Multitalented EspB of enteropathogenic Escherichia coli (EPEC) enters cells autonomously and induces programmed cell death in human monocytic THP-1 cells

• Published in: International journal of medical microbiology 2018-04, Vol.308 (3), p.387-404
• Main Authors: Baumann, Doreen, Salia, Helen, Greune, Lilo, Norkowski, Stephanie, Körner, Britta, Uckeley, Zina M., Frankel, Gad, Guenot, Marianne, Rüter, Christian, Schmidt, M. Alexander
• Format: Article
• Language: English
• Subjects: Bacterial Adhesion; Bacterial Outer Membrane Proteins - genetics; Bacterial Outer Membrane Proteins - metabolism; Bacterial Proteins - genetics; Caco-2 Cells; Cell Death - genetics; Cell-penetrating proteins; Enteropathogenic E. coli; Enteropathogenic Escherichia coli - genetics; Enteropathogenic Escherichia coli - metabolism; Enteropathogenic Escherichia coli - pathogenicity; Epithelial Cells - microbiology; Epithelial Cells - pathology; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; EspB homologes; HeLa Cells; Humans; L-Lactate Dehydrogenase - analysis; Monocytes - microbiology; Monocytes - pathology; Multitalented EspB; Programmed cell death; Propidium - metabolism; Protein Transport; Salmonella - genetics; THP-1 Cells
• ISSN: 1438-4221; 1618-0607
• DOI: 10.1016/j.ijmm.2018.03.005

Enteropathogenic Escherichia coli (EPEC) subvert host cell signaling pathways by injecting effector proteins via a Type 3 Secretion System (T3SS). The T3SS-dependent EspB protein is a multi-functional effector protein, which contributes to adherence and translocator pore formation and after injection exhibits several intracellular activities. In addition, EspB is also secreted into the environment. Effects of secreted EspB have not been reported thus far. As a surrogate for secreted EspB we employed recombinant EspB (rEspB) derived from the prototype EPEC strain E2348/69 and investigated the interactions of the purified protein with different human epithelial and immune cells including monocytic THP-1 cells, macrophages, dendritic cells, U-937, epithelial T84, Caco-2, and HeLa cells. To assess whether these proteins might exert a cytotoxic effect we monitored the release of lactate dehydrogenase (LDH) as well as propidium iodide (PI) uptake. For comparison, we also investigated several homologs of EspB such as IpaD of Shigella, and SipC, SipD, SseB, and SseD of Salmonella as purified recombinant proteins. Interestingly, cytotoxicity was only observed in THP-1 cells and macrophages, whereas epithelial cells remained unaffected. Cell fractionation and immune fluorescence experiments showed that rEspB enters cells autonomously, which suggests that EspB might qualify as a novel cell-penetrating effector protein (CPE). Using specific organelle tracers and inhibitors of signaling pathways we found that rEspB destroys the mitochondrial membrane potential – an indication of programmed cell death induction in THP-1 cells. Here we show that EspB not only constitutes an essential part of the T3SS-nanomachine and contributes to the arsenal of injected effector proteins but, furthermore, that secreted (recombinant) EspB autonomously enters host cells and selectively induces cell death in immune cells.