Production of an endo-beta-N-acetylglucosaminidase activity mediates growth of Enterococcus faecalis on a high-mannose-type glycoprotein

Abstract

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 (Man(5)- to Man(9)-GlcNAc(2)), 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-beta-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.

FIG. 1

Glycan structures present in the different glycoforms of RNase B. M, mannose; G, N-acetylglucosamine; Asn, asparagine residue in the polypeptide. Mannose residues are linked via α(1→2), α(1→3), α(1→6), and β(1→4) linkages, which are indicated by the numbers 2, 3, 6, and 4, respectively.

FIG. 2

SDS-PAGE of residual RNase B during the growth of E. faecalis. Lanes 1 to 6, RNase B following 0, 1, 2, 3, 4, and 5 h of bacterial growth, respectively. Molecular mass markers are indicated by arrows.

FIG. 3

MALDI-TOF analysis of residual RNase B in culture supernatant during the growth of E. faecalis BC002. (a) Spectra of RNase B following 0, 1, 2, 3, and 4 h of incubation with E. faecalis (BC002). Culture supernatants were analyzed by MALDI-TOF mass spectrometry with sinapinic acid as the matrix. The 13,885- and 14,088-Da peaks correspond to the RNase B peptide backbone with one and two N-acetylglucosamine residues bound, respectively. (b) Relative proportions of RNase B glycoforms during the first 5 h of growth. Relative amounts of each glycoform were estimated from the peak height on MALDI-TOF spectra, with the 0-h amount taken as 100%. Amounts of Man₅ (⧫), Man₆ (■), Man₇ (▴), Man₈, (●), and Man₉ (×) were measured.

FIG. 4

HPAEC analyses of free RNase B glycans present in the culture supernatant during growth of E. faecalis. (a) Glycans from heat-inactivated culture supernatant after 2 h of incubation were resolved by HPAEC using a gradient of 10 to 60 mM sodium acetate (0 to 30 min) in 100 mM sodium hydroxide. ∗, resolution of two Man₇ isoforms by HPAEC as previously described by Fu et al. (27). (b) Changes in the abundance of free glycans in the culture supernatant during exponential growth of E. faecalis as determined by HPAEC. Glycans were quantified by peak integration of detector response. Values for Man₅-GlcNAc (⧫), Man₆-GlcNAc (■), Man₇-GlcNAc (▴), Man₈-GlcNAc (●), and Man₉-GlcNAc (×) are shown.

FIG. 5

MALDI-TOF analysis of free RNase B glycans in the culture supernatant after 2 h of E. faecalis BC002 growth. The culture supernatant was subjected to reverse-phase chromatography to remove hydrophobic contaminants and was desalted by ion exchange. Samples were analyzed using 2,5-dihydroxybenzoic acid as the matrix. ∗, molecular ions plus phosphate (98 mass units).