In this study, we discovered a unique mechanism of intracellular toxin neutralization by an antibody to the A subunit of Stx2. Although both Stx2 A-subunit- and B-subunit-specific HuMAbs 5C12 and 5H8, respectively, neutralized Stx2, the neutralizing mechanisms were distinct. The HuMAb 5H8 inhibited cytotoxicity by preventing binding of the unbound toxin to the cells but was ineffective against the cell bound toxin. In contrast, the A-subunit-specific 5C12 intercepted the cell-bound toxin and prevented toxin-mediated cell death.
Since Stx2 must be endocytosed and translocated to the cytosol by retrograde transport to exert its cytotoxic effect, a block in any step in this process of toxin transport by 5C12 may lead to inhibition of cytotoxicity. Before investigating this possibility, we determined the route and time course of Stx2 transport through the cellular organelles of HeLa cells because the sequence of retrograde transport has been studied for Stx (8
) and Stx1 (29
) but not for Stx2. Our results indicate that in HeLa cells Stx2 follows the same direct route of retrograde transport as that followed by Stx B subunit in these cells (8
). Stx2 starts accumulating in the EE soon after incubation at 37°C. It accumulates in the Golgi compartment after 1 h of incubation and in the ER after 4 h of incubation at 37°C; the results are in agreement with the time required for Stx B subunit to accumulate in these organelles in HeLa cells (8
). Stx B subunits have been shown to bypass the late endocytic pathway on their way to the Golgi apparatus in HeLa cells (8
). We also could not locate Stx2 in LE/lysosomes in HeLa cells (results not shown).
5C12 does not interfere with the endocytosis of Stx2, since Stx2/5C12 complexes were detected in the EE. However, 5C12 blocks further passage of the toxin into other cellular organelles, since even after incubation for 5 h at 37°C, the toxin was mainly located in the EE. The same results were obtained after 8 h of incubation at 37°C (results not shown). We were not able to locate Stx2-AF/5C12 complex in the LE and/or lysosomes since it was not found in LAMP3-positive structures (results not shown). It was clear from these results that 5C12 interfered with retrograde transport of the toxin. Further studies were performed to determine the fate of the internalized Stx2/5C12 complex.
Internalized membrane molecules are either targeted for degradation or recycled back to the plasma membrane (18
). In contrast, endocytosed nonmembrane molecules are usually targeted for degradation, but some molecules evade that route completely and enter the RE in order to be released outside the cell (18
). These studies suggested to us that the Stx2/5C12 complex may be in the RE. The two types of RE compartments identified thus far include those that recycle molecules rapidly to the plasma membrane and those that recycle molecules slowly (the PNRC) to the plasma membrane (18
). It seems that the 5C12-treated Stx2 localized in both the compartments alongside the plasma membrane (presumably rapid RE) and the PNRC (Fig. and Video S2 in the supplemental material). This suggests that the endocytosed complex follows two routes of repeated recycling; one involves rapid transport to and then endocytosis from the plasma membrane, and another involves slow transport from the PNRC to and then endocytosis from the plasma membrane. Videos S1 and S2 in the supplemental material also suggest that toxin/5C12 complex accumulates in endocytic recycling compartments. In some cells, the complex may preferentially recycle either through the PNRC or through the rapid RE, whereas in other cells the recycling seems to be distributed equally among the two compartments.
During fusion of RE with the cell membrane, the Stx2/5C12 complex may come off because the toxin detaches from Gb3. The toxin could then reattach to any Gb3 receptor, leading to the endocytosis of the complex again. We investigated this hypothesis by incubating the cells that had endocytosed Stx2-AF/5C12 complex with B subunit-specific 5H8. Since 5H8 blocks the binding of Stx2 with the cells and can bind to Stx2/5C12 complex (enzyme-linked immunosorbent assay results [not shown]), we anticipated 5H8 to block the binding of detached Stx2-AF/5C12 complex with the Gb3. The presence of 5H8 caused a marked reduction in overall fluorescence (Fig. and ), which clearly suggests that Stx2/5C12 complex is transported to the cell membrane, where a new cycle of its internalization is blocked by 5H8. The results also showed that the fluorescence, although present inside the cell (presumably in the PNRC), almost completely disappeared from alongside the plasma membrane, which suggests that rapidly recycled complex had more chances for neutralization by 5H8 than the slowly recycled complex from the PNRC because they were trapped there for a longer time.
Although the mechanism by which 5C12 makes Stx2 quit the retrograde transport and follow the recycling transport needs to be investigated, it is tempting to speculate and also exclude some possibilities. Major histocompatibility complex class I-like Fcγ receptor (FcRn) is known to bind serum IgG and recycle it back to the plasma membrane, rescuing it from lysosomal degradation (15
). Since HeLa cells do not express FcRn (34
), 5C12/Stx2 recycling cannot be FcRn mediated. It is well understood that TfR accumulates in recycling compartments after endocytosis and recycles back to the plasma membrane (18
). However, the intracellular transport of Gb3
has not been studied. It is possible that Gb3
, like TfR and some other host cell surface molecules, may be destined to be recycled back to the cell surface. Since Stx2 is known to bind to Gb3
at low affinity (32
), it may come off at low endosomal pH to follow the retrograde transport. However, binding of 5C12 may confer conformational changes in the Stx2 molecule, leading to slightly stronger binding of the Stx2 with the Gb3
. We have observed that the binding of Stx2 with its receptor in the presence of 5C12 is somewhat stronger than in its absence (Fig. ).
The present study is the first to report intracellular neutralization of a toxin by an antibody. Also, the mechanism of antibody neutralization described here has not been reported earlier, although several studies have shown that antibodies can neutralize pathogens intracellularly (3
). We speculate that 5C12 will also protect Stx2-bound cells in vivo. This would make 5C12 effective for patients where a toxin dose sufficient to initiate kidney damage has been absorbed systemically from the gut. Our recent study in which 5C12 protected piglets against Stx2-mediated lethal neurological complications wherein 5C12 was administered 48 h after oral infection with STEC supports this hypothesis (30
). Furthermore, 5C12 protects 20 to 40% mice when administered after 1 to 2 h of intravenous challenge with a lethal dose of Stx2 (unpublished results). We believe that the 5C12/Stx2 complex will ultimately be removed by the reticuloendothelial system in vivo and will not keep recycling in the cells. Studies are currently being performed to investigate the in vivo sites of Stx2 neutralization by 5C12 and the removal of 5C12/Stx2 complexes from the body.