castellanii cells were performed four hours after infection with fluorescein-labelled T. equigenitalis (Figure 2A) or T. asinigenitalis check details (Figure 2B). For both taylorellae, we observed exclusively intracellular bacteria, mainly grouped in clusters. No bacterium was observed attached to the cell surface of the amoeba. Our data show that the persistent amoeba-associated taylorellae are located within the cytoplasm of A. castellanii. Figure 2 Location of T. equigenitalis and T. asinigenitalis within A. castellanii . Confocal laser scanning micrographs of A. castellanii

cells at 4 h post-infection with fluorescein-labelled T. equigenitalis (A) or T. asinigenitalis (B). Similar results were observed in two independent

experiments. Actin polymerisation and phosphoinositide 3-kinase play a key role in taylorellae internalisation To investigate the uptake mechanism involved in taylorellae internalisation, two chemical inhibitors were used: Cytochalasin D (CytoD), a potent inhibitor of actin polymerisation, and Wortmannin (Wort), an inhibitor of phosphoinositide 3-kinases (PI3K). Bacterial uptake in amoebae was measured by trypan blue quenching of fluorescein-labelled T. equigenitalis, T. asinigenitalis, E. coli or L. pneumophila. Fluorescein-labelled Navitoclax in vitro bacteria were used to infect A. castellanii when CytoD or Wort were present, as indicated. After contact, trypan blue was added to quench the fluorescence of non-internalised bacteria and the fluorescence, which was representative of bacterial internalisation by amoebae, was measured (Figure 3). For the four selleck products tested bacterial species, amoebae exposed to CytoD and Wort show a decrease in fluorescence compared to untreated amoebae. The decrease in fluorescence was comparable for all four bacterial species and for both phagocytosis inhibitors. These results suggest that taylorellae are internalised by an uptake mechanism such as phagocytosis, which is dependent upon actin polymerisation and PI3K. Figure 3 Taylorellae are actively phagocytised by A. castellanii . Bacterial uptake assay by trypan blue

quenching. Acanthamoeba castellanii cells were infected with fluorescein-labelled E. coli, T. equigenitalis, T. asinigenitalis or L. pneumophila at an MOI of 50, in the presence, when indicated, of either 10 μM of cytochalasin D—an actin polymerization inhibitor (+CytoD)—or 2 μM of Wortmanin—a PI3K inhibitor (+Wort). After 30 min of incubation, the medium was replaced by trypan blue solution to quench the fluorescence of non-internalised bacteria. The fluorescence of internalised bacteria was measured using an excitation level of 485 nm and an emission of 530 nm. Fluorescence data were corrected for differences in labelling efficiency between the tested strains. Each bar represents the mean of triplicate wells and error bars represent the standard deviations.