Ingestion and digestion studies in Tetrahymena pyriformis based on chemically modified microparticles

Abstract

Recognition of food and, in consequence, ingestion of digestible particles is a prerequisite for energy metabolism in Tetrahymena pyriformis. Understanding why some particles are ingested and digested, whereas others are not, is important for many fields of research, e.g. survival of pathogens in single-celled organisms or establishment of endosymbiotic relationships. We offered T. pyriformis synthetical bovine-serum-albumin (BSA)-methacrylate microparticles of approximately 5.5 μm diameter and studied the ciliates’ ingestion and digestion behaviour. Different staining techniques as well as co-feeding with a transformant strain of Escherichia coli revealed that T. pyriformis considers these particles as natural food source and shows no feeding preference. Further, they are ingested at normal rates and may serve as sole food source. A pivotal advantage of these particles is the convenient modification of their surface by binding different ligands resulting in defined surface properties. Ingestion rate of modified microparticles either increased (additional BSA, enzymes) or decreased (amino acids). Furthermore, we investigated glycosylation patterns by lectin binding. By binding different substances to the surface in combination with various staining techniques, we provide a versatile experimental tool for elucidating details on food recognition and digestion that may allow to study evading digestion by pathogens or potential endosymbionts, too.

Introduction

Recognition of potential food sources and, as a logical consequence, ingestion of food particles, are important prerequisites for many processes in a ciliate cell. A suitable model organism for studying ingestion and digestion in protozoa is Tetrahymena pyriformis (e.g. Nelson et al., 2003, Nilsson, 1977, Ricketts, 1971a). Also selective feeding has been studied in some detail (e.g. Boenigk and Novarino, 2004, Thurman et al., 2010). Tetrahymena pyriformis seems to depend on particles in ambient media as a stimulus for initiating the formation of food vacuoles. The ciliates’ uptake and growth rates in filter-sterilized and consequently particle-free medium are reduced drastically in comparison to autoclaved medium or medium containing particles (Rasmussen and Kludt, 1970, Rasmussen and Modeweg-Hansen, 1973), which recommends this organism for studying the effects of different food particles on ingestion and digestion.

In this study, we offered T. pyriformis artificial microparticles with approximately 5.5 μm diameter, based on bovine-serum-albumin (BSA), embedded and stabilized by a methacrylate polymer matrix. These microparticles can easily be stained with different dyes. In addition, the surface properties can be defined by covalent binding of different substances, thus opening wide possibilities for studying effects of chemical and physical properties on the fate of such particles.

Little is known about food recognition in T. pyriformis. Filter-feeding ciliates as T. pyriformis seem to show less discriminative selective feeding than other free living protists (Boenigk and Novarino, 2004, Jacobs et al., 2006). Other studies suggest differing interpretations (Thurman et al. 2010). By an unknown mechanism, these organisms are capable of distinguishing between biotic and abiotic particles, since starved T. pyriformis cultures do not ingest colloidal gold when food bacteria are offered in the same medium, whereas gold particles are observed within T. pyriformis in the absence of bacteria (Elliott and Clemmons, 1966). Comparable observations were made by Ricketts (1971a) and Seaman (1961), showing that starved cultures of T. pyriformis ingest exclusively useful particles. On the other hand, the addition of proteose-peptone-yeast extract medium (PPY) leads to passive ingestion of latex particles (Ricketts 1971a). Furthermore, different substances reveal various efficiency in stimulation of food vacuole formation. Whereas proteins or peptides like proteose peptone or bovine serum albumin, as well as polypeptides or RNA are highly effective in stimulating food vacuole formation, glutamate, low molecular weight substances like amino acid mixtures, polysaccharides or glucose stimulate vacuole formation only moderately. Sodium acetate is completely ineffective (Ricketts 1972). Since the tested substances vary in size as well as in charge, the author concluded that these traits play only a minor role in stimulating food vacuole formation. Further experiments by Rasmussen and Modeweg-Hansen (1973) pointed out that molecule net charge plays only a minor role, since they provided T. pyriformis with particles of uniform size, but different net charges (positive, negative and uncharged) and each of the offered particles enhanced growth similarly. Based on these experiments, it seems reasonable to assume a complex food recognition site at the cytostome of T. pyriformis, since biotic and abiotic particles are distinguished and different substances influence uptake rates.

