Evaluation of bacterial ligands involved in receptor-mediated phagocytosis in Tetrahymena pyriformis

Boboc, Adela and Parry, Jackie (2020) Evaluation of bacterial ligands involved in receptor-mediated phagocytosis in Tetrahymena pyriformis. Masters thesis, Lancaster University.

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Abstract

This study examined the feeding of Tetrahymena pyriformis on eleven strains of bacteria evaluating the importance of receptor-ligand interactions in prey uptake and food vacuole formation. Three prey ‘types’ were employed: live cells (full complement of surface ligands), heat-killed (dead) cells (disrupted receptor ligands) and, latex beads (no ligands). The ciliates were fed for five minutes with each prey type in monocultures (2x107 cells/ml) and in 50:50 mixtures and the number of vacuoles/cell, prey/cell and prey/vacuole were determined after fixation of the ciliates. Prey uptake and vacuole formation in T. pyriformis was highest with live cells, followed by dead cells and then beads; except Synechococcus sp., Serratia marcescens and Staphylococcus aureus which behaved like beads after heat-treatment. There was no evidence of T. pyriformis actively selecting one prey type over another in any of the 50:50 mixtures, and the ciliate showed to deposit different prey types in the same vacuole, suggesting only one route of internalization of different types of prey. Significant trends regarding vacuole formation were observed in prey mixtures: (i) live and dead cells always controlled vacuole formation in mixture with beads, and (ii) live and dead cells together showed a synergistic effect on vacuoles/cell (but did not show synergy with regards to prey uptake). The latter observation led to the proposal that different receptor-ligand interactions might be in place for vacuole formation (involving a vacuole formation factor, VFF) and prey uptake (involving a prey uptake factor, PUF). Moreover, when heat-stabilities of PUF and VFF were analysed, data for Pseudomonas aeruginosa (VFF heat-stable, PUF heat-labile) and Salmonella enterica 12694, Pseudomonas fluorescens, Klebsiella pneumoniae (VFF heat-labile, PUF heat-stable) also suggested distinct PUF and VFF factors, even if similar heat-stabilities were recorded for most bacteria tested (either both intact, partially or completely destroyed). Removal of A. hydrophila, S. marcescens and Synechococcus sp. S-layers by lithium chloride showed no direct implication of this ligand in prey uptake by T. pyriformis. However, experiments involving trypsin and formaldehyde fixation, which damage cell-associated proteins but not carbohydrates, suggested that the uptake could be mediated by a carbohydrate ligand (for Gramnegative bacteria) or by a protein (for Gram-positive bacteria). Analysis of capsular heat stability of all strains tested suggested that capsules could be positively involved in the uptake of Gramnegative bacteria by the ciliate, but not Gram-positive. Finally, the presence of sugar receptors and scavenger receptors in T. pyriformis was examined. Pre-incubation of T. pyriformis with up to 200mM sugar concentration of mannose, Nacetylgalactosamine (GalNAc) or N-acetylglucosamine (GlcNAc) suggested that receptors with binding affinities for all three sugars are involved in uptake of live and dead S. enterica 74. The uptake of live S. enterica 74 was higher than dead cells and beads until a particular sugar concentration which led them to become equivalent to dead cells (Mannose at 20mM, GalNAc at 50mM and GlcNAc at 125mM), and then at an even higher sugar concentration, when the uptake became equivalent to beads (Mannose at 175mM; GalNAc/GlcNAc at 150mM). A similar trend was observed for vacuole formation, suggesting a sequential blocking of receptors for theattachment to live cells. No evidence was found for the involvement of scavenger receptors in the uptake of beads or live and dead Gram-negative S. enterica 74 by T. pyriformis.