Discussion Cytotoxicity of haemolytic Listeria spp. in ciliates and amoebae was originally demonstrated by Chau Ly and Müller [7]. They have shown that haemolytic L. monocytogenes and L. seeligeri induce lysis of T. pyriformis and Acanthamoeba castellani during 8-15 days while only few protozoa underwent lysis in the presence of non-haemolytic L. innocua. Our results demonstrated that a L. monocytogenes mutant strain deficient in L. monocytogenes haemolysin, listeriolysin O (LLO) was incapable of impairing T. pyriformis growth compared to the isogenic wild type strain. A saprophytic species of L. innocua expressing LLO acquired toxicity in protozoa and caused their mortality and encystment. Thus, obtained results
suggested that it is LLO that is responsible for L. monocytogenes cytotoxicity in protozoa. Another observed NVP-HSP990 LLO activity was stimulation of T. pyriformis encystment. Both cell death and encystment were responsible for decrease of trophozoite counts in the presence of L. monocytogenes. Here our results were in contradiction with previously published [7]. Although cited above authors found that L. monocytogenes accelerates encystment of A. castellani, they did not observe T. pyriformis encystment independently
on bacterial presence [7]. This contradiction is related to the protozoan ability to encyst rather than LLO activity and might be due to different AZD9291 mouse sources of a protozoan culture. Cyst formation by ciliates was described earlier [21] and cysts that we observed for the used T. pyriformis culture were similar to cysts depicted there (see Figure 1). In contrast to wild type L. monocytogenes, LLO-expressing L.
innocua caused a rapid decrease in counts not only trophozoites but as well cysts (see Figure 5). The constitutive LLO expression driven by PrfA* protein, which gene was inserted into the pHly/PrfA* plasmid, might be responsible for higher toxicity of L. innocua transformed with the plasmid. Wild Ureohydrolase type PrfA protein activity is regulated by co-factor binding, while the PrfA* protein is locked in the active conformation by a Gly145Ser substitution [19]. Obtained results suggested that PrfA activity and LLO expression by intracellular L. monocytogenes might be switch off after host cell encystment but this is not possible for PrfA* protein. Further studies with using L. monocytogenes prfA* [19] are needed to get evidences in support of this suggestion. Another pathogenic bacterium, a AR-13324 purchase common representative of natural ecosystems, L. pneumophila was demonstrated to be cytotoxic for amoeba and to kill A. polyphaga via induction of necrosis due to Legionella pneumophila pore-forming activity [25]. A similar mechanism might be responsible for the cytotoxic effect of LLO. LLO belongs to the family of cholesterol-dependent haemolysins, which includes streptolysin O and pneumolysin O [13, 14]. Proteins of this family can form oligomeric rings that plunge into membrane and generate pores [26].
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