K. Sandvig

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.

Delivery into cells: lessons learned from plant and bacterial toxins

A number of protein toxins of bacterial and plant origin have cytosolic targets, and knowledge about these toxins have provided us with essential information about mechanisms that can be used to gain access to the cytosol as well as detailed knowledge about endocytosis and intracellular sorting. Such toxins include those that have two moieties, one (the B-moiety) that binds to cell surface receptors and another (the A-moiety) with enzymatic activity that enters the cytosol, as well as molecules that only have the enzymatically active moiety and therefore are inefficient in cell entry. The toxins discussed in the present article include bacterial toxins such as Shiga toxin and diphtheria toxin, as well as plant toxins such as ricin and ribosome-inactivating proteins without a binding moiety, such as gelonin. Toxins with a binding moiety can be used as vectors to translocate epitopes, intact proteins, and even nucleotides into the cytosol. The toxins fall into two main groups when it comes to cytosolic entry. Some toxins enter from endosomes in response to low endosomal pH, whereas others, including Shiga toxin and ricin, are transported all the way to the Golgi apparatus and the ER before they are translocated to the cytosol. Plant proteins such as gelonin that are without a binding moiety are taken up only by fluid-phase endocytosis, and normally they have a low toxicity. However, they can be used to test for disruption of endosomal membranes leading to cytosolic access of internalized molecules. Similarly to toxins with a binding moiety they are highly toxic when reaching the cytosol, thereby providing the investigator with an efficient tool to study endosomal disruption and induced transport to the cytosol. In conclusion, the protein toxins are useful tools to study transport and cytosolic translocation, and they can be used as vectors for transport to the interior of the cell.

Clathrin-coated pits with long, dynamin-wrapped necks upon expression of a clathrin antisense RNA

To investigate the role of clathrin in coated vesicle formation, a cell line with inducible expression of clathrin heavy chain (CHC) antisense RNA was produced. After 18 h of CHC antisense RNA expression, the internalization of transferrin was inhibited by 90%. Although the amount of CHC was reduced by only 10%, the frequency of clathrin-coated pits at the cell surface increased by a factor of 3–5, and clathrin-coated structures also accumulated on a pleiomorphic, multivesicular, endosomal compartment. Remarkably, the coated pits were connected to the cell surface by long, tubular necks wrapped by dynamin rings, and the level of dynamin in the CHC antisense RNA-expressing cells was up-regulated 10-fold. In contrast, the amount of several other proteins associated with clathrin coat formation was unaffected. Thus, this study demonstrates that CHC antisense RNA causes accumulation of clathrin-coated pits with dynamin rings around the neck in intact cells not transfected with dynamin mutants, suggesting the existence of a previously uncharacterized functional interplay between clathrin and dynamin.

Receptor-mediated endocytosis of a ricin-colloidal gold conjugate in vero cells. Intracellular routing to vaculoar and tubulo-vesicular portions of the endosomal system

We have prepared a conjugate (Ri-Au) of the toxic plant protein ricin and colloidal gold (particle size 5 nm) and used it for internalization studies in monolayer cultures of Vero cells. The Ri-Au conjugate was very stable, with only little release of ricin ([125I]Ri) from the gold particles within a pH range of 4.5-8.0. Within 2 h at 37 degrees C, only very little intracellular degradation of the ricin preparation ([125I]Ri-Au) occurred. The cells bound the same proportion of native ricin ([125I]Ri) and Ri-Au from the medium, and the kinetics of toxicity (decrease in cellular incorporation of [3H]leucine) of [125I]Ri and [125I]Ri-Au were also comparable. At 4 degrees C, the cell-surface binding of Ri-Au was continuous and distinct, as revealed by electron microscopy. This binding was specific, since almost no Ri-Au surface binding occurred at 4 degrees C in the presence of 0.1 M lactose or 1 mg/ml native (unlabelled) ricin. Within the first 30 min of warming prelabelled cells to 37 degrees C, the amount of surface-associated Ri-Au decreased considerably (from 150 to 60 gold particles per micron cell surface in 40 nm sections). Coated pits and vesicles were involved in the internalization of Ri-Au, and within 5-30 min at 37 degrees C Ri-Au had been delivered to vacuolar and tubulo-vesicular portions of the endosomal system, and later also to lysosomes. Analysis of very thin (ca 20 nm) serial sections revealed that most of the tubulo-vesicular elements were separate structures not connected to the membrane of the vacuolar portion. Data here presented indicate that our ricin conjugate, like many physiological ligands and viruses, is internalized by receptor-mediated endocytosis via the coated pit-endosomal pathway.

Delivery of internalized ricin from endosomes to cisternal Golgi elements is a discontinuous, temperature-sensitive process.

