Since the discovery that Legionella pneumophila can infect and replicate in free-living amoebae , there has been an increasing interest in these professional phagocytes which have been used as an alternative host model to study various aspects of host-pathogen interactions and to characterise Selleck Entospletinib bacterial PI3K inhibitor virulence mechanisms . Among the bacteria that have evolved to resist destruction by free-living
amoebae (hereinafter called ARB for amoeba-resistant bacteria)  we can distinguish (i) true symbionts, which cohabit with the amoeba and maintain a stable host-parasite ratio over a specific period and (ii) pathogens able to lyse the amoebae . As a protective environment for ARB, free-living protozoa represent a potential bacterial reservoir and may act as a vector for bacterial dissemination and colonisation of new niches . In this study, we examined the potential of the bactivorous amoeba A. castellanii as a host model for T. equigenitalis and T. asinigenitalis. We assessed (i) the survival capacity of taylorellae in the presence of A. castellanii, (ii) the internalisation of taylorellae by A. castellanii and (iii) the impact of taylorellae on Acanthamoeba castellanii cultures. Methods Bacterial this website strains and growth conditions The bacterial strains used in this study were as follows: Escherichia coli strain DH5α (Invitrogen),
L. pneumophila serogroup 1 strain Lens  and the two recently-sequenced strains T. equigenitalis MCE9  and T. asinigenitalis MCE3 .
The axenic A. castellanii strain used in this study was derived from an environmental isolate . Escherichia coli was grown at 37°C in Luria-Bertani (LB) medium. Legionella pneumophila was grown at 30°C either on buffered charcoal yeast extract (BCYE) agar [10 g.L-1 ACES (N-(2-Acetamido)-2-aminoethanesulfonic acid); 10 g.L-1 Yeast extract; 2 g.L-1 Charcoal; 15 g.L-1 why agar; 0.4 g.L-1 L-cystein; 0.25 g.L-1 FeNO3; pH 6.9] or in BYE liquid medium. Taylorella equigenitalis and T. asinigenitalis were grown at 37°C in 5% (v/v) CO2 in air for 48 h and 72 h respectively on ready-to-use chocolate agar media (AES Chemunex, Combourg, France). Acanthamoeba castellanii cells were grown at 30°C on PYG medium [0.75% (w/v) proteose peptone, 0.75% (w/v) yeast extract and 1.5% (w/v) glucose]  and split once a week. Bacterial survival following A. castellanii co-infection Acanthamoeba castellanii cells were infected with E. coli, L. pneumophila, T. equigenitalis or T. asinigenitalis at an MOI (multiplicity of infection) of 50. Infections were synchronised by spinning the bacteria (880 × g, 10 min) and extracellular bacteria removed by washing. Extracellular bacteria were quantified by plating the supernatant, while amoeba-associated bacteria were quantified by plating once the amoebae were lysed (Triton X-100 0.