Protein toxins from plants and bacteria: probes for intracellular transport and tools in medicine.

A number of protein toxins produced by bacteria and plants enter eukaryotic cells and inhibit protein synthesis enzymatically. These toxins include the plant toxin ricin and the bacterial toxin Shiga toxin, which we will focus on in this article. Although a threat to human health, toxins are valuable tools to discover and characterize cellular processes such as endocytosis and intracellular transport. Bacterial infections associated with toxin production are a problem worldwide. Increased knowledge about toxins is important to prevent and treat these diseases in an optimal way. Interestingly, toxins can be used for diagnosis and treatment of cancer.

ß-arrestins attenuate p38-mediated endosome to Golgi transport.

Shiga toxin (Stx) is after endocytosis transported via early endosomes to the Golgi apparatus and endoplasmic reticulum. It is then translocated to the cytosol where it exerts its toxic effect. We recently reported that p38 is required for endosome to Golgi transport of Stx. In the present study, we investigated whether ß-arrestins are effectors of this pathway. ß-arrestin knockdown led to enhanced Stx transport. A similar phenotype was achieved upon p38 activation. We demonstrate that p38 and ß-arrestin act on the same pathway. ß-arrestin colocalized with internalized Stx and, interestingly, was recruited to endosomes upon p38 activation. After Stx treatment, p38 and ß-arrestin formed a transient complex. From these data we propose that ß-arrestin negatively regulates Stx transport via an interaction with activated p38 and attenuation of its signalling. Interestingly, also mannose 6-phosphate receptor transport was regulated by p38 and ß-arrestin. ß-arrestins therefore seem to regulate an endosome to Golgi pathway used by multiple cargo proteins.

Nanowire-based delivery of Escherichia coli O157 shiga toxin 1 A subunit into human and bovine cells.

Silica nanowires (NWs) were used to introduce the Shiga toxin type 1 A subunit (StxA1) into cultured bovine and human epithelial cells. We extended technology developed in our laboratories that employs fibronectin (Fn) to induce integrin-mediated uptake of NWs by coating NWs with StxA1 and Fn. The bonding strengths of Fn and StxA1 to the surface of NWs were measured by X-ray photoelectron spectroscopy. This technique demonstrated complex interactions between Fn, StxA1, and the NWs. Neutral red cytotoxicity assays and field emission scanning electron microscopy confirmed that the NW-StxA1-Fn complexes were effectively internalized and caused cell death. This indicates that NWs can carry StxA1 and potentially other toxic or therapeutic agents into eukaryotic cells. Ongoing studies include improved functionalizing of NWs aimed at increasing internalization efficiency and substituting ligands for specific cell targeting.

Identification and characterization of small molecules that inhibit intracellular toxin transport.

Shiga toxin (Stx), cholera toxin (Ctx), and the plant toxin ricin are among several toxins that reach their intracellular destinations via a complex route. Following endocytosis, these toxins travel in a retrograde direction through the endosomal system to the trans-Golgi network, Golgi apparatus, and endoplasmic reticulum (ER). There the toxins are transported across the ER membrane to the cytosol, where they carry out their toxic effects. Transport via the ER from the cell surface to the cytosol is apparently unique to pathogenic toxins, raising the possibility that various stages in the transport pathway can be therapeutically targeted. We have applied a luciferase-based high-throughput screen to a chemical library of small-molecule compounds in order to identify inhibitors of Stx. We report two novel compounds that protect against Stx and ricin inhibition of protein synthesis, and we demonstrate that these compounds reversibly inhibit bacterial transport at various stages in the endocytic pathway. One compound (compound 75) inhibited transport at an early stage of Stx and Ctx transport and also provided protection against diphtheria toxin, which enters the cytosol from early endosomes. In contrast, compound 134 inhibited transport from recycling endosomes through the Golgi apparatus and protected only against toxins that access the ER. Small-molecule compounds such as these will provide insight into the mechanism of toxin transport and lead to the identification of compounds with therapeutic potential against toxins routed through the ER.

The epithelial cell cytoskeleton and intracellular trafficking. I. Shiga toxin B-subunit system: retrograde transport, intracellular vectorization, and more.

Many intracellular transport routes are still little explored. This is particularly true for retrograde transport between the plasma membrane and the endoplasmic reticulum. Shiga toxin B subunit has become a powerful tool to study this pathway, and recent advances on the molecular mechanisms of transport in the retrograde route and on its physiological function(s) are summarized. Furthermore, it is discussed how the study of retrograde transport of Shiga toxin B subunit allows one to design new methods for the intracellular delivery of therapeutic compounds.

Entry of ricin and Shiga toxin into cells: molecular mechanisms and medical perspectives.

A large number of plant and bacterial toxins with enzymatic activity on intracellular targets are now known. These toxins enter cells by first binding to cell surface receptors, then they are endocytosed and finally they become translocated into the cytosol from an intracellular compartment. In the case of the plant toxin ricin and the bacterial toxin Shiga toxin, this happens after retrograde transport through the Golgi apparatus and to the endoplasmic reticulum. The toxins are powerful tools to reveal new pathways in intracellular transport. Furthermore, knowledge about their action on cells can be used to combat infectious diseases where such toxins are involved, and a whole new field of research takes advantage of their ability to enter the cytosol for therapeutic purposes in connection with a variety of diseases. This review deals with the mechanisms of entry of ricin and Shiga toxin, and the attempts to use such toxins in medicine are discussed.