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Journal of Bacteriology, February 2000, p. 882-890, Vol. 182, No. 4
Joint Microbiology Research Unit, Guy's,
King's & St. Thomas' Dental Institute, Kings College London,
London SE5 9RW, United Kingdom
Received 4 August 1999/Accepted 22 November 1999
Enterococcus faecalis is associated with a high
proportion of nosocomial infections; however, little is known of the
ability of this organism to proliferate in vivo. The ability of RNase B, a model glycoprotein with a single N-glycosylation site occupied by
a family of high-mannose-type glycans (Man5- to
Man9-GlcNAc2), to support growth of E. faecalis was investigated. Sodium dodecyl sulfate-polyacrylamide
gel electrophoresis of RNase B demonstrated a reduction in the
molecular mass of this glycoprotein during bacterial growth. Further
analysis by matrix-assisted laser desorption ionization-time of flight
(MALDI-TOF) mass spectrometry revealed that this mass shift was due to
the degradation of all high-mannose-type glycoforms to a single
N-linked N-acetylglucosamine residue. High-pH anion-exchange chromatography analysis during exponential growth demonstrated the presence of RNase B-derived glycans in the culture supernatant, indicating the presence of an endoglycosidase activity. The free glycans were eluted with the same retention times as those
generated by the action of Streptomyces plicatus
endo-
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Production of an
Endo-
-N-Acetylglucosaminidase Activity Mediates Growth of
Enterococcus faecalis on a High-Mannose-Type
Glycoprotein
-N-acetylglucosaminidase H on RNase B. The cleavage
specificity was confirmed by MALDI-TOF analysis of the free glycans,
which showed glycan species containing only one
N-acetylglucosamine residue. No free glycans were
detectable after 5 h of bacterial growth, and we have subsequently
demonstrated the presence of mannosidase activity in E. faecalis, which releases free mannose from RNase B-derived
glycans. We propose that this deglycosylation of glycoproteins
containing high-mannose-type glycans and the subsequent degradation of
the released glycans by E. faecalis may play a role in the
survival and persistence of this nosocomial pathogen in vivo.
*
Corresponding author. Mailing address: Joint
Microbiology Research Unit, GKT Dental Institute, Caldecot Rd., London
SE5 9RW, United Kingdom. Phone: (44) 171 346 3272. Fax: (44) 171 3073. E-mail: gretta.roberts{at}kcl.ac.uk.
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