Essential role of Helicobacter pylori gamma-glutamyltranspeptidase for the colonization of the gastric mucosa of mice.
Constitutive expression of gamma-glutamyltranspeptidase (GGT) activity is common to all Helicobacter pylori strains, and is used as a marker for identifying H. pylori isolates. Helicobacter pylori GGT was purified from sonicated extracts of H. pylori strain 85P by anion exchange chromatography. The N-terminal amino acid sequences of two of the generated endo-proteolysed peptides were determined, allowing the cloning and sequencing of the corresponding gene from a genomic H. pylori library. The H. pylori ggt gene consists of a 1681 basepair (bp) open reading frame encoding a protein with a signal sequence and a calculated molecular mass of 61 kDa. Escherichia coli clones harbouring the H. pylori ggt gene exhibited GGT activity at 37 degrees C, in contrast to E. coli host cells (MC1061, HB101), which were GGT negative at 37 degrees C. GGT activity was found to be constitutively expressed by similar genes in Helicobacter felis, Helicobacter canis, Helicobacter bilis, Helicobacter hepaticus and Helicobacter mustelae. Western immunoblots using rabbit antibodies raised against a His-tagged-GGT recombinant protein demonstrated that H. pylori GGT is synthesized in both H. pylori and E. coli as a pro-GGT that is processed into a large and a small subunit. Deletion of a 700 bp fragment within the GGT-encoding gene of a mouse-adapted H. pylori strain (SS1) resulted in mutants that were GGT negative yet grew normally in vitro. These mutants, however, were unable to colonize the gastric mucosa of mice when orally administered alone or together (co-infection) with the parental strain. These results demonstrate that H. pylori GGT activity has an essential role for the establishment of the infection in the mouse model, demonstrating for the first time a physiological role for a bacterial GGT enzyme.
Chevalier
C
C
Unité INSERM U389, Institut Pasteur, Paris, France.
Thiberge
J M
JM
Ferrero
R L
RL
Labigne
A
A
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005753:Gastric Mucosa / Q000382:microbiology*; D005822:Genetic Vectors; D016480:Helicobacter pylori / Q000201:enzymology* / Q000472:pathogenicity*; D051379:Mice; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D005723:gamma-Glutamyltransferase / Q000302:isolation & purification / Q000502:physiology*
D005723:gamma-Glutamyltransferase