Free-living amebae are ubiquitous in the environment and can be isolated from a variety of habitats including water, soil, air, hospital water systems, dental units, contact lens cases, and cooling towers. The interaction of amebae with other microorganisms in their environment is varied. Bacteria are a major food source for free-living amebae. However, some bacteria have established a stable symbiotic relationship with amebae. Recent reports indicate an association of amebae with intracellular bacterial pathogens. Such amebae may serve as reservoirs for maintaining and dispersing pathogenic bacteria in the environment or as vectors of bacterial disease in humans.

Amoebae of the xD strain of Amoeba proteus that arose from the D strain by spontaneous infection of Legionella-like X-bacteria are now dependent on their symbionts for survival. Each xD amoeba contains about 42,000 symbionts within symbiosomes, and established xD amoebae die if their symbionts are removed. Thus, harmful infective bacteria changed into necessary cell components. As a result of harboring X-bacteria. xD amoebae exhibit various physiological and genetic characteristics that are different from those of symbiont-free D amoebae. One of the recent findings is that bacterial symbionts control the expression of a host's house-keeping gene. Thus, the expression of the normal amoeba sams gene (sams1) encoding one form of S-adenosylmethionine synthetase is switched to that of sams2 by endosymbiotic X-bacteria. Possible mechanisms for the switching of sams genes brought about by endosymbionts and its significance are discussed.

The occurrence of bacterial endosymbionts in free-living amoebae has been known for decades, but their obligate intracellular lifestyle hampered their identification. Application of the full cycle rRNA approach, including 16S rRNA gene sequencing and fluorescence in-situ hybridization with 16S rRNA-targeted oligonucleotide probes, assigned the symbionts of Acanthamoeba spp. and Hartmannella sp. to five different evolutionary lineages within the Proteobacteria, the Bacteroidetes, and the Chlamydiae, respectively. Some of these bacterial symbionts are most closely related to bacterial pathogens of humans, and it has been suggested that they should be considered potential emerging pathogens. Complete genome sequence analysis of a chlamydia-related symbiont of Acanthamoeba sp. showed that this endosymbiont uses similar mechanisms for interaction with its eukaryotic host cell as do the well-known bacterial pathogens of humans. Furthermore, phylogenetic analysis suggested that these mechanisms have been evolved by the ancestor of these amoeba symbionts in interplay with ancient unicellular eukaryotes.

Pore-forming polypeptides have been purified from several amoeboid protozoans that are well-known human pathogens. Obligate enteric parasites, such as Entamoeba histolytica, and free-living but potentially highly pathogenic species, such as Naegleria fowleri, contain these cytolytic molecules inside cytoplasmic granules. Comprehensive functional and structural studies have been conducted that include isolation of the proteins from their natural sources, monitoring of their biological activity towards different targets, and molecular cloning of the genes of their precursors. In the case of the most prominent member of the protein family, with respect to protozoans, the three-dimensional structure of amoebapore A was solved recently. The amoebic pore-forming polypeptides can rapidly perforate human cells. The antibacterial activity of amoebapores and of related polypetides from free-living protozoa points to a more vital function of these molecules: inside the digestive vacuoles they combat growth of phagocytosed bacteria which are killed when their cytoplasmic membranes are permeabilized. The concommitant activity of these proteins towards host cells may be due to a coincidental selection for an efficient effector molecule. Nonetheless, several lines of evidence indicate that these factors are involved in pathogenesis of fatal diseases induced by amoeboid protozoa.

Found in soil and freshwater habitats, Naegleria fowleri are free-living amebae that cause a fatal disease in humans called Primary Amebic Meningoencephalitis. In the natural environment, amebae feed on bacteria. In the infected host, the amebae lyse and ingest nerve tissue. Recently, we have established that N. fowleri expresses a “CD59-like” surface protein, but the function of this protein in the ameba has not been elucidated. In mammalian cells, CD59 is a complement-regulatory protein that inhibits complement-mediated lysis of cells expressing this protein. In the present study, expression of the “CD59-like” protein in response to bacteria and bacterial toxins was investigated by Western immunoblot analysis. Co-culture of N. fowleri with log phase Escherichia coli or Pseudomonas aeruginosa resulted in differential expression of the “CD59-like” protein. Co-cultures of amebae and bacteria were examined by electron microscopy. The results of our study implicate a possible protective role of the “CD59-like” protein in response to bacterial predators and bacterial toxins, because amebae remained intact after co-culture with bacteria.

