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Identification of novel agents for bladder cancer treatment is highly desirable due to the high incidence of tumor recurrence and the risk of progression to muscle-invasive disease. The key feature of the cholesterol-dependent toxin listeriolysin O mutant (LLO Y406A) is its preferential activity at pH 5.7, which could be exploited either directly for selective targeting of cancer cells or the release of accumulated therapeutics from acidic endosomes. Therefore, our goal was to compare the cytotoxic effect of LLO Y406A on cancer cells (RT4) and normal urothelial cells (NPU), and to identify which cell membranes are the primary target of LLO Y406A by viability assays, life-cell imaging, fluorescence, and electron microscopy. LLO Y406A decreased viability, altered cell morphology, provoked membrane blebbing, and induced apoptosis in RT4 cells, while it did not affect NPU cells. LLO Y406A did not cause endosomal escape in RT4 cells, while the plasma membrane of RT4 cells was revealed as the primary target of LLO Y406A. It has been concluded that LLO Y406A has the ability to selectively eliminate cancer urothelial cells through pore-forming activity at the plasma membrane, without cytotoxic effects on normal urothelial cells. This promising selective activity merits further testing as an anti-cancer agent.

Aerolysin-like pore-forming proteins are an important family of proteins able to efficiently damage membranes of target cells by forming transmembrane pores. They are characterized by a unique domain organization and mechanism of action that involves extensive conformational rearrangements. Although structures of soluble forms of many different members of this family are well understood, the structures of pores and their mechanism of assembly have been described only recently. The pores are characterized by well-defined β-barrels, which are devoid of any vestibular regions commonly found in other protein pores. Many members of this family are bacterial toxins; therefore, structural details of their transmembrane pores, as well as the mechanism of pore formation, are an important base for future drug design. Stability of pores and other properties, such as specificity for some cell surface molecules, make this family of proteins a useful set of molecular tools for molecular recognition and sensing in cell biology.This article is part of the themed issue 'Membrane pores: from structure and assembly, to medicine and technology'., (© 2017 The Author(s).)