteric and nondysenteric colitis, as well as amoebic liver abscess. Amoebic virulence factors are essential for the invasion and development of lesions. There is evidence showing that the association of enterobacteria with trophozoites contributes to increased gene expression of amoebic virulence factors. Enteropathogenic Escherichia coli is an important bacterium causing diarrhea, with high incidence rates in the world population, allowing it to interact with Entamoeba sp. in the same host. In this context, this study aims to evaluate the influence of enteropathogenic Escherichia coli on ACFN and ADO Entamoeba dispar strains by quantifying the gene expression of virulence factors, including galactose/N-acetyl-D-galactosamine-binding lectin, cysteine proteinase 2, and amoebapores A and C. Additionally, the study assesses the progression and morphological aspect of amoebic liver abscess and the profile of inflammatory cells. Our results demonstrated that the interaction between EPEC and ACFN Entamoeba dispar strains was able to increase the gene expression of virulence factors, as well as the lesion area and the activity of the inflammatory infiltrate. However, the association with the ADO strain did not influence the gene expression of virulence factors. Together, our findings indicate that the interaction between EPEC, ACFN, and ADO Entamoeba dispar strains resulted in differences in vitro and in vivo gene expression of Gal/GalNAc-binding lectin and CP2, in enzymatic activities of MPO, NAG, and EPO, and consequently, in the ability to cause lesions.

Identification of Mamu-DRB1 gene as a susceptibility factor for Entamoeba nuttalli infection in Chinese Macaca mulatta.

Entamoeba nuttalli infection is highly prevalent in captive and wild macaques. A recent study suggested that the genetic factor of host macaques was correlated with the genotypes of E. nuttalli isolates. This study focused on the correlation between the rhesus macaque host major histocompatibility complex gene and E. nuttalli infection. Thirty-nine stool samples were obtained from Mount Qing-ling (Guizhou Province, China). Polymerase chain reaction analysis detected the infection rate of E. nuttalli, Entamoeba coli, and Entamoeba chattoni as 69.23%, 69.23%, and 87.18%, respectively. A new Serine-rich Protein genotype was detected, and the rRNA of E. nuttalli isolates from Mount Qian-ling was completely identical to the GY4 strain. In the distance-based neighbor-joining tree, Mamu-DRB1, not Mamu-DPB or Mamu-B gene, was related to E. nuttalli infection. Mamu-DRB1 genes of rhesus macaques in Mounts Qian-ling and Long-hu were highly polymorphic, and the rhesus macaques with two major types of Mamu-DRB1 showed susceptibility to E. nuttalli infection. The Mamu-DRB1 gene analysis in this study indicated that the Mamu-DRB1 gene is an important factor that influences the susceptibility of E. nuttalli infection in Chinese Macaca mulatta. This study contributes to a better understanding of host susceptibility to Entamoeba.

Morphological and Motility Features of the Stable Bleb-Driven Monopodial Form of Entamoeba and Its Importance in Encystation.

Entamoeba histolytica and its reptilian counterpart and encystation model Entamoeba invadens formed a polarized monopodial morphology when treated with pentoxifylline. This morphology was propelled by retrograde flow of the cell surface resulting from a cyclic sol-gel conversion of cytoplasm and a stable bleb at the leading edge. Pentoxifylline treatment switched the unpolarized, adherent trophozoites to the nonadherent, stable bleb-driven form and altered the motility pattern from slow and random to fast, directionally persistent, and highly chemotactic. Interestingly, exogenously added adenosine produced multiple protrusions and random motility, an opposite phenotype to that of pentoxifylline. Thus, pentoxifylline, an adenosine antagonist, may be inducing the monopodial morphology by preventing lateral protrusions and restricting the leading edge to one site. The polarized form of E. invadens was aggregation competent, and time-lapse microscopy of encystation revealed its appearance during early hours, mediating the cell aggregation by directional cell migration. The addition of purine nucleotides to in vitro encystation culture prevented the formation of polarized morphology and inhibited the cell aggregation and, thus, the encystation, which further showed the importance of the polarized form in the Entamoeba life cycle. Cell polarity and motility are essential in the pathogenesis of Entamoeba parasites, and the stable bleb-driven polarized morphology of Entamoeba may also be important in invasive amoebiasis.

