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Background

Purpose of making an adhesion system between Yersinia and probiotic bacteria

While creating a biological machine that can efficiently kill specific pathogens without disturbing other cells in its environment, an adhesion system could play a vital role. Getting our Bactissile to attach to its target, in our case Yersinia enterocolitica , could give important advantages in target specificity and delivering medicine in a way that ensures it reaches Y. enterocolitica in a concentration as high as possible.

Technical Approach

The idea was to manipulate our Bactissiles bonding to any of Y. enterocoliticas surface structures and thus preventing the pathogen from ever attaching to the gut wall. This could be done with a DNA construct coding for a membrane protein that either resembles the bonding that Y. enterocolitica makes to our cells or one that itself binds to Y. enterocolitica.

We studied the ways of infection of Yersinia enterocolitica and could determine that its main strategies of entering through the gut wall is by using its diverse membrane proteins YadA, invasin and Ail. YadA and invasin work together to bind to our cell membranes. While YadA binds to collagen, invasin structurally resembles fibronectin, that is present in the extracellular matrix, which is recognized by the fibronectin binding membrane protein integrin alpha-5-beta-1 in our cell membranes.^[1] Y. enterocolitica uses our own cell-to-cell binding mechanism to find a target cell and trick it to endocytose by binding to the integrin and to the collagen outside and in between the cells.^[2] At first glance we thought of manipulating our probiotics to also use the same bioorganic systems. That could be done by expressing collagen-like proteins and fusing them with an anchor protein that could transport the whole construct to the outer membrane and keep the collagen attached to the probiotic surface.^[3]

By using collagen, and especially putting it on the surface of a bacterial outer membrane that is meant to enter the human body, is an extremely risky procedure. “As antibodies would be produced to target the collagen and anchor protein complex this could lead to an autoimmune reaction as they would also react on the collagen naturally present in our body” says Lars Hellman, a professor in immunology at Uppsala University. More about problems with Yersinia-adhesion and the development of autoimmune disease can be found in this text.

Instead we took inspiration from our teams killing system which secretes a bacteriocin, CFY, which specifically binds to and ruptures the outer cell membrane of Y. enterocolitica.^[4] The main focus of the adhesion system drastically changed from expressing human like proteins to expressing the bacteriocin fused with the anchor protein.

If the bacteriocin would survive degradation in stomach like environments and later on find Y. enterocolitica, the bacteriocin, while attaching to the membrane, would fix the probiotic to its target. Even though this connection would be much weaker than a system with collagen, it is safe and still an advantage to the Bactissile.

System Design

The Anchor

Parts

• [1] (2012) “Bacterial Cell Surface Structures of Yersinia”, N. Bialas
• [2] (2011) “Unique Cell Adhesion and Invasion Properties of Yersinia enterocolitica” O:3, the Most Frequent Cause of Human Yersiniosis” by F. Piscano et.al.
• [3] (2001) “YadA, the multifaceted Yersinia adhesin” by J. El Tahir, M. Skurnik.
• [4] (2012) “Novel Colicin FY of Yersinia frederiksenii Inhibits Pathogenic Yersinia Strains via YiuR-Mediated Reception, TonB Import, and Cell Membrane Pore Formation“ by J. Bosak et al.