Galactose-terminating membrane glycoproteins and glycolipids on two established human breast carcinoma cell lines were tagged at 4 degrees C with a ricin-horseradish peroxidase conjugate (Ri-HRP). The cells were then incubated for various periods of time at 37 or 18 degrees C. After fixation and diaminobenzidine cytochemistry, the compartments reached by Ri-HRP were studied by analyzing thin serial sections. In both cell types a highly pleomorphic endosomal system comprising vacuolar elements as well as smaller, sometimes branched, tubular elements (tubular endosomes) was revealed at both 37 and 18 degrees C. At 37 degrees C Ri-HRP was consistently observed in flattened cisterns of the Golgi region in 30-40% of the Golgi complexes examined after 30-60 min of incubation. However, no Ri-HRP reached such Golgi elements at 18 degrees C, even after incubation for 180 min. Moreover, at 18 degrees C the ability of ricin to inhibit protein synthesis was virtually abolished, whereas the effect of diphtheria toxin was reduced much less. Following incubation with a monovalent transferrin-HRP conjugate or with unconjugated HRP, no labeling of cisternal Golgi elements was detected. These data indicate that delivery of galactose-terminating membrane molecules from endosomes to the Golgi complex is a discontinuous, temperature-sensitive process and that this process may be required for optimal ricin A-chain translocation.

Ricin transport into cells: Studies of endocytosis and intracellular transport

The plant toxin ricin binds to both glycoproteins and glycolipids with terminal galactose, and the toxin will therefore be endocytosed by the different mechanisms operating in a given cell. After endocytosis the toxin is transported to the Golgi apparatus by a process that differs from the Rab9-dependent transport of mannose-6-phosphate receptors. Retrograde toxin transport from the Golgi apparatus to the endoplasmic reticulum (ER) seems to be a requirement for subsequent toxin translocation to the cytosol where the toxin inhibits protein synthesis enzymatically. By using ricin we have characterized different types of endocytosis and the transport steps used by this toxin.

Intracellular Transport and Processing of Protein Toxins Produced by Enteric Bacteria

Bacterial toxins are associated with disease in humans and animals. Toxins can either be preformed in food or produced by bacteria in the intestine. There are two types of toxins: heat-labile protein toxins and heat stabile toxins. Heat labile toxins are produced by Bacillus cereus, Clostridium perfringens, Escherichia coli, and Vibrio cholerae, and heat-stabile enterotoxins consisting of relatively few amino acids are produced by Escherichia coli and acts by activation of guanylate cyclase. Similarly, heat-stabile entero-toxins are also produced by Staphylococcus aureus, a common cause of food poisoning in the United States, and Yersenia enterocolitica. Protein toxins produced by enteric bacteria can intoxicate intestinal cells and can also be taken up from the gut and reach other cells in the body. For example the Shiga-like toxins (vero-toxins) can intoxicate endothelial cells in the kidney and cause kidney failure. Intracellular transport and processing of a few of the protein toxins produced by enteric bacteria, namely Clostridium difficile toxin A and B, cholera toxin and the related heat-labile toxin produced by Escherichia coli, and Shiga toxin and Shiga-like toxins are presented.

Endocytic Uptake of Ricin and Shiga Toxin

Protein toxins which efficiently kill eukaryotic cells are found in plants and produced by bacteria. Examples of such toxins are the plant toxins ricin, abrin, modeccin, viscumin and volkensin, and the bacterial toxins diphtheria toxin and Shiga toxin (for review, see Olsnes and Sandvig 1988). Schematic structures of toxins are shown in Fig. 1. All these toxins kill cells in the following manner: They bind to cell surface receptors by their B-chains, they are internalized by endocytosis, and then an enzymatically active part of the molecule, the A-chain, enters the cytosol where it inhibits protein synthesis, either by inactivation of the 60 S subunit of the ribosome or by inactivation of elongation factor 2. In spite of their structural similarities, these protein toxins enter the cytosol from different intracellular compartments, and they have different requirements for entry.

Protein Uptake and Cytoplasmic Access in Animal Cells

Cell membranes are the major barriers to protein delivery into cells. As a great deal of pharmaceutical and biotechnological research attempts to find ways of delivering drugs into cells—for instance, with the purpose of irreversibly inhibiting the protein synthesis machinery of cancer cells—mechanisms by which various protein ligands are internalized by cells and subsequently translocated across the membrane of intracellular compartments are coming into focus.

Shigella Toxin and Related Proteins — Translocation to the Cytosol and Mechanism of Action

Shigella species and certain E. coli strains produce toxins that are exceedingly toxic to many mammalian cells (van Heyningen and Gladstone, 1953; Olsnes and Eiklid, 1980; Karmali et al., 1985; O’Brian and Holmes, 1987). Thus, as little as 0.1 pg/ml Shigella toxin is enough to kill a culture of sensitive HeLa cells. Shigella toxin and the related Shiga-like toxins have been cloned and sequenced (Calderwood et al., 1987; Kozlov et al., 1987; Strockbine et al., 1988). The toxins act by inactivating the ribosomes and thereby block protein synthesis (Reisbig, Olsnes and Eiklid, 1981). A necessary step in their mechanism of action is to translocate to the cytosol an enzymatically active polypeptide chain.