Entamoeba histolytica and Entamoeba dispar: Morphological and Behavioral Differences Induced by Fibronectin through GTPases Activation and Actin-Binding Proteins.

Early steps of tissue invasion by Entamoeba histolytica are mediated by adhesion and migration through matrix components such as fibronectin with the participation of the actin cytoskeleton. Striking differences in their produced structures, movement, and migration were found. These observations suggest differential changes in their ability to organize the actin cytoskeleton and, therefore, to modify its morphology after adhesion to fibronectin. To understand these observations, we explore deeper the cytoskeleton pathway of E. histolytica compared to Entamoeba dispar, analyzing the activation and involvement of actin cytoskeleton regulatory proteins such as small GTPases (Rho, Rac1 and Cdc42), myosin IB, paxillin, alpha-actinin, and ARP2/3 during interaction with fibronectin. Results showed a higher activation of Rac1 in E. histolytica compared to E. dispar, while Cdc42 and RhoA were equally activated in both amebae; besides, variations in the amount of myosin IB, paxillin, and ARP2/3 were detected among these species, coinciding and reflected in formation of lamellipodia in E. histolytica and filopodia in E. dispar. These could partially explain the higher invasive capacity of E. histolytica compared to E. dispar, due to its pleomorphic ability, high motility, migration, activation, and abundance of proteins involved in the cytoskeleton arrangement.

Nuclear Factor Y (NF-Y) Modulates Encystation in Entamoeba via Stage-Specific Expression of the NF-YB and NF-YC Subunits.

Nuclear factor Y (NF-Y) is a heterotrimeric transcription factor composed of three subunits, namely, NF-YA, NF-YB, and NF-YC, which are conserved throughout evolution. In higher eukaryotes, NF-Y plays important roles in several cellular processes (development, cell cycle regulation, apoptosis, and response to growth, stress, and DNA damage) by controlling gene expression through binding to a CCAAT promoter motif. We demonstrated that NF-Y subunits in the protist Entamoeba, while significantly divergent from those of higher eukaryotes, have well-conserved domains important for subunit interactions and DNA binding and that NF-YB and NF-YC are developmentally expressed during encystation. Electrophoretic mobility shift assays confirmed that the NF-Y protein(s) from Entamoeba cysts binds to a CCAAT motif. Consistent with a role as a transcription factor, the NF-Y proteins show nuclear localization during development. Additionally, we demonstrated that NF-YC localizes to the chromatoid body (an RNA processing center) during development, indicating that it may have a role in RNA processing. Finally, silencing of the NF-YC subunit resulted in reduced stability of the NF-Y complex and decreased encystation efficiency. We demonstrated that the NF-Y complex functions at a time point subsequent to the NAD+ flux and expression of the transcription factor encystation regulatory motif-binding protein, both of which are early regulators of Entamoeba development. Taken together, our results demonstrate that the NF-Y complex plays an important role in regulating encystation in Entamoeba and add to our understanding of the transcriptional networks and signals that control this essential developmental pathway in an important human pathogen.IMPORTANCE The human parasite Entamoeba histolytica is an important pathogen with significant global impact and is a leading cause of parasitic death in humans. Since only the cyst form can be transmitted, blocking encystation would prevent new infections, making the encystation pathway an attractive target for the development of new drugs. Identification of the genetic signals and transcriptional regulatory networks that control encystation would be an important advance in understanding the developmental cascade. We show that the Entamoeba NF-Y complex plays a crucial role in regulating the encystation process in Entamoeba.

Discrimination Experiments in Entamoeba and Evidence from Other Protists Suggest Pathogenic Amebas Cooperate with Kin to Colonize Hosts and Deter Rivals.