Chlorarachniophytes are marine amoeboflagellate protists that have acquired their plastid (chloroplast) through secondary endosymbiosis with a green alga. Like other algae, most of the proteins necessary for plastid function are encoded in the nuclear genome of the secondary host. These proteins are targeted to the organelle using a bipartite leader sequence consisting of a signal peptide (allowing entry in to the endomembrane system) and a chloroplast transit peptide (for transport across the chloroplast envelope membranes). We have examined the leader sequences from 45 full-length predicted plastid-targeted proteins from the chlorarachniophyte Bigelowiella natans with the goal of understanding important features of these sequences and possible conserved motifs. The chemical characteristics of these sequences were compared with a set of 10 B. natans endomembrane-targeted proteins and 38 cytosolic or nuclear proteins, which show that the signal peptides are similar to those of most other eukaryotes, while the transit peptides differ from those of other algae in some characteristics. Consistent with this, the leader sequence from one B. natans protein was tested for function in the apicomplexan parasite, Toxoplasma gondii, and shown to direct the secretion of the protein.

In Tetrahymena thermophila, an “antisense ribosome” technology has been developed for inhibiting gene expression and generating novel mutants. Short segments of genes are inserted in antisense orientation into an rDNA vector in a region corresponding to an external loop of the folded rRNA. DNA segments derived from the 5′-ends of genes have proven most effective in reducing cognate gene expression. To investigate the efficacy of other genic regions, we generated Tetrahymena cell lines with antisense ribosome constructs containing 100-bp DNA segments derived from the 5′-ends, 3′-ends, and internal coding regions of two non-essential genes, granule lattice protein 1 and macronuclear histone H1. The 5′- and 3′-end constructs inhibited gene expression, but antisense ribosomes derived exclusively from coding regions had little effect.

This study was undertaken to assess whether amoebae commonly found in mesohaline environments are in fact stages in the life cycles of Pfiesteria and Pfiesteria-like dinoflagellates. Primary isolations of amoebae and dinoflagellates were made from water and sediment samples from five tributaries of the Chesapeake Bay. Additional amoebae were also cloned from bioassay aquaria where fish mortality was attributed to Pfiesteria. Electron microscopy and small subunit (SSU) rRNA gene sequence analysis of these isolates clearly demonstrated that the commonly depicted amoeboid form of Pfiesteria is very likely a species of Korotnevella and is unrelated to Pfiesteria or Pfiesteria-like dinoflagellates. We have determined that the Pfiesteria and Pfiesteria-like dinoflagellates examined in this study undergo a typical homothallic life cycle without amoeboid stages. Furthermore, we have demonstrated that cloned amoebae sharing morphological characteristics described for stages in the life cycle of Pfiesteria do not transform into dinozoites. The strict clonal isolation and cultivation techniques used in this study substantially support the conclusion that the amoebae and some of the flagellates depicted in the life cycle of Pfiesteria are environmental contaminants of the Pfiesteria culture system and that the Ambush Predator Hypothesis needs to be rigorously reevaluated.

Ultraviolet light is being considered as a disinfectant by the water industry because it appears to be very effective for inactivating pathogens, including Cryptosporidium parvum. However, many organisms have mechanisms for repairing ultraviolet light-induced DNA damage, which may limit the utility of this disinfection technology. Inactivation of C. parvum was assessed by measuring infectivity in cells of the human ileocecal adenocarcinoma HCT-8 cell line, with an assay targeting a heat shock protein gene and using a reverse transcriptase polymerase chain reaction to detect infections. Oocysts of five different isolates displayed similar sensitivity to ultraviolet light. An average dosage of 7.6 mJ/cm² resulted in 99.9% inactivation, providing the first evidence that multiple isolates of C. parvum are equally sensitive to ultraviolet disinfection. Irradiated oocysts were unable to regain pre-irradiation levels of infectivity, following exposure to a broad array of potential repair conditions, such as prolonged incubation, pre-infection excystation triggers, and post-ultraviolet holding periods. A combination of data-mining and sequencing was used to identify genes for all of the major components of a nucleotide excision repair complex in C. parvum and Cryptosporidium hominis. The average similarity between the two organisms for the various genes was 96.4% (range, 92–98%). Thus, while Cryptosporidum spp. may have the potential to repair ultraviolet light-induced damage, oocyst reactivation will not occur under the standard conditions used for storage and distribution of treated drinking water.

