Use of Tetrahymena thermophila To Study the Role of Protozoa in Inactivation of Viruses in Water

Abstract

The ability of a ciliate to inactivate bacteriophage was studied because these viruses are known to influence the size and diversity of bacterial populations, which affect nutrient cycling in natural waters and effluent quality in sewage treatment, and because ciliates are ubiquitous in aquatic environments, including sewage treatment plants. Tetrahymena thermophila was used as a representative ciliate; T4 was used as a model bacteriophage. The T4 titer was monitored on Escherichia coli B in a double-agar overlay assay. T4 and the ciliate were incubated together under different conditions and for various times, after which the mixture was centrifuged through a step gradient, producing a top layer free of ciliates. The T4 titer in this layer decreased as coincubation time increased, but no decrease was seen if phage were incubated with formalin-fixed Tetrahymena. The T4 titer associated with the pellet of living ciliates was very low, suggesting that removal of the phage by Tetrahymena inactivated T4. When Tetrahymena cells were incubated with SYBR gold-labeled phage, fluorescence was localized in structures that had the shape and position of food vacuoles. Incubation of the phage and ciliate with cytochalasin B or at 4°C impaired T4 inactivation. These results suggest the active removal of T4 bacteriophage from fluid by macropinocytosis, followed by digestion in food vacuoles. Such ciliate virophagy may be a mechanism occurring in natural waters and sewage treatment, and the methods described here could be used to study the factors influencing inactivation and possibly water quality.

Ingestion and Inactivation of Bacteriophages by Tetrahymena

“…If phages are ingested, it should be possible to localize them to food vacuoles. Indeed, in T4‐ Tetrahymena co‐incubations, Pinheiro et al (2007) showed SYBR gold‐labeled T4 in structures that appeared to be food vacuoles. Our approach was complementary, but employed a post‐ingestion staining strategy rather than a pre‐ingestion strategy.…”

“…Their loss from the environment is thought to be primarily due to abiotic factors, such as UV‐ or chemically induced damage, but some loss by biotic factors also occurs (Vincent and Neale 2000; Weinbauer 2004). González and Suttle (1993) and Pinheiro et al (2007) have demonstrated that feeding by heterotrophic flagellates and ciliates, respectively, may be two biotic factors reducing phage population densities. In this paper, we confirmed and extended the results of Pinheiro et al (2007) by showing that several species of Tetrahymena ingest and inactivate phage T4, and that T. thermophila inactivates not only T4, but also phages T5 and λ.…”

Separation of Bacteria, Protozoa and Carbon Nanotubes by Density Gradient Centrifugation

“…Eukaryotic microbes, however, have been reported to become destroyed in concentrated sucrose gradients and thus require either fixation before centrifugation in sucrose or use of a different density gradient medium for separation of live cells. For example, polysucrose has been used to separate T. thermophila from virus particles [19], and colloidal silica suspensions have been used to separate the green alga Desmodesmus subspicatus from unbound MWCNTs [20]. However, neither polysucrose nor colloidal silica solutions are ideal for density gradient separation of eukaryotic cells, especially ciliates, because of the high viscosity and hyperosmotic properties of the former [14], and because the latter is comprised of particles that are ingestible by phagocytosing ciliates [21,22].…”

Indirect interactions in the microbial world: specificities and similarities to plant–insect systems

“…Although the direct predation rate of HNF on free-living viral particles accounts for \10% of HNF predation on bacteria (i.e., Suttle and Cheng 1992;Gonzáles and Suttle 1993;Bettarel et al 2005), it could have nonnegligible effects on the size distribution of viral assemblages (Demuth et al 1993;Weinbauer 2004). In addition, it is also worth noting that viruses within bacterial host cells are killed indirectly by HNF predation of infected bacterial cells (e.g., Pinheiro et al 2007), and this effect has been incorporated into theoretical models (Binder 1999;Miki and Yamamura 2005). It is also interesting to note that infected bacterial cells seem to be more vulnerable to predation than uninfected cells (Evans and Wilson 2008).…”