Entamoeba histolytica is one of the least understood protists in terms of taxa, clone, and kin discrimination/recognition ability. However, the capacity to tell apart same or self (clone/kin) from different or nonself (nonclone/nonkin) has long been demonstrated in pathogenic eukaryotes like Trypanosoma and Plasmodium, free-living social amebas (Dictyostelium, Polysphondylium), budding yeast (Saccharomyces), and in numerous bacteria and archaea (prokaryotes). Kin discrimination/recognition is explained under inclusive fitness theory; that is, the reproductive advantage that genetically closely related organisms (kin) can gain by cooperating preferably with one another (rather than with distantly related or unrelated individuals), minimizing antagonism and competition with kin, and excluding genetic strangers (or cheaters = noncooperators that benefit from others' investments in altruistic cooperation). In this review, we rely on the outcomes of in vitro pairwise discrimination/recognition encounters between seven Entamoeba lineages to discuss the biological significance of taxa, clone, and kin discrimination/recognition in a range of generalist and specialist species (close or distantly related phylogenetically). We then focus our discussion on the importance of these laboratory observations for E. histolytica's life cycle, host infestation, and implications of these features of the amebas' natural history for human health (including mitigation of amebiasis).

Entamoeba gingivalis - prevalence and correlation with dental caries in children from rural and urban regions of Lublin Province, Eastern Poland.

INTRODUCTION: Entamoeba gingivalis, as the name implies. typically lives around the gumline of the teeth in the tartar and gingival pockets of the oral cavity. Pathogenicity of protozoa in the oral cavity is not completely understood. The occurrence in patients with a good state of the immune system usually does not cause any pathological changes. In the presentede survey, an increased incidence of Entamoeba gingivalis in conjunction with pathological changes in the oral cavity was found. OBJECTIVES: The aim of the study was to evaluate the prevalence of Entamoeba gingivalis in children treated in Department of Paediatric Dentistry of the Medical University in Lublin and its correlation with dental caries. MATERIAL AND METHODS: Diagnostic materials in the form of swabs were obtained from 154 children in the age 2-18 years old, treated in the Department of Paediatric Dentistry of the Medical University in Lublin. The main index for epidemiological surveys in oral health - DMFT (decayed, missed, and filled teeth) for permanent teeth and DMFT for deciduous teeth was calculated. RESULTS: The average number of Entamoeba gingivalis in urban children was 12.84. The DMFT number in the deciduous teeth was lower in urban patients (5.0) than from rural patients (5.4). The DMFT number in the permanent teeth was lower in urban patients (3.155) than rural patients (3.98). There was no statistically significant correlation between the DMFT value and the number of Entamoeba gingivalis. CONCLUSIONS: 1) Entamoeba gingivalis occurs in the oral cavity of children. 2) In the presented survey, both DMFT and dmft indexes were high which proves high activity of dental carious. 3) There was no significant statistical correlation observed between dental caries and the presence of Entamoeba gingivalis.

Identification and functional analysis of a stress-responsive MAPK15 in Entamoeba invadens.

E. histolytica, a protozoan parasite is the causative agent of amoebiasis in human beings. It exists in two different forms - the motile trophozoite form which undergoes encystation under starvation conditions to form the non-motile, osmotically resistant cyst form. Cellular stresses stimulate several signaling cascades which assist the parasite in counter-attacking such conditions thereby, promoting cell survival. To study the stress-associated pathways activated during encystation, we have used Entamoeba invadens, a reptilian parasite as a model organism because of its ability to undergo encystation under in vitro conditions. In this study, we have identified a stress-responsive MAPK which gets upregulated under different stress conditions, including encystation. Sequence analysis and phylogenetic classification show that the MAPK belongs to the atypical MAPK15 family (henceforth, named EiMAPK15), which does not require an upstream MAPKK for its phosphorylation and activation. The in vitro kinase activity of recombinant EiMAPK15 exhibits its auto-phosphorylation ability. Immunolocalization studies reveal that the protein is mainly cytosolic under normal growing conditions but gets translocated into the nucleus under stress conditions. Knockdown of EiMAPK15 using double-stranded RNA was found to reduce the expression of other encystation-specific genes which in turn, resulted in the decline of the overall encystation efficiency of the cells. Overall, the present work has laid the platform for further characterization of this important MAPK gene in Entamoeba invadens.

