competed with av subunit containing integrins for binding to Vn by interacting with domains located at the heparin binding regions of Vn [39]. Moreover, the high molecular weight form of kinninogen confers a strong anti-adhesive function upon inte- grin-mediated cell interaction with Vn [40]. Nevertheless, we reported in this study that the anti-adhesive factor(s) found in SbS is a (are) heat-sensitive compound (s) with a molecular weight higher than 9 kDa. It is now clear that Vn plays a crucial role in many biological processes including cell migration, adhesion, tissue repair and angiogenesis [41]. Interestingly, other studies have suggested potential roles of Vn in microbial colonization and serum resistance. According to this, recent findings unveiled that many bacterial species, including C. difficile and H. Pylori interact with Vn [32]. The functionality of these interactions in pathogenesis has not been fully elucidated, although Vn most likely functions as a bridge between bacteria and epithelial cells [32]. Therefore we could postulate that SbS contains compounds that block Vn- enterocyte interactions and then inhibits adhesion of pathogens to host cells. Further work is needed to explore this putative antimicrobial activity in more depth. Sb Modulates avb5 Integrin Activity Activation of avb5 integrin leads to remodeling of the actin cytoskeleton through the formation and activation of signaling complexes called ‘‘adhesion complexes’’. These multi-molecular complexes contain enzymes and scaffolding molecules including FAK and paxillin [42]. Activation of these signaling molecules, following av integrin activation leads to the modulation of cell migration [42]. Several arguments suggest that SbS promotes dynamic changes in the avb5 integrin affinity for its ligand. Firstly, immunolocalization of both av integrin subunit and paxillin showed that SbS decreased the area of focal adherence structures. Secondly, this SbS-induced reorganization of focal adherence structures was associated with both a decrease in FAK tyrosine phosphorylation on the Y397 residue upon cell adhesion to Vn and an increase in avb5 dependent migration. FAK is a key mediator of intracellular signaling by integrins and may serve as conduits for the transmission of the force necessary for cell migration and bidirectional signaling between the cell interior and its environment [43]. FAK activation leads to the stimulation of other signaling proteins such as paxillin, thereby activating various signaling pathways crucial in the regulation of cell adhesion and migration [43]. Moreover, signals that modify tyrosine phosphor- Figure 9. Sb secretes several molecules that differentially regulate both cell adhesion and migration. (A) Isolated HCT-8/E11 cells were treated with or without SbS (SbS), boiled SbS (SbS boiled) and plated on 3.15 mg/ml vitronectin. Cell-vitronectin adhesion was evaluated as described in Methods. Results are expressed as the percentage of cell adhesion. Data represent the mean+SD of 3 separate experiments. (B) Isolated HCT-8/E11 cells were treated with or without SbS (SbS), or fractionated SbS (.9 kDa or ,9 kDa) and plated on 3.15 mg/ml vitronectin. Cell-vitronectin adhesion was evaluated as described in Methods. Results are expressed as the percentage of cell adhesion. Data represent the mean+SD of 3 separate experiments. (C) HCT-8/E11 cell monolayers were wounded and incubated without (Ctrl) or with SbS (SbS), boiled SbS (SbS boiled) or fractionated Sb supernatant (.9 kDa or ,9 kDa) for 5 h. Wound closure was determined as described in Methods. Results are expressed as the percentage of cell migration compared to control. Data represent the mean+SD of 2 separate experiments. *** P,0.001. doi:10.1371/journal.pone.0045047.g009 PLOS ONE | www.plosone.org 10 September 2012 | Volume 7 | Issue 9 | e45047