We first reported here that the harmful alga Cochlodinium polykrikoides, which had been previously known as an autotrophic dinoflagellate, was a mixotrophic species. We investigated the kinds of prey species and the effects of the prey concentration on the growth and ingestion rates of C. polykrikoides when feeding on an unidentified cryptophyte species (Equivalent Spherical Diameter, ESD = 5.6 μm). We also calculated grazing coefficients by combining field data on abundances of C. polykrikoides and co-occurring cryptophytes with laboratory data on ingestion rates obtained in the present study. Cocholdinium polykrikoides fed on prey cells by engulfing the prey through the sulcus. Among the phytoplankton prey offered, C. polykrikoides ingested small phytoplankton species that had ESD's ≤ 11 μm (e.g. the prymnesiophyte Isochrysis galbana, an unidentified cryptophyte, the cryptophyte Rhodomonas salina, the raphidophyte Heterosigma akashiwo, and the dinoflagellate Amphidinium carterae). It did not feed on larger phytoplankton species that had ESD's ≥ 12 μm (e.g. the dinoflagellates Heterocapsa triquetra, Prorocentrum minimum, Scrippsiella sp., Alexandrium tamarense, Prorocentrum micans, Gymnodinium catenatum, Akashiwo sanguinea, and Lingulodinium polyedrum). Specific growth rates of C. polykrikoides on a cryptophyte increased with increasing mean prey concentration, with saturation at a mean prey concentration of approximately 270 ng C ml⁻¹ (i.e. 15,900 cells ml⁻¹). The maximum specific growth rate (mixotrophic growth) of C. polykrikoides on a cryptophyte was 0.324 d⁻¹, under a 14:10 h light-dark cycle of 50 μE m⁻² s⁻¹, while its growth rate (phototrophic growth) under the same light conditions without added prey was 0.166 d⁻¹. Maximum ingestion and clearance rates of C. polykrikoides on a cryptophyte were 0.16 ng C grazer⁻¹d⁻¹ (9.4 cells grazer⁻¹d⁻¹) and 0.33 μl grazer⁻¹h⁻¹, respectively. Calculated grazing coefficients by C. polykrikoides on cryptophytes were 0.001–0.745 h⁻¹ (i.e. 0.1–53% of cryptophyte populations were removed by a C. polykrikoides population in 1 h). The results of the present study suggest that C. polykrikoides sometimes has a considerable grazing impact on populations of cryptophytes.

Traditionally, monoxenous trypanosomatid protozoa are not believed to infect vertebrate cells. Using light and electron microscopy, we show that the monoxenous trypanosomatids Crithidia deanei and Herpetomonas roitmani are able to infect dermal mouse fibroblasts in vitro. We present experimental evidence of phagocytosis of these trypanosomatids, and demonstrate their survival in vertebrate cells. This paper raises the question about the role of C. deanei and H. roitmani, and perhaps other monoxenous trypanosomatid species, in opportunistic infections of immunocompromised individuals and cutaneos lesions in vertebrate hosts.

Percolomonas cosmopolitus is a common free-living flagellate of uncertain phylogenetic position that was placed within the Heterolobosea on the basis of ultrastructure studies. To test the relationship between Percolomonas and Heterolobosea, we analysed the primary structure of the actin and small-subunit ribosomal RNA (SSU rRNA) genes of P. cosmopolitus as well as the predicted secondary structure of the SSU rRNA. Percolomonas shares common secondary structure patterns of the SSU rRNA with heterolobosean taxa, which, together with the results of actin gene analysis, confirms that it is closely related to Heterolobosea. Phylogenetic reconstructions based on the sequences of the SSU rRNA gene suggest Percolomonas belongs to the family Vahlkampfiidae. The first Bayesian analysis of a large taxon sampling of heterolobosean SSU rRNA genes clarifies the phylogenetic relationships within this group.

Soil protozoa, and ciliates in particular, represent a microbial group abundant in the rhizosphere with an influential role on nutrient cycling. Under laboratory conditions, ciliates regulate the size and the composition of bacterial communities, and appear to stimulate ammonification and nitrification. In spite of their important ecological role, our understanding about the factors that control their diversity and abundance in natural forest ecosystems is still rudimentary. Plant species-specific interactions have been demonstrated between plants and soil bacteria and mycorrhizal fungi, due in part to the release of phytohormones and C- and N-rich exudates. We tested the hypothesis that the rhizosphere environments of different plant species also influence the species richness and abundance of soil ciliates. Plant effect, soil pH, moisture content, microbial biomass C, and inorganic nitrogen were measured among five plant species to determine the best predictor variables for soil ciliate species richness and total abundance in a subtropical moist forest in Puerto Rico. Based on an analysis of variance, we rejected the hypothesis that there was a plant species-specific effect on soil ciliates, unlike other microbial groups mentioned above. Using multiple regression analysis, we demonstrated that the flush of total inorganic nitrogen was the best predictor variable for both species richness and abundance of ciliates.