Streptococcus pyogenes glycoprotein-binding strepadhesin activity is mediated by a surface-associated carbohydrate-degrading enzyme, pullulanase.
The interactions between pathogenic bacteria and the host need to be resolved at the molecular level in order to develop novel antiadhesive drugs and vaccines. We have previously identified strepadhesin, a novel glycoprotein-binding activity in Streptococcus pyogenes binding to thyroglobulin, submaxillar mucin, fetuin, and asialofetuin. The activity is known to be regulated by Mga, a regulator of streptococcal virulence factors, and is carried by the surface-associated streptococcal cysteine protease, SpeB. In the present study, we focused on the high strepadhesin activity in an S. pyogenes strain (NZ131rgg) lacking SpeB expression. By extracting surface proteins from the bacteria, a new strepadhesin protein was identified, and mass spectrometric analysis and database search identified it as a putative pullulanase. The gene was cloned, and the recombinant pullulanase (PulA) exhibited pullulanase and starch hydrolyzing activity, as well as strepadhesin activity. Sequencing of the pulA gene revealed an open reading frame with 3,498 bp encoding a protein of 1,165 amino acids with a predicted molecular mass of 129 kDa. PulA exhibited properties typical for a gram-positive surface protein with a putative signal sequence and LPKTGE cell wall anchoring motif and contained the four highly conserved regions common to pullulanases. Mutant bacteria deficient in PulA expression showed diminished strepadhesin activity on bacterial dot blot assay and reduced adherence to thyroglobulin immobilized on microtiter plates. Thus, S. pyogenes strepadhesin activity is carried by a surface-bound pullulanase, which combines glycoprotein-binding and carbohydrate-degrading activities in the same molecule.
Hytönen
Jukka
J
Department of Medical Biochemistry and Molecular Biology, University of Turku, Finland. jukka.hytonen@utu.fi
Haataja
Sauli
S
Finne
Jukka
J
D018829:Adhesins, Bacterial / Q000378:metabolism*; D000595:Amino Acid Sequence; D050260:Carbohydrate Metabolism; D006023:Glycoproteins / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009154:Mutation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013297:Streptococcus pyogenes / Q000201:enzymology* / Q000235:genetics; D013954:Thyroglobulin
D018829:Adhesins, Bacterial; D006023:Glycoproteins; D011994:Recombinant Proteins; D013954:Thyroglobulin; D006026:Glycoside Hydrolases; C100181:pullulanase