B. Trophozoite (left) and cyst (right) RG7112 concentrations related to LLO production: while columns – L. innocua NCTC11288 strain; black columns – LLO-expressing L. innocua NCTC11288 (pHly/PrfA*) strain. Data represent mean ± SE of two experiments made in triplicate. * p < 0,05; **p < 0,005. Introduction of the LLO-expressing plasmid produced a dramatic effect on the outcome of interactions

between L. innocua and T. pyriformis. In 48 h in co-culture, trophozoite concentration diminished by a factor of four in the presence of recombinant L. innocua in comparison with a control, which was T. pyriformis co-cultivated with the parental L. innocua NCTC 2188 strain. Moreover, trophozoites totally disappeared in co-culture with LLO-expressing L. innocua after 72 h (Figure 5B). LLO-expressing L. innocua accelerated T. pyriformis encystment as it was previously observed with L. monocytogenes. At 48 h cyst concentration was about 7 fold higher in the presence of LLO-expressing L. innocua compared to the wild type strain.

Interestingly, the cyst concentration diminished by a factor 5.6 between 48 h and 72 h, the effect was not observed in the presence of wild type L. monocytogenes. Obtained results supported a suggestion about a leading role of LLO in L. monocytogenes toxicity for protozoa. LLO supports L. monocytogenes survival in the presence of T. pyriformis The next issue addressed was the L. monocytogenes survival in the presence of bacteriovorous T. pyriformis and its dependence on LLO production. Bacterial growth was measured in the sterile LB broth and in the presence of T. pyriformis. Similar growth rates were observed for the wild Vistusertib in vitro type L. monocytogenes EGDe strain grown both alone or in association with T. pyriformis until end of week 1 (Figure 6). Later, bacterial population was stabilized in the association with T. pyriformis and higher bacterial concentrations were observed in the co-culture with T. pyriformis as compared with the control culture where L. monocytogenes grew alone.

By the end of week 2 in the association with protozoa bacterial cell numbers exceeded the concentration of control bacteria by a factor Methane monooxygenase of ten. Figure 6 Bacterial growth in dependence on the presence of T. pyriformis and LLO production. White and solid symbols show L. monocytogenes grown alone and in the presence of T. pyriformis, respectively; triangles and squares are correspondent to the EGDe and EGDeΔhly strains, respectively. Bacterial concentrations were determined by plating of corresponding dilutions. A representative experiment from two replicates with similar results is shown. Deletion of the hly gene did not affect bacterial growth rates in the sterile LB broth. In contrast, T. pyriformis impaired the EGDe Δhly growth especially during the first 5 days (Figure 6). By day 14, EGDeΔhly concentration was higher in co-culture with protozoa than in the sterile LB broth. In whole, LLO deficiency deteriorated L.