Nuclear trafficking of bacterial effector proteins.

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 epigenetic-modulating 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.

Campylobacter jejuni extracellular vesicles harboring cytolethal distending toxin bind host cell glycans and induce cell cycle arrest in host cells.

Cytolethal distending toxins (CDTs) are released by Gram-negative pathogens into the extracellular medium as free toxin or associated with extracellular vesicles (EVs), commonly known as outer membrane vesicles (OMVs). CDT production by the gastrointestinal pathogen Campylobacter jejuni has been implicated in colorectal tumorigenesis. Despite CDT being a major virulence factor for C. jejuni, little is known about the EV-associated form of this toxin. To address this point, C. jejuni mutants lacking each of the three CDT subunits (A, B, and C) were generated. C. jejuni cdtA, cdtB, and cdtC bacteria released EVs in similar numbers and sizes to wild-type bacteria, ranging from 5 to 530 nm (mean ± SEM = 118 ±6.9 nm). As the CdtAC subunits mediate toxin binding to host cells, we performed "surface shearing" experiments, in which EVs were treated with proteinase K and incubated with host cells. These experiments indicated that CDT subunits are internal to EVs and that surface proteins are probably not involved in EV-host cell interactions. Furthermore, glycan array studies demonstrated that EVs bind complex host cell glycans and share receptor binding specificities with C. jejuni bacteria for fucosyl GM1 ganglioside, P1 blood group antigen, sialyl, and sulfated Lewisx. Finally, we show that EVs from C. jejuni WT but not mutant bacteria induce cell cycle arrest in epithelial cells. In conclusion, we propose that EVs are an important mechanism for CDT release by C. jejuni and are likely to play a significant role in toxin delivery to host cells. IMPORTANCE: Campylobacter jejuni is the leading cause of foodborne gastroenteritis in humans worldwide and a significant cause of childhood mortality due to diarrheal disease in developing countries. A major factor by which C. jejuni causes disease is a toxin, called cytolethal distending toxin (CDT). The biology of this toxin, however, is poorly understood. In this study, we report that C. jejuni CDT is protected within membrane blebs, known as extracellular vesicles (EVs), released by the bacterium. We showed that proteins on the surfaces of EVs are not required for EV uptake by host cells. Furthermore, we identified several sugar receptors that may be required for EV binding to host cells. By studying the EV-associated form of C. jejuni CDT, we will gain a greater understanding of how C. jejuni intoxicates host cells and how EV-associated CDT may be used in various therapeutic applications, including as anti-tumor therapies.

A new isolation method for bacterial extracellular vesicles providing greater purity and improved proteomic detection of vesicle proteins.

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.