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Helicobacter pylori, as many other bacterial pathogens, contains within its virulence arsenal the Cag Type IV Secretion System. Epidemiological studies have revealed a correlation of the severity of gastric H. pylori-associated pathologies with the presence of CagA and VacA, but not with the presence of a functional Cag apparatus. The effect of CagA on the host cells has been related with its translocation into the cell by the T4SS. During the evaluation of possible effects of CagA in the motility of neutrophils-like cells, it was discovered that not only the presence of CagA was disturbing the motility towards a gradient, but also the possibility was opened that this effect seen is independent of the successful translocation and phosphorylation of CagA. If that would be the case, a new immunomodulating activity of CagA could be added to the many functions associated to this multifacetted toxin. It could explain why only CagA presence, and not a functional secretion system, is associated with disease.

While establishing the effect of the Cag T4SS on motility, the essentiality of integrin β1 for the translocation and phosphorylation of the CagA by the Cag apparatus was discovered. In previous studies, performed by the team of Prof. Dr. Rainer Haas, the theory of the necessity of a receptor on the cell membrane interacting with the T4SS for translocation of CagA was formulated. During this doctoral study, it has been determined that integrin β1 is the receptor for the Cag Type IV Secretion System and the signaling related to the integrin β1 is responsible for the IL-8 induction. In the case of CagA translocation, only the extracellular and transmembrane domains play an important role, what gives the integrin β1 the function of a “docking” molecule. It seems that the capability of integrin β1 to change conformations from extend to bend has made it a perfect “docking” molecule for the apparatus. Even though, neither the intracellular domains nor the ILK-related pathways of integrin β1 are relevant for CagA translocation, functions related with clustering seem to be relevant for the CagA translocation. Both functions, the capacity of bending and forming clusters, could play an independent but important role in the CagA translocation. While the binding to an activated form of the integrin β1 increases the efficiency of CagA translocation, as seen in the presence of MnCl2, clustering of integrins could stabilize the binding of the apparatus to the cells. At

the same time, the capacity of “bending” of the extracellular domain of integrin β1 could reduce the distance between the tip of the pilus-like structure of the apparatus and the cell membrane and facilitate the translocation of CagA. How this translocation occurs, is one of the many questions that have to be answered in future research.

Another discovery made was the involvement of clathrin dependent endocytosis in CagA phosphorylation. The participation of clathrin or clathrin associated proteins open the possibility of an endocytic process necessary for translocation. However, the failure to inhibit CagA translocation by disrupting endocytic transport by microtubules or actin, as well as endosome maturation related processes, as acidification and changes in membrane potential, makes it difficult to accept an uptake of Cag components necessary for CagA translocation. Another possibility to explain the involvement of clathin or clathrin associated proteinscould be the necessity of these proteins for the stabilization of the pore created by the Cag apparatus in order to get CagA inside the cell. Both theories should be considered in following studies relating the Cag apparatus interaction with the eukaryotic cell.

Knowing a little bit more about the behavior of H. pylori on the first minutes of infection and under different conditions, a way to stimulate the formation of a pilus-like structure without the presence of eukaryotic cells was found. The presence of such structures has been identified in past research to be related with the Cag apparatus. Knowing a way to induce their formation can help to produce enough material for Cryo-EM studies realized in conjunction with Dr. Valério Mathias at the Max-Planck Institute in Martinsried. These studies will not only determine the structure of the Cag apparatus and the components seen, they will also help to determine which proteins from the Cag apparatus are associated with its formation. Equally interesting will be to discover the molecule or molecules produced by the AGS cells that can induce the formation of the structure associated with the Cag T4SS.

It is only to hope, that the efforts done here to learn more about the interaction of H. pylori’s Cag T4SS will allow in the future a better understanding of the biomechanics by which pathogens, especially Helicobacter pylori, are able to deliver substrates into the cells. By understanding the minimal requirements for their success, the mechanism could be copied and used for targeted delivery of compounds to cancer cells, or even parasites hidden in the depths of an organism.