Intravital imaging of Helicobacter pylori protein CagA in cells of the stomach lining.
Zusammenfassung der Projektergebnisse
The aim of the study was to develop a novel imaging tool, which potentially would allow us to detect precancerous lesions in the gastrointestinal tract. The study design was based on fluorescence confocal endomicroscopy, which allows taking ‘optical in vivo biopsies’ in living organisms. At the end of the 24 months project phase none of the specific aims could be reached. The course of the project was full of ‘surprises’ and unexpected technical difficulties. Based on our preliminary data, we did not expect the difficulties in establishing the TVA/RCAS mouse model for membrane-bound CagA. We were able to infect the TVA-expressing mouse stomach lining with GFP-RCAS viruses, which could be detected in vivo using confocal endomicroscopy. In our work we discovered that the N-terminal membrane-binding domain of CagA is not delivered correctly to the plasma membrane in vitro and in vivo in the TVA/RCAS system, but accumulates in vesicles in the cytoplasm. The second membrane-binding domain of CagA at the C- terminus contains signaling motifs, which prevents its expression in RCAS viruses. Because the CagA TVA/RCAS model was critical to the overall project, much time and effort was spent in finding a solution. The solution maybe is a novel H. pylori strain, which is able to inject CagA into the stomach lining of infected mice. However, these experiments were started at the end of the project phase and results are not yet available. The second finding of the project was that the delivery of peptides across the stomach lining is complex and remains a challenge. None of the published CPPs (cell penetrating peptides) was able to penetrate polarized epithelial cells in vitro and in vivo except for Penetratin. However, Penetratin accumulates in vesicles in the cytoplasm and it is unclear if its cargo could reach the plasma membrane. In specific aim 3, we were able to establish two peptide phage display libraries in the laboratory. The panning experiments worked correctly using positive and negative controls. However, repeating the experiments using CagA mutant proteins did not reveal specific peptide hits. We adjusted the experimental conditions to increase the probability of positive results, but we were without success despite extensive efforts. It is unclear at this point, if CagA lacks binding structures at its surface for the peptide sequences in the heptapeptide and dodecapeptide phage library. During the project phase we gained much experience and insight into various methods, but we were unsuccessful in producing positive results for future publications. The work may continue after the funding period after establishing a new laboratory at the Universitätsmedizin in Mannheim.
