Raman Spectroscopic Characterization of Packaged L. pneumophila Strains Expelled by T. thermophila.

The intracellular lifestyle of L. pneumophila within protozoa is considered to be a fundamental process that supports its survival in nature. However, after ingesting the cells of L. pneumophila, some protozoa expel them as compressed live cells in the form of small rounded pellets. The pellets of tightly packaged viable but not culturable forms (VBNCFs) as well as highly infectious mature intracellular forms (MIFs) of L. pneumophila are considered as infectious particles most likely capable to cause human infection. Since L. pneumophila cells are hardly culturable from these pellets, detection methods for packaged live L. pneumophila forms remaining in water should be cultivation free. Hence, we demonstrate the potential of Raman microspectroscopy to directly sort pellets containing L. pneumophila cells, expelled by T. thermophila, and to characterize them on the basis of their Raman spectra.

Carbon nanotube compared with carbon black: effects on bacterial survival against grazing by ciliates and antimicrobial treatments.

The ingestion and digestion of Escherichia coli by the ciliated protozoan, Tetrahymena thermophila, was investigated after an initial exposure to either water-soluble single-walled carbon nanotubes (SWNT) or to carbon black (CB). Both SWNT and CB were internalised and visible in food vacuoles of ciliates. When presented with E. coli expressing green-fluorescent protein (GFP), these ciliates internalised bacteria as well. However, ciliates that had first internalised SWNT but not CB subsequently externalised or egested vesicle-like structures with fluorescent bacteria inside. These egested bacteria were viable and less susceptible than planktonic E. coli to killing either by the antibiotic, chloramphenicol or the disinfectant, glutaraldehyde. These results suggest that SWNT can alter the intracellular trafficking of vesicles within ciliates, leading to bacterial prey being packaged externally and protected for a time from environmental killing, which could have implications for sewage treatment and for public health.

Development of an Aeromonas hydrophila infection model using the protozoan Tetrahymena thermophila.

Aeromonas hydrophila is a motile bacterium present in numerous freshwater habitats worldwide and is frequently the cause of infections in fish and numerous terrestrial vertebrates including humans. Because A. hydrophila is also a component of the normal intestinal flora of healthy fish, virulence mechanisms are not well understood. Considering that fish models used for the examination of A. hydrophila genes associated with virulence have not been well defined, we established an infection model using the free-living, ciliate protozoa Tetrahymena thermophila. The expression of A. hydrophila virulence genes following infection of T. thermophila was assessed by reverse transcription-PCR and demonstrated that the aerolysin (aerA) and Ahe2 serine protease (ahe2) genes (not present in the avirulent A. hydrophila NJ-4 strain) in the virulent J-1 strain were upregulated 4-h postinfection. Furthermore, the presence of intact A. hydrophila J-1 within T. thermophila suggested that these bacteria could interfere with phagocytosis, resulting in the death of the infected protozoan 48-h postinfection. Conversely, A. hydrophila NJ-4-infected T. thermophila survived the infection. This study established a novel T. thermophila infection model that will provide a novel means of examining virulence mechanisms of A. hydrophila.

Biomagnification of cadmium selenide quantum dots in a simple experimental microbial food chain.

Previous studies have shown that engineered nanomaterials can be transferred from prey to predator, but the ecological impacts of this are mostly unknown. In particular, it is not known if these materials can be biomagnified-a process in which higher concentrations of materials accumulate in organisms higher up in the food chain. Here, we show that bare CdSe quantum dots that have accumulated in Pseudomonas aeruginosa bacteria can be transferred to and biomagnified in the Tetrahymena thermophila protozoa that prey on the bacteria. Cadmium concentrations in the protozoa predator were approximately five times higher than their bacterial prey. Quantum-dot-treated bacteria were differentially toxic to the protozoa, in that they inhibited their own digestion in the protozoan food vacuoles. Because the protozoa did not lyse, largely intact quantum dots remain available to higher trophic levels. The observed biomagnification from bacterial prey is significant because bacteria are at the base of environmental food webs. Our findings illustrate the potential for biomagnification as an ecological impact of nanomaterials.

Shiga toxin as a bacterial defense against a eukaryotic predator, Tetrahymena thermophila.

Bacterially derived exotoxins kill eukaryotic cells by inactivating factors and/or pathways that are universally conserved among eukaryotic organisms. The genes that encode these exotoxins are commonly found in bacterial viruses (bacteriophages). In the context of mammals, these toxins cause diseases ranging from cholera to diphtheria to enterohemorrhagic diarrhea. Phage-carried exotoxin genes are widespread in the environment and are found with unexpectedly high frequency in regions lacking the presumed mammalian "targets," suggesting that mammals are not the primary targets of these exotoxins. We suggest that such exotoxins may have evolved for the purpose of bacterial antipredator defense. We show here that Tetrahymena thermophila, a bacterivorous predator, is killed when cocultured with bacteria bearing a Shiga toxin (Stx)-encoding temperate bacteriophage. In cocultures with Tetrahymena, the Stx-encoding bacteria display a growth advantage over those that do not produce Stx. Tetrahymena is also killed by purified Stx. Disruption of the gene encoding the StxB subunit or addition of an excess of the nontoxic StxB subunit substantially reduced Stx holotoxin toxicity, suggesting that this subunit mediates intake and/or trafficking of Stx by Tetrahymena. Bacterially mediated Tetrahymena killing was blocked by mutations that prevented the bacterial SOS response (recA mutations) or by enzymes that breakdown H(2)O(2) (catalase), suggesting that the production of H(2)O(2) by Tetrahymena signals its presence to the bacteria, leading to bacteriophage induction and production of Stx.

Impact of carbon nanotubes on the ingestion and digestion of bacteria by ciliated protozoa.

Research on the toxicity of carbon nanotubes has focused on human health risks, and little is known about their impact on natural ecosystems. The ciliated protozoan Tetrahymena thermophila has been widely studied by ecotoxicologists because of its role in the regulation of microbial populations through the ingestion and digestion of bacteria, and because it is an important organism in wastewater treatment and an indicator of sewage effluent quality. Here we show that single-walled carbon nanotubes are internalized by T. thermophila, possibly allowing the nanotubes to move up the food chain. The internalization also causes the protozoa to aggregate, which impedes their ability to ingest and digest their prey bacteria species, although it might also be possible to use nanotubes to improve the efficiency of wastewater treatment.

Intracellular multiplication of Legionella pneumophila in Tetrahymena thermophila.

It has been shown that Legionella pneumophila proliferates intracellularly in more than ten species of protozoa, but the fate of the bacteria in Tetrahymena thermophila has not been reported. We investigated the multiplication of L. pneumophila Philadelphia-1 strain in micronucleated T. thermophila, and the effects of temperature and numbers of the bacteria ingested by the protozoa after in vitro feeding were studied. T. thermophila preyed actively upon the bacteria. After being ingested, the fate of the bacteria was affected by both temperature and the number of bacteria ingested. When the number of ingested bacteria was 30 per protozoon, the bacteria proliferated intracellularly at 35 degrees C. The bacteria, however, could not proliferate at 28 degrees C or 32 degrees C though they survived in the protozoa. When the ingested bacteria was 10 per protozoon, the bacteria were killed in the protozoa at all of the temperatures tested. Electron microscopic examination revealed that the protozoa ingesting the bacteria remarkably swelled and that protozoan food vacuoles which contained L. pneumophila were studded with ribosomes.