castellanii infected monolayers Acanthamoeba castellanii monolayers were infected at an MOI of 50 with E. coli, L. pneumophila, T. equigenitalis or T. asinigenitalis. Cell-bacterium contact was initiated by centrifugation (880 × g,
10 min) and incubated at 37°C in 5% (v/v) CO2 in air. Monolayers were observed with a Nikon inverted microscope coupled with an Olympus camera (DP120). Influence PI3K inhibitor of heat-killed A. castellanii and A. castellanii culture supernatant on taylorellae growth Microfiltered (0.22 μm) supernatant of A. castellanii cultured in PYG medium for 5 days and heat-killed A. castellanii cells (100°C, 30 min) were inoculated with a T. equigenitalis or T. asinigenitalis strain at an OD600 of 0.1, 0.2 and 0.5. These cultures were incubated for 5 days at 37°C, either in 5% (v/v) CO2 in air in a static state or aerobically under agitation (200 rpm). Bacterial growth was measured over time by optical density measurement and
plate counts. Results Evolution of taylorellae concentrations in co-culture with A. castellanii To characterise the capacity of T. equigenitalis and T. asinigenitalis to persist within the free-living amoeba A. castellanii, we performed A. castellanii-taylorellae co-cultures and determined the evolution of extracellular (Figure 1A) and amoeba-associated (Figure 1B) bacterial concentrations over time. Escherichia coli MLN8237 in vivo was used as an amoeba-sensitive control bacterium and L. pneumophila, which is able to replicate and evade amoebae, was used as an amoeba-resistant control bacterium. The same evolution of T. equigenitalis and T. asinigenitalis concentrations was observed over the 7 days of co-culture with A. castellanii: the extracellular taylorellae concentrations decreased about one fold over the experiment period, while the amoeba-associated taylorellae concentrations remained strikingly
constant throughout. By comparison, the extracellular and amoeba-associated concentrations Thymidylate synthase of L. pneumophila rapidly rose after two days of incubation and then declined as expected up to and including day 7, due to the nutrient limitation of the culture medium. As expected, the amount of extracellular and amoeba-associated E. coli declined drastically over time during co-culture with A. castellanii. These results show that T. equigenitalis and T. asinigenitalis persist in association with A. castellanii over time. Figure 1 Taylorella equigenitalis and T. asinigenitalis persist within A. castellanii over time. Evolution of extracellular (A) and amoeba-associated (B) bacterial concentrations https://www.selleckchem.com/products/lazertinib-yh25448-gns-1480.html following co-cultures with A. castellanii of T. equigenitalis, T. asinigenitalis, E. coli or L. pneumophila. Amoebae were infected at an MOI of 50 and at indicated time, extracellular and amoeba-associated bacteria following lysis were quantified by plating. The results are expressed in CFU/ml and each bar represents the geometric mean of triplicate wells. The standard deviations are represented by error bars.