Supervisors info:
Μαυρή-Βαβαγιάννη Μαίρη Αναπλ. Καθηγ.(Επιβλέπουσα), Σγούρας Διονύσιος Ερευνητής Β' , Γαλανοπούλου Ντία Αναπλ. Καθηγ.
Summary:
Helicobacter pylori (H. pylori) is a gram-negative bacterium which is endemic
in the human gastric mucosa. It has been proven to be the determining factor
for the development of chronic gastric inflammation (gastritis) and peptic
ulcer and has been shown to increase the risk for the occurrence of gastric
adenocarcinoma in adults. The bacteria manifest its pathogenic role through the
activity of a variety of virulence factors and the CagA protein is one of the
major virulence determinants. Following H. pylori adhesion to gastric
epithelial cells, CagA protein is translocated endocellularly, through a type
IV bacterial secretion system and deregulates key signaling pathways related to
cellular polarity and the induction of a scattering phenotype that resembles to
the epithelial to mesenchymal transition. Pivotal role in the induction of such
phenomena by CagA protein, plays its hierarchic phosphorylation by
intracellular kinases at tyrosine phosphorylation sites (Y) involved in
repetitive glutamic acid-proline-isoleucine-tyrosine-alanine (EPIYA) peptide
sequences. The type and number of EPIYA motifs at the carboxy-terminus of CagA
has been observed to vary in clinical strains and has been reported to be
associated with more severe histopathological lesions in adults. In this study,
we investigated the potential involvement of H. pylori CagA protein, in the
transcriptional activation of matrix metalloproteinase-3 (MMP-3) in an in vitro
experimental infection model of gastric epithelial cells (AGS). A number of
isogenic mutants of H. pylori P12 reference strain were utilized, expressing
CagA protein with a variable number of functional EPIYA and
phosphorylation-deficient EPIFA motifs. MMP-3 gene activation was determined by
quantitative Real Time PCR. Furthermore, the expression of MMP-3 protein in
total H. pylori-infected epithelial cell lysates as well as secreted MMP-3 in
the culture supernatants was determined by Western Blot. In addition, secreted
MMP-3 caseinolytic activity in the supernatants of H. pylori-infected
epithelial cells was determined by zymography. An increase of the
transcriptional activity of the MMP-3 gene was observed in the presence of CagA
protein and found to be proportional to the number of terminal functional EPIYA
motifs. In contrast, phosphorylation-deficient CagA induced MMP-3 activation at
background levels. The expression of total MMP-3 protein, in H. pylori-infected
gastric epithelial cells, appeared to be in part, depended upon CagA
expression, but not on the phosphorylation on EPIYA motifs, as bacterial
mutants with CagA phosphorylation-functional EPIYA or phosphorylation-deficient
EPIFA domains induced the same levels of total MMP-3 protein. In contrast,
levels of secreted MMP-3 in the supernatants of H. pylori-infected gastric
epithelial cells, was observed to depend upon EPIYA phosphorylation of CagA.
MMP-3 levels of caseinolytic activity measured in the H. pylori-infected
epithelial cell supernatants showed that indeed, MMP-3 caseinolytic activity
depended upon phosphorylation of the functional EPIYA motifs of CagA protein.
These results point out to a new potential tumorigenic role of the bacterial
CagA protein.
Keywords:
Helicobacter pylori, Stromelysin-1 , Matrix metalloproteinases, CagA, Phosphorylation
