Unit:
ΠΜΣ Μικροβιακή ΒιοτεχνολογίαLibrary of the School of Science
Supervisors info:
Διαλλινάς Γεώργιος Καθηγητής
Original Title:
Μελέτη του ρόλου της πρωτεΐνης CagA στην επαγωγή Ιντερλευκίνης-8 μέσω μεταγραφικής ενεργοποίησης του NF-κΒ στη λοίμωξη από H. pylori.
Translated title:
The role of CagA protein in the IL-8 induction through NF-κB transcriptional activation in the H. pylori infection.
Summary:
Helicobacter pylori is a Gram negative bacteria that colonizes the human
gastric mucosa, with a world-wide mean prevalence of 50%. The persistent
pathogen can cause a variety of damages, ranging from persistent chronic
gastritis to duodenal ulcer and gastric cancer. H. pylori pathogenesis is
manifested through a combined effect of bacterial virulence factors, host
genetics, and environmental factors. One of the most important virulence
factors is the protein CagA (Cytotoxin-associated gene A) which has been
proposed to act as a bacterial oncoprotein. H. pylori injects CagA into the
host gastric epithelial cells, through its needle-like structure, type IV
secretion system (T4SS). Injected CagA hijacks physiological signal
transduction associated with cell polarity, inflammation and cancer. Tyrosine
(Y) phosphorylation of CagA occurs at the EPIYA motif; a five amino acid
sequence (Glu-Pro-Ile-Tyr-Ala: glutamic acid-
proline-isoleucine-tyrosine-alanine) that is present in the carboxyl-terminal
variable region of the protein and may affect the prevalence of gastric cancer.
In the present study H. pylori isogenic strains, expressing CagA protein with a
variable number of EPIYA and the respective non-phosphorylatable EPIFA motifs,
labeled at the carboxyl-terminal region with tetracysteines (Cys), for FlAsH
labeling, were evaluated. The ultimate aim was to investigate CagA involvement
and the contribution of CagA-EPIYA motifs in the secretion of interleukin 8
(IL-8) from infected gastric epithelial cells, following NF-κB activation.
The gastric epithelial cell line AGS was infected with the aforementioned
series of isogenic CagA mutants based on H. pylori P12 reference strain,
expressing CagA protein with differing number of functional (EPIYA) and
non-phosphorylatable (EPIFA) motifs. All strains were evaluated beforehand for
their efficiency to adhere equally well to AGS, induce pilus formation, and
finally, functionally translocate CagA protein into the AGS cells. The
characteristic “scattering and elongation” hummingbird phenotype was evaluated.
Also, levels of secreted IL-8 after 24 h infection of AGS cells with all
strains were determined. Furthermore, the role of CagA in the induction of IL-8
through NF-κB pathway was investigated.The results of this study highlighted
the important role of the phosphorylated CagA protein (CagAPY) in the induction
of cytoskeletal rearrangement and the hummingbird phenotype.With reference to
the induction of IL-8 secretion, the importance of a functional T4SS and the
successful translocation of CagA protein into the AGS cells were confirmed.
These findings are consistent with existing bibliography and highlight the
multifactorial nature of bacterium-host interactions. Finally, image analysis
from confocal microscopy of AGS cells infected with the aforementioned H.
pylori strains stained with CFDA-SE or FlAsH, detected the presence of CagA on
the cell surface as well as inside the cells. The successful application of the
FlAsH labeling technique in H. pylori will be an important tool for further
determination of the intracellular localization of CagA protein utilizing Live
Imaging and confocal microscopy.
Keywords:
H. pylori, CagA, NF-κΒ, IL-8, Gastric Cancer
Number of index pages:
IV-VII
Number of references:
274
Number of pages:
I-XI, 98
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