Hemolytic uremic syndrome is a leading cause of acute renal failure in children, and is typically associated with enteric infections by shiga and shiga-like toxins. Intensive research into the pathogenic mechanisms of these toxins has resulted in them being a model system of elucidating the actions of many bacterial toxins that share a similar mode of action. To reach its target host protein, shiga toxin must be internalized and transported through a series of organelles within the host cell, and ultimately be translocated into the host cytosolic space. Although perturbing this intracellular itinerary abrogates toxin action, the precise pathways that are required for toxin action remains ill-defined. In this regard, we have recently elucidated mechanisms of transport regulation at two critical junctures of intracellular transport within mammalian cells. The small GTPase maned ADP- Ribosylation Factor 6 (ARF6) regulates transport between the plasma membrane and the early endosome, while the intracellular KDEL receptor regulates retrograde transport from the Golgi complex to the endoplasmic reticulum (ER). Thus, we will test whether the action of shiga toxin requires endocytosis through the early endosome by determining whether overexpression of a mutant ARF6 that blocks endocytosis abrogates toxin action. We will also test whether toxin action requires transport to the ER by determining whether overexpression of a mutant KDEL receptor that blocks transport from the Golgi complex to the ER abrogates toxin action. These results will provide more precise information regarding the transport itinerary of shiga within the host cell. As many bacterial toxins share a similar intracellular itinerary, our findings will likely be applicable to understanding how a variety of bacterial toxins interface with their host to cause disease. Moreover, as different intracellular compartments possess characteristics that may be unique, knowledge derived from our proposed studies may be applied to a rational design of therapeutic intervention against shiga toxin.
