Making something requires connecting parts in a particular order. Baking: dry ingredients must be mixed before adding wet ingredients. Puzzles: much easier if the border is assembled first. Legos. Ikea products. Similarly, the order of biological interactions at the molecular level can determine whether a complex has the correct structure or activity for its function: translation is a simple example. If the small and large ribosomal subunits formed before interacting with an mRNA, there would be no message to scan for instructions on amino acid incorporation into proteins.
The molecular order of events is important in virulence, too, and a new paper published in the Journal of Bacteriology looks at the order of events necessary for bacterial toxin delivery to a host cell. Specifically, the authors looked at the assembly of Shiga toxin, produced by Shigella dysenteriae and shigatoxigenic E. coli (STEC), including enterohemorrhagic E. coli and the infamous O157:H7 E. coli serogroup. Shiga toxin is an AB5 toxin that uses a pentamer of B subunits to mediate translocation of the A subunit into host cells, where the A subunit can have its toxic effects (see schematic, left).
Since there are six subunits involved total, the 5-part B subunit pentamer and the single A subunit, first author Christine Pellino, working with senior author Alison Weiss, wanted to investigate how assembly might influence toxin internalization. While some AB toxins, such as anthrax toxin, can assemble at the cell receptor, some AB5 toxins, like cholera toxin, are secreted from the bacterial cell fully assembled. Unlike other AB5 toxin-producing bacteria, Shiga toxin is released when the bacterial cells lyse, which suggests its assembly might differ as well.
The authors wanted to test whether Shiga toxin assembly occurred before or after the B unit bound its receptor, globotriaosylceramide (Gb3). The researchers first determined that only 1% of the total released toxin was preassembled at the expected 72 kDa. Monomers of A and B subunits were incubated and then spun through a size-exclusion filter. Here, only unassembled toxin could flow through; because of its large size, assembled toxin was retained above the filter (see schematic, right). About half the toxin filtered through, unassembled. This result differed from incubated cholera toxin monomers, which fully assembled and had no toxin components filter through as monomers.
To test whether assembly affects toxicity, the researchers then tested the assembled and nonassembled toxins for activity in mice. All mice injected with Shiga toxin lost weight, but those injected with unassembled toxin lost more weight than those with assembled toxin. All 12 of the unassembled toxin-treated mice fully succumbed to toxin activity, while only half of the 12 assembled toxin-treated mice died. From this, the authors concluded that Shiga toxin preassembly is not required for toxicity, differentiating Shiga toxin from AB5 toxins like cholera toxin. They further showed that toxin subunits, injected separately into different locations in a mouse, could assemble in vivo to cause toxicity.
Why does the order of assembly matter? Bacteria that produce Shiga toxins (and Shiga-like toxins) cause severe disease, including bloody diarrhea and hemolytic uremic syndrome, a type of kidney failure that is especially dangerous in young children. Around 265,000 STEC infections occur annually in the U.S., and serious complications occur in about 10% of these. Antibiotics use to treat infection is controversial, because the drugs can limit bacterial growth but can also increase toxin production. Interrupting toxin assembly can help alleviate disease symptoms for a disease that currently consists of supportive therapy.
The scientists tested this last idea by adding only a partial A subunit peptide, the A2 peptide. Interaction of this peptide with the B subunits allows toxin assembly and internalization but eliminates the toxic enzymatic activity of the A subunit. When injected into mice, the A2 peptide protected mice from lethal toxin injections, demonstrating an immediate potential clinical application for these exciting basic research findings.
-- Julie Wolf