Bacterial Surface Traits Influence Digestion by Tetrahymena pyriformis and Alter Opportunity to Escape from Food Vacuoles.

Endosymbiotic interactions are frequently found in nature, especially in the group of protists. Even though many endosymbioses have been studied in detail, little is known about the mechanistic origins and physiological prerequisites of endosymbiont establishment. A logical step towards the development of endocytobiotic associations is evading digestion and escaping from the host's food vacuoles. Surface properties of bacteria are probably involved in these processes. Therefore, we chemically modified the surface of a transformant strain of Escherichia coli prior to feeding to Tetrahymena pyriformis. N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide allows any substance carrying amino- or carboxyl groups to be bound covalently to the bacterial surface by forming a peptide bond, thus, altering its properties biochemically and biophysically in a predictable manner. The effect of different traits on digestion of T. pyriformis was examined by fluorescence and transmission electron microscopy. The efficiency of digestion differs considerably depending on the coupled substances. Alkaline substances inhibit digestion partially, resulting in incomplete digestion and slightly enhanced escape rates. Increasing hydrophobicity leads to much higher escape frequencies. Both results point to possible mechanisms employed by pathogenic bacteria or potential endosymbionts in evading digestion and transmission to the host's cytoplasm.

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

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.

A model for endosymbiosis: interaction between Tetrahymena pyriformis and Escherichia coli.

Endosymbiosis in ciliates is a common and highly diverse phenomenon in nature, but its development at the mechanistic level and the origins are not easy to understand, since these associations may have arisen at any time during evolution. Therefore a laboratory model is helpful. It could be provided by the interaction of Tetrahymena pyriformis and Escherichia coli. Microscopic analyses with a genetically manipulated fluorescent strain of E. coli show single bacteria leaving food vacuoles and escaping digestion, an important prerequisite for further experiments. Under selective conditions, beneficial for T. pyriformis, the ciliate was shown to internalize E. coli cells. After feeding, bacteria, transformed with the plasmids pBS-neoTet or pNeo4, provide T. pyriformis with the ability to handle toxic conditions, caused by the aminoglykoside antibiotic paromomycin. Axenic cultures or cocultures with untransformed bacteria show lower cell numbers and survival rates compared to cocultures with transformed bacteria after transfer to paromomycin containing media. PCR detects bacterial DNA inside T. pyriformis cells. Additionally, microscopical analysis of selectively grown cocultures reveals fluorescing particles in the cytoplasm of T. pyriformis containing DNA and lipids, corresponding in size to E. coli. This system could be a reasonable model for understanding mechanisms of endosymbiosis establishment in ciliates.