Bacterial pathogens can subvert host responses by producing effector proteins that directly target the nucleus of eukaryotic cells in animals and plants. Nuclear-targeting proteins are categorised as either: "nucleomodulins," which have epigeneticmodulating activities; or "cyclomodulins," which specifically interfere with the host cell cycle. Bacteria can deliver these effector proteins to eukaryotic cells via a range of strategies. Despite an increasing number of reports describing the effects of bacterial effector proteins on nuclear processes in host cells, the intracellular pathways used by these proteins to traffic to the nucleus have yet to be fully elucidated. This review will describe current knowledge about how nucleomodulins and cyclomodulins enter eukaryotic cells, exploit endocytic pathways and translocate to the nucleus. We will also discuss the secretion of nuclear-targeting proteins or their release in bacterial membrane vesicles and the trafficking pathways employed by each of these forms. Besides their importance for bacterial pathogenesis, some nuclear-targeting proteins have been implicated in the development of chronic diseases and even cancer. A greater understanding of nuclear-targeting proteins and their actions will provide new insights into the pathogenesis of infectious diseases, as well as contribute to advances in the development of novel therapies against bacterial infections and possibly cancer.

Contaminants within cell culture media often co-isolate with eukaryotic extracellular vesicles (EVs) thus affecting their biological properties. It has yet to be investigated if this is also true for bacterial EVs (BEVs), especially for organisms grown in complex culture media containing animal-derived products. To address this question, we isolated BEVs from the fastidious bacterium Helicobacter pylori grown in either standard Brain Heart Infusion (BHI) medium or BHI depleted of animal-derived products (D-BHI). We show that BEVs prepared from bacteria grown in D-BHI medium have similar morphologies, size ranges and yields to those prepared from standard medium. Similarly, no differences were found in the ability of H. pylori BEVs to induce IL-8 responses in epithelial cells. However, H. pylori BEVs prepared from D-BHI medium were of higher purity than those prepared from standard medium. Importantly, proteomic analyses detected 3.4-fold more H. pylori proteins and 10-fold fewer bovine-derived proteins in BEV samples prepared from D-BHI rather than the standard method. Fifty-seven H. pylori proteins were uniquely detected in BEV samples prepared from D-BHI. In conclusion, we have described an improved method for BEV isolation. Furthermore, we demonstrate how animal-derived products in bacteriological culture media may adversely affect proteomic analyses of BEVs. K E Y WO R D S bacterial extracellular vesicles, Helicobacter pylori, outer membrane vesicles, proteomics  INTRODUCTIONCells belonging to all three domains of life release spherical membrane-bound nanostructures, known as extracellular vesicles (EVs) (Gill et al., 2019). The EVs produced by bacteria (BEVs) are 20-400 nm in size and contain a range of products, such as proteins, nucleic acids, lipopolysaccharide and peptidoglycan, which may contribute to infection and to horizontal gene transfer (Gao & van der Veen, 2020;Kaparakis-Liaskos & Ferrero, 2015) BEVs are generally isolated by either ultracentrifugation or ultrafiltration, though both techniques result in relatively crude preparations (Klimentová & Stulík, 2015). Ultracentrifugation has been reported to promote vesicle and extra-vesicle proteinThis is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.