Phosphorylation of Eukaryotic Initiation Factor-2α during Stress and Encystation in Entamoeba Species.

Entamoeba histolytica is an enteric pathogen responsible for amoebic dysentery and liver abscess. It alternates between the host-restricted trophozoite form and the infective environmentally-stable cyst stage. Throughout its lifecycle E. histolytica experiences stress, in part, from host immune pressure. Conversion to cysts is presumed to be a stress-response. In other systems, stress induces phosphorylation of a serine residue on eukaryotic translation initiation factor-2α (eIF2α). This inhibits eIF2α activity resulting in a general decline in protein synthesis. Genomic data reveal that E. histolytica possesses eIF2α (EheIF2α) with a conserved phosphorylatable serine at position 59 (Ser59). Thus, this pathogen may have the machinery for stress-induced translational control. To test this, we exposed cells to different stress conditions and measured the level of total and phospho-EheIF2α. Long-term serum starvation, long-term heat shock, and oxidative stress induced an increase in the level of phospho-EheIF2α, while short-term serum starvation, short-term heat shock, or glucose deprivation did not. Long-term serum starvation also caused a decrease in polyribosome abundance, which is in accordance with the observation that this condition induces phosphorylation of EheIF2α. We generated transgenic cells that overexpress wildtype EheIF2α, a non-phosphorylatable variant of eIF2α in which Ser59 was mutated to alanine (EheIF2α-S59A), or a phosphomimetic variant of eIF2α in which Ser59 was mutated to aspartic acid (EheIF2α-S59D). Consistent with the known functions of eIF2α, cells expressing wildtype or EheIF2α-S59D exhibited increased or decreased translation, respectively. Surprisingly, cells expressing EheIF2α-S59A also exhibited reduced translation. Cells expressing EheIF2α-S59D were more resistant to long-term serum starvation underscoring the significance of EheIF2α phosphorylation in managing stress. Finally, phospho-eIF2α accumulated during encystation in E. invadens, a model encystation system. Together, these data demonstrate that the eIF2α-dependent stress response system is operational in Entamoeba species.

Entamoeba Clone-Recognition Experiments: Morphometrics, Aggregative Behavior, and Cell-Signaling Characterization.

Studies on clone- and kin-discrimination in protists have proliferated during the past decade. We report clone-recognition experiments in seven Entamoeba lineages (E. invadens IP-1, E. invadens VK-1:NS, E. terrapinae, E. moshkovskii Laredo, E. moshkovskii Snake, E. histolytica HM-1:IMSS and E. dispar). First, we characterized morphometrically each clone (length, width, and cell-surface area) and documented how they differed statistically from one another (as per single-variable or canonical-discriminant analyses). Second, we demonstrated that amebas themselves could discriminate self (clone) from different (themselves vs. other clones). In mix-cell-line cultures between closely-related (E. invadens IP-1 vs. E. invadens VK-1:NS) or distant-phylogenetic clones (E. terrapinae vs. E. moshkovskii Laredo), amebas consistently aggregated with same-clone members. Third, we identified six putative cell-signals secreted by the amebas (RasGap/Ankyrin, coronin-WD40, actin, protein kinases, heat shock 70, and ubiquitin) and which known functions in Entamoeba spp. included: cell proliferation, cell adhesion, cell movement, and stress-induced encystation. To our knowledge, this is the first multi-clone characterization of Entamoeba spp. morphometrics, aggregative behavior, and cell-signaling secretion in the context of clone-recognition. Protists allow us to study cell-cell recognition from ecological and evolutionary perspectives. Modern protistan lineages can be central to studies about the origins and evolution of multicellularity.

