BAHL, Christopher D. The mechanism of Stx1 incorporation in outer membrane vesicles of Escherichia coli O157:H7 and its role in bacterial predation

Gram negative bacteria secrete vesicles that are formed when a portion of the outer membrane “blebs off” [1]. The lumen of these outer membrane vesicles (OMVs) contains a small portion of the periplasm. However, it appears that the quantity of most proteins found within OMVs does not reflect their periplasmic abundances [3]. This suggests the existence of a novel mechanism for the selection, concentration, and packaging of proteins for secretion inside OMVs. In the case of pathogenic bacteria, toxins are often secreted in association with OMVs, which can aid in delivery to other cells [4]. Infection by the pathogenic Escherichia coli strain O157:H7 is an international health concern, and the production of Shiga-like toxin (Stx) by these bacteria is a major contributor to its pathogenicity [5]. Stx1 produced by E. coli O157:H7 has been shown to localize within OMVs [6]. Very little is known about how OMVs are formed [7] or how the protein cargo they carry is loaded into them. In all cases that have been investigated, protein toxins that are packaged into OMVs are enriched [8-11]. Stx1 has yet to be directly shown to be enriched in OMVs. We propose that Stx1 is enriched in OMVs, and that this is achieved by associating with an as of yet unidentified outer membrane protein (OMP) while in the periplasm. This protein sequesters it for packaging and leaves the cell with Stx1 in the OMV. We will identify any proteins that are able to bind Stx1 within purified OMVs. The goal is to identify the mechanism by which virulence factors that are packaged into OMVs are selected, using Stx1 produced by E. coli O157:H7 as a model for exploration. In addition to pathogenesis, OMVs are effective vehicles for delivery of antimicrobial factors used in bacterial predation [12]. Since it has been shown that Stx1 can enter mammalian cells without the aid of OMVs [13], we propose that Stx1 may not be packaged into OMVs for delivery to mammalian tissue. Instead, Stx1 is packaged into OMVs to for delivery to bacteria that may be competing for nutrients, and again will be used block protein synthesis [14]. We will test this hypothesis by treating selected bacteria species with Stx1 containing OMVs. In doing so, we are seeking to elucidate an alternate role for Stx1 that does not involve direct pathogenic effects to mammals. Specific Aim 1: Test the hypothesis that Shiga-like toxin 1 packaging into OMVs is both a selective process and is mediated by protein factor(s). We will purify OMVs and periplasm from E. coli O157:H7 and characterize the quantity of Stx1 contained within them by Western blot. To identify protein components that provide packaging specificity for Stx1, we will first identify proteins that interact with Stx1 inside OMVs by mass spectrometry. Next, we will use gene deletions to test the role of these proteins in packaging