Studies on selective feeding of other free living protists reveal more details about reception mechanisms (Montagnes et al. 2008). Different lectins may be involved in food recognition and binding in Oxyrrhis marina (Wootton et al. 2007) and Hartmanella vermiformis (Venkataraman et al. 1997). Allen and Dawidowicz (1990) identified a mannose-binding receptor protein involved in attaching and internalizing yeast by Acanthamoeba castellanii. How Tetrahymena distinguishes between different food particles before ingestion remains unknown. Also a complete phagosome proteome of T. thermophila reveals no clues for food perception prior to ingestion (Jacobs et al. 2006). To identify structures involved in food perception, we performed in vivo lectin labelling of T. pyriformis. Glycosylated lipids and proteins are often involved in recognition processes, but are also important as target sequences for transport, self- and non-self-recognition and as protection against self-digestion. Therefore, we used the lectins concanavalinA (conA) and wheat-germ-agglutinin (WGA), coupled with a fluorescent dye. WGA binds to terminal, non-reducing N-acetyl-glucosamine and to sialic acid (Wright 1984), which are both found in N- as well as O-glycosylated proteins. ConA labels internal and terminal, non-reducing α-d-manno-pyranosyl- and α-d-gluco-pyranosyl-residues as well as α-d-glucose and α-d-mannose (Goldstein et al. 1974).

The digestion process of T. pyriformis was studied in some detail (Nilsson, 1977, Nilsson, 1987, Nilsson and van Deurs, 1983). After food vacuoles are formed and detached from the cytostome, the vacuoles are acidified, which is an important prerequisite for lysosome fusion (Nilsson 1977). In Paramecium the acidification is realized by fusion of acidosomes with the food vacuole membrane, a process, resulting in integration of specialized proton pumps (Ishida et al. 1997). How acidification is realized in T. pyriformis remains unclear, but vesicles of unknown function formed at parasomal sacks were observed to fuse with early food vacuoles (Nilsson, 1987, Nilsson and van Deurs, 1983). A phagosome proteome analysis of T. thermophila identified three subunits of a membrane associated, vacuolar ATPase that is possibly involved in acidification (Jacobs et al. 2006). After acidification lysosomes fuse with food vacuoles. The maximal acidic pH of 3.5–4.0 is reached after 1 h. Before defecation of food vacuole contents, they are neutralized. The whole digestion process requires about 2 h (Nilsson 1977).

Since ingestion alone is not inducing digestion, an additional recognition system seems to act inside food vacuoles. By measuring acid phosphatase activity as marker for digestion, Ricketts (1971a) showed that food vacuoles containing indigestible material, i.e. latex particles, do not result in increased acid phosphatase activity, whereas vacuoles containing bacteria, yeast or PPY exhibit increased acid phosphatase activity. Similar findings were made by Boenigk et al. (2001) with nanoflagellates. Food vacuoles, containing indigestible particles, were egested 2–3 min after uptake by Spumella and Ochromonas, whereas vacuoles containing bacteria remained longer within the cells. This points to selective digestion behaviour and consequently to recognition sites within food vacuoles.

Understanding the process of digestion in detail is essential for studying why and how some ingested bacteria are able to evade digestion and in consequence survive or even divide within protists. Especially pathogenic microorganisms were shown to survive the digestive process in T. pyriformis and even to propagate inside the cells (Barker and Brown 1994). Some of them lyse the host after propagation or induce putative encystment, like Listeria monocytogenes. For L. monocytogenes the virulence factor listeriolysin O was identified as one of the causative factors, since defective mutants do not show this effect (Pushkareva and Ermolaeva 2010). Other pathogens multiply inside of T. pyriformis food vacuoles without killing the host, e.g. Mycobacterium sp. (Strahl et al. 2001), whereas Campylobacter jejuni only evades digestion and, thus, survives in T. pyriformis without propagation (Snelling et al. 2005).

Recently, it has been shown that also a non-pathogenic strain of Escherichia coli K12 is able to escape digestion and food vacuoles and, in consequence, persists in the cytoplasm of T. pyriformis (Siegmund et al. 2013). Evading digestion is a logical prerequisite for establishing endosymbiotic interactions. Endosymbiosis is a widespread and very common phenomenon in protozoa and therefore developed into a highly attractive field of research for decades (for review see Görtz 2010). A lot of these interactions are studied in detail, but until now little is known about the mechanistic and physiological prerequisites for intracellular establishment of bacteria. Understanding the specificity of digestive processes will help to identify physiological conditions for escaping food vacuoles and to define those points during digestion, where the potential symbiont interferes. For elucidating the effects of special chemical groups at the surface, we decided to develop a versatile system, allowing to add essentially any chemical side chain to a digestible protein matrix. Studying the influence of microparticles with different, defined surface traits on ingestion and digestion, combined with appropriate staining techniques, will help to understand more details of digestive processes in T. pyriformis and, in the long run, may possibly also facilitate the identification of bacterial surface properties allowing to evade digestion.