Exploring NAD+ metabolism in host-pathogen interactions.

Nicotinamide adenine dinucleotide (NAD(+)) is a vital molecule found in all living cells. NAD(+) intracellular levels are dictated by its synthesis, using the de novo and/or salvage pathway, and through its catabolic use as co-enzyme or co-substrate. The regulation of NAD(+) metabolism has proven to be an adequate drug target for several diseases, including cancer, neurodegenerative or inflammatory diseases. Increasing interest has been given to NAD(+) metabolism during innate and adaptive immune responses suggesting that its modulation could also be relevant during host-pathogen interactions. While the maintenance of NAD(+) homeostatic levels assures an adequate environment for host cell survival and proliferation, fluctuations in NAD(+) or biosynthetic precursors bioavailability have been described during host-pathogen interactions, which will interfere with pathogen persistence or clearance. Here, we review the double-edged sword of NAD(+) metabolism during host-pathogen interactions emphasizing its potential for treatment of infectious diseases.

Development of RNA Interference Trigger-Mediated Gene Silencing in Entamoeba invadens.

Entamoeba histolytica, a protozoan parasite, is an important human pathogen and a leading parasitic cause of death. The organism has two life cycle stages, trophozoites, which are responsible for tissue invasion, and cysts, which are involved in pathogen transmission. Entamoeba invadens is the model system to study Entamoeba developmental biology, as high-grade regulated encystation and excystation are readily achievable. However, the lack of gene-silencing tools in E. invadens has limited the molecular studies that can be performed. Using the endogenous RNA interference (RNAi) pathway in Entamoeba, we developed an RNAi-based trigger gene-silencing approach inE. invadens We demonstrate that a gene's coding region that has abundant antisense small RNAs (sRNAs) can trigger silencing of a gene that is fused to it. The trigger fusion leads to the generation of abundant antisense sRNAs that map to the target gene, with silencing occurring independently of trigger location at the 5' or 3' end of a gene. Gene silencing is stably maintained during development, including encystation and excystation. We have used this approach to successfully silence two E. invadens genes: a putative rhomboid protease gene and a SHAQKY family Myb gene. The Myb gene is upregulated during oxidative stress and development, and its downregulation led, as predicted, to decreased viability under oxidative stress and decreased cyst formation. Thus, the RNAi trigger silencing method can be used to successfully investigate the molecular functions of genes inE. invadens Dissection of the molecular basis of Entamoeba stage conversion is now possible, representing an important technical advance for the system.

Expanding the Entamoeba Universe: New Hosts Yield Novel Ribosomal Lineages.

Removing the requirement for cell culture has led to a substantial increase in the number of lineages of Entamoeba recognized as distinct. Surveying the range of potential host species for this parasite genus has barely been started and it is clear that additional sampling of the same host in different locations often identifies additional diversity. In this study, using small subunit ribosomal RNA gene sequencing, we identify four new lineages of Entamoeba, including the first report of Entamoeba from an elephant, and extend the host range of some previously described lineages. In addition, examination of microbiome data from a number of host animals suggests that substantial Entamoeba diversity remains to be uncovered.

Entamoeba mitosomes play an important role in encystation by association with cholesteryl sulfate synthesis.

