Shiga Toxin

Shiga toxin (STx) is encoded and expressed by the bacterium Shigella dysenteriae and largely contributes to the pathogenesis of shigellosis, otherwise known as bacillary dysentery [1]. Among the three species of Shigella that are capable of causing the condition, S. dysenteriae is regarded as the most worrisome owing particularly to its expression of Shiga toxin. The disease shigellosis is characterized by severe bloody, mucosal diarrhea and is endemic to developing nations with overcrowding and little or no sanitation. Correspondingly, the amplified bacteria are transmitted through fecal contamination of water or food, although only a remarkably small cell number is required for infection. Recent estimates indicate that Shigellosis is responsible for over 100,000 deaths each year, predominately among children in the developing world [2].
Irrespective of Shiga toxin, Shigella bacteria invade an infected host’s colonic epithelial cells by means of surreptitious M-cell transcytosis, followed by macrophage lytic activity. After successful entry into the cells, the bacteria proliferate and spread to adjacent cells, often killing them and inducing inflammatory responses [1]. The effect of such activity assists in the further invasion and ulceration of the intestinal mucosa; instances of low-volume diarrhea associated with dysentery are an attribute of the bacteria-induced inflammatory response. In spite of this toxin-independent aspect of shigellosis pathogenesis, the Shiga toxin can ultimately be held accountable for much of the devastating effects of Shigella dysenteriae infection [3].
The Shiga toxin is composed of an A subunit connected to a pentameric B moiety. The B subunit acts as a binding domain by preferentially binding the cell-surface ganglioside globotriaosylceramide (Gb3) [4]. Gb3 is most prevalent in a select few epithelial and endothelial cell types, which will become important later on in the discussion of STx use in research. Once bound to the Gb3 receptor, the toxin-receptor complex enters the cell via either clathrin-dependent or calveolae-dependent (lipid raft) endocytosis, the precise mechanisms of which are still not entirely known. The early endosome-encased toxin is then described as being shuttled
directly to the trans Golgi network (TGN), circumventing the late endosome pathway that leads to degradation in a lysosome. During its transport in the early endosome, the A subunit of STx undergoes proteolytic cleavage by furan, a crucial step in activating toxicity. In this manner the A subunit is split into two components (A1 and A2) held together by an internal disulfide bond. The endosome is transported in retrograde fashion beginning with the TGN through the Golgi apparatus and subsequently on to the ER. In the ER, the A1 component of the STx A subunit is cleaved and translocated into the cytosol by what is considered to be STx interactions with
and manipulations of the sec61 membrane protein translocator in the ER. As strongly evidenced, the movement of Shiga toxin through a cell is quite valuable for the study of retrograde trafficking of intracellular materials from the endosome to the Golgi, ER, and cytosol [5],[6].
Essentially tantamount to the toxic mechanism of ricin, the enzymatically active A1 subunit of Shiga is an N-glycosidase that acts by removing adenine 4324 from the 28s rRNA. This blocks binding of elongation factor 1-dependent aminoacyl-tRNA to the 60s ribosomal subunit, leading to inhibition of protein synthesis and ultimately cell death. In relation to shigellosis, Shiga toxin can rapidly induce cell death in the intestinal epithelium, which is peppered with Gb3 [5]. STx therefore participates in the apoptotic death of intestinal cells,
while also possessing the ability to affect the vascular endothelium of capillaries in the gut leading to hemorrhaging [6]. STx has furthermore been discovered to act systemically in a variety of ways that makes Shigella dysenteriae infection even more complicated and dangerous than other non-Shiga expressing Shigellae [7].
Studies have shown that red blood cells, platelets, neutrophils, and monocytes all carry Gb3 receptors for the Shiga toxin, however, they are much less sensitive to its toxic nature. These cell types are proposed to bind the toxin and transport it to other regions of the body, in particular the renal glomerular endothelium and tubular epithelium, where Gb3 can be found in abundance. The irreversible ribosome-inhibiting action of STx can then work its course, inducing apoptosis and inflammation of the kidney and its microvascular network. As
a result, Shiga toxin can cause hemolytic-uremic syndrome (HUS), most notably in children who have higher Gb3 receptors in these tissues [7]. HUS is often characterized by acute renal failure, to which the severity of Shigella dysenterae infection is attributed [2].
Shiga and analogous protein toxins such as the plant-produced ricin toxin have been studied vigorously in attempts to understand retrograde vesicular transport. In fact, STx is apparently the first molecule noted to be transported from the cell surface through the Golgi to the ER. Not only have toxins played a crucial role in elucidating many gray areas of cell biology, but they have also been used in medical applications such as engineering of immunotoxins to help combat various cancers and HIV [8]. In the case of Shiga toxin, recent efforts are being made to manipulate the B subunit alone as a means of targeting tumors for imaging purposes. By removing the toxic component of STx, researchers have been exploring the potential uses of the B subunit. In one study by Janssen et al [9], the markedly high proportion of Gb3 expression in tumors of the digestive tract prompted the researchers to consider the STx B subunit (STxB) as a valuable tool for not only imaging/detection purposes, but chemotherapeutic agents as well. STx is especially viable for these purposes on account of its propensity to move along a retrograde path and thus avoid recycling or degradation in the cell.
In order to test the efficacy of STxB for tumor targeting, Janssen et al developed several transgenic mouse models to which they subjected STxB with a fluorescent probe, either orally or intravenously. An initial Gb3 analysis indicated a stark contrast between high levels of Gb3 expression in tumor cells compared to relatively low levels in normal cells. In the orally-fed mouse assay, fluorophore-labeled STxB was found to significantly accumulate in adenocarcinomas of the digestive tract, whereas the STxB marker was not strongly
persistent in most normal cells, nearly disappearing within 3 hours of those few normal cells it did happen to target. Moreover, STxB was found in tumor cells for up to 24 hours and in some cases even longer. Immunofluorescent techniques affirmed that STxB traveled in a retrograde manner through the cells, providing an explanation for its persistence. PET imaging was utilized to monitor STxB coupled with another contrast agent in its ability to target carcinoma cells, and an equivalent set of results was turned over.
Viel et al. [10] conducted a similar experiment in 2008 using two mouse models with human colorectal carcinoma xenografts. STxB was found to accumulate in these high Gb3-expressing tumors, and in monocytes and macrophages. In contrast to the previous study mentioned, Viel et al found intravenous injection much more successful than oral administration of STxB. They also discovered a heterogeneous distribution of Gb3 in tumor cells, however, this did not affect the affinity of these cells for STxB adhesion. Again, STxB was markedly localized to grafted human colorectal carcinoma cells as opposed to normal cells in the mice models. Ultimately both of these in vivo research attempts have elucidated the potential use for STx B
subunit as drug vector for various cancers, in particular colorectal cancers that are known to express the STx receptor in high degrees. Accumulation of STxB in immune cells furthermore suggests a possibility for immunotherapy drug targeting. At the very least, STxB may be developed for use as an efficient tumor imagingdevice which would be crucial for early diagnosis.
Further research into the efficacy of Shiga toxin B subunit for the purposes of early tumor detection and tumor cell-specific drug delivery may well have promising discoveries. This has been evidenced by Shiga toxin’s ability to evade intracellular degradation and recycling, along with its specificity for Gb3-overexpressing carcinomas.


References
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