Cloning and purification of the Streptococcus suis serotype 2 glyceraldehyde-3-phosphate dehydrogenase and its involvement as an adhesin.
Streptococcus suis serotype 2 is a swine pathogen responsible for diverse diseases and may be present in the tonsils of pigs which show no sign of illness. Because adhesion to host cells may be important in the carrier state, this study was undertaken to characterize a 39 kDa surface protein identified as a glyceraldehyde-3-phosphate dehydrogenase (GAPDH), possibly implicated in the adhesion of the bacteria. The gene encoding for the GAPDH of S. suis was cloned and sequenced. The DNA sequence contained an open reading frame encoding for a 336 amino acid polypeptide exhibiting 95% sequence identity with the GAPDH from Streptococcus pyogenes and from other streptococci. Using the Qiaexpress expression plasmids, the gapdh gene was inducibly overexpressed in E. coli to produce GAPDH with a hexahistidyl N-terminus to permit its purification. The (His)6GAPDH protein was found to possess functional GAPDH enzymatic activity after the purification. An adherence assay with S. suis and porcine tracheal rings pre-incubated with (His)6GAPDH and non-incubated rings was showed a significant reduction in the adhesion of S. suis in the (His)6GAPDH pre-incubated rings compared to the non-incubated rings. The GAPDH protein of S. suis seems to be involved in the first steps of the bacterial adhesion to host cells.
GREMIP, Département de Pathologie et Microbiologie, Faculté de médecine vétérinaire, Université de Montréal, CP 5000, Saint-Hyacinthe, Quebec, Canada J2S 7C6.
Julie
J
Brassard
Marcelo
M
Gottschalk
Sylvain
S
Quessy
D000595:Amino Acid Sequence; D000818:Animals; D001422:Bacterial Adhesion / Q000502:physiology*; D001483:Base Sequence; D002846:Chromatography, Affinity / Q000662:veterinary; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology*; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000662:veterinary; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D016415:Sequence Alignment; D013290:Streptococcal Infections / Q000382:microbiology / Q000662:veterinary*; D017011:Streptococcus suis / Q000201:enzymology* / Q000235:genetics; D013552:Swine; D013553:Swine Diseases / Q000382:microbiology*; D014132:Trachea / Q000382:microbiology*
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+)