Hydrogenosomes and mitosomes are mitochondrion-related organelles (MROs) that have highly reduced and divergent functions in anaerobic/microaerophilic eukaryotes. Entamoeba histolytica, a microaerophilic, parasitic amoebozoan species, which causes intestinal and extraintestinal amoebiasis in humans, possesses mitosomes, the existence and biological functions of which have been a longstanding enigma in the evolution of mitochondria. We previously demonstrated that sulfate activation, which is not generally compartmentalized to mitochondria, is a major function of E. histolytica mitosomes. However, because the final metabolites of sulfate activation remain unknown, the overall scheme of this metabolism and the role of mitosomes in Entamoeba have not been elucidated. In this study we purified and identified cholesteryl sulfate (CS) as a final metabolite of sulfate activation. We then identified the gene encoding the cholesteryl sulfotransferase responsible for synthesizing CS. Addition of CS to culture media increased the number of cysts, the dormant form that differentiates from proliferative trophozoites. Conversely, chlorate, a selective inhibitor of the first enzyme in the sulfate-activation pathway, inhibited cyst formation in a dose-dependent manner. These results indicate that CS plays an important role in differentiation, an essential process for the transmission of Entamoeba between hosts. Furthermore, we show that Mastigamoeba balamuthi, an anaerobic, free-living amoebozoan species, which is a close relative of E. histolytica, also has the sulfate-activation pathway in MROs but does not possess the capacity for CS production. Hence, we propose that a unique function of MROs in Entamoeba contributes to its adaptation to its parasitic life cycle.

MALDI-TOF mass spectrometry for the detection and differentiation of Entamoeba histolytica and Entamoeba dispar.

Detection of Entamoeba histolytica and its differentiation from Entamoeba dispar is an important goal of the clinical parasitology laboratory. The aim of this study was the identification and differentiation of E. histolytica and E. dispar by MALDI-TOF MS, in order to evaluate the application of this technique in routine diagnostic practice. MALDI-TOF MS was applied to 3 amebic reference strains and to 14 strains isolated from feces that had been differentiated by molecular methods in our laboratory. Protein extracts from cultures of these strains (axenic cultures for the 3 reference strains and monoxenic cultures for the 14 field isolates) were analyzed by MALDI-TOF MS and the spectra obtained were analyzed by statistical software. Five peaks discriminating between E. histolytica and E. dispar reference strains were found by protein profile analysis: 2 peaks (8,246 and 8,303 Da) specific for E. histolytica and 3 (4,714; 5,541; 8,207 Da) for E. dispar. All clinical isolates except one showed the discriminating peaks expected for the appropriate species. For 2 fecal samples from which 2 strains (1 E. histolytica and 1 E. dispar) out of the 14 included in this study were isolated, the same discriminating peaks found in the corresponding isolated amebic strains were detected after only 12h (E. histolytica) and 24h (E. dispar) of incubation of the fecal samples in Robinson's medium without serum. Our study shows that MALDI-TOF MS can be used to discriminate between E. histolytica and E. dispar using in vitro xenic cultures and it also could have potential for the detection of these species in clinical samples.

Iron-sulphur clusters, their biosynthesis, and biological functions in protozoan parasites.

Fe-S clusters are ensembles of sulphide-linked di-, tri-, and tetra-iron centres of a variety of metalloproteins that play important roles in reduction and oxidation of mitochondrial electron transport, energy metabolism, regulation of gene expression, cell survival, nitrogen fixation, and numerous other metabolic pathways. The Fe-S clusters are assembled by one of four distinct systems: NIF, SUF, ISC, and CIA machineries. The ISC machinery is a house-keeping system conserved widely from prokaryotes to higher eukaryotes, while the other systems are present in a limited range of organisms and play supplementary roles under certain conditions such as stress. Fe-S cluster-containing proteins and the components required for Fe-S cluster biosynthesis are modulated under stress conditions, drug resistance, and developmental stages. It is also known that a defect in Fe-S proteins and Fe-S cluster biogenesis leads to many genetic disorders in humans, which indicates the importance of the systems. In this review, we describe the biological and physiological significance of Fe-S cluster-containing proteins and their biosynthesis in parasitic protozoa including Plasmodium, Trypanosoma, Leishmania, Giardia, Trichomonas, Entamoeba, Cryptosporidium, Blastocystis, and microsporidia. We also discuss the roles of Fe-S cluster biosynthesis in proliferation, differentiation, and stress response in protozoan parasites. The heterogeneity of the systems and the compartmentalization of Fe-S cluster biogenesis in the protozoan parasites likely reflect divergent evolution under highly diverse environmental niches, and influence their parasitic lifestyle and pathogenesis. Finally, both Fe-S cluster-containing proteins and their biosynthetic machinery in protozoan parasites are remarkably different from those in their mammalian hosts. Thus, they represent a rational target for the development of novel chemotherapeutic and prophylactic agents against protozoan infections.

Dysfunctions at human intestinal barrier by water-borne protozoan parasites: lessons from cultured human fully differentiated colon cancer cell lines.

Some water-borne protozoan parasites induce diseases through their membrane-associated functional structures and virulence factors that hijack the host cellular molecules and signalling pathways leading to structural and functional lesions in the intestinal barrier. In this Microreview we analyse the insights on the mechanisms of pathogenesis of Entamoeba intestinalis, Giardia and Cryptosporidium observed in the human colon carcinoma fully differentiated colon cancer cell lines, cell subpopulations and clones expressing the structural and functional characteristics of highly specialized fully differentiated epithelial cells lining the intestinal epithelium and mimicking structurally and functionally an intestinal barrier.

Discrimination, crypticity, and incipient taxa in entamoeba.

Persistent difficulties in resolving clear lineages in diverging populations of prokaryotes or unicellular eukaryotes (protistan polyphyletic groups) are challenging the classical species concept. Although multiple integrated approaches would render holistic taxonomies, most phylogenetic studies are still based on single-gene or morphological traits. Such methodologies conceal natural lineages, which are considered "cryptic." The concept of species is considered artificial and inadequate to define natural populations. Social organisms display differential behaviors toward kin than to nonrelated individuals. In "social" microbes, kin discrimination has been used to help resolve crypticity. Aggregative behavior could be explored in a nonsocial protist to define phylogenetic varieties that are considered "cryptic." Two Entamoeba invadens strains, IP-1 and VK-1:NS are considered close populations of the same "species." This study demonstrates that IP-1 and VK-1:NS trophozoites aggregate only with alike members and discriminate members of different strains based on behavioral and chemical signals. Combined morphological, behavioral/chemical, and ecological studies could improve Archamoebae phylogenies and define cryptic varieties. Evolutionary processes in which selection acted continuously and cumulatively on ancestors of Entamoeba populations gave rise to chemical and behavioral signals that allowed individuals to discriminate nonpopulation members and, gradually, to the emergence of new lineages; alternative views that claim a "Designer" or "Creator" as responsible for protistan diversity are unfounded.

Differences in cap formation between invasive Entamoeba histolytica and non-invasive Entamoeba dispar.

The rapid redistribution of surface antigen-antibody complexes in trophozoites of the human protozoan parasite Entamoeba histolytica, in a process known as capping, has been considered as a means of the parasite to evade the host immune response. So far, capping has been documented in the invasive E. histolytica, whereas the mobility of surface components in the non-invasive Entamoeba dispar is not known. E. dispar does not induce liver lesions in rodent experimental models, in contrast to the liver abscesses produced by E. histolytica in the same animal model. We have therefore analyzed the mobility of surface receptors to the lectin concanavalin A and of Rab11, a membrane-associated protein, in both species of Entamoebae by confocal fluorescence microscopy and transmission and scanning electron microscopy. The great majority of E. histolytica trophozoites became morphologically polarized through the formation of well-defined caps at the posterior pole of the parasite. Actin colocalized with the lectin caps. Antibodies against the membrane protein Rab 11 also produced capping. In striking contrast, in E. dispar, the mobility of concanavalin A surface receptors was restricted to the formation of irregular surface patches that did no progress to constitute well-defined caps. Also, anti-Rab 11 antibodies did not result in capping in E. dispar. Whether the failure of E. dispar to efficiently mobilize surface molecules in response to lectin or antibodies as shown in the present results is related to its non-invasive character represents an interesting hypothesis requiring further analysis.