EndoE from <i>Enterococcus faecalis</i> Hydrolyzes the Glycans of the Biofilm Inhibiting Protein Lactoferrin and Mediates Growth

<div><p>Glycosidases are widespread among bacteria. The opportunistic human pathogen <i>Enterococcus faecalis</i> encodes several putative glycosidases but little is known about their functions. The identified endo-β-<i>N</i>-acetylglucosaminidase EndoE has activity on the N-linked glycans of the human immunoglobulin G (IgG). In this report we identified the human glycoprotein lactoferrin (hLF) as a new substrate for EndoE. Hydrolysis of the N-glycans from hLF was investigated using lectin blot, UHPLC and mass spectrometry, showing that EndoE releases major glycoforms from this protein. hLF was shown to inhibit biofilm formation of <i>E. faecalis in vitro</i>. Glycans of hLF influence the binding to <i>E. faecalis,</i> and EndoE-hydrolyzed hLF inhibits biofilm formation to lesser extent than intact hLF indicating that EndoE prevents the inhibition of biofilm. In addition, hLF binds to a surface-associated enolase of <i>E. faecalis</i>. Culture experiments showed that the activity of EndoE enables <i>E. faecalis</i> to use the glycans derived from lactoferrin as a carbon source indicating that they could be used as nutrients <i>in vivo</i> when no other preferred carbon source is available. This report adds important information about the enzymatic activity of EndoE from the commensal and opportunist <i>E. faecalis</i>. The activity on the human glycoprotein hLF, and the functional consequences with reduced inhibition of biofilm formation highlights both innate immunity functions of hLF and a bacterial mechanism to evade this innate immunity function. Taken together, our results underline the importance of glycans in the interplay between bacteria and the human host, with possible implications for both commensalism and opportunism.</p></div

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p

Growth of <i>Enterococcus faecalis</i> in the presence of lactoferrin.

<p>Growth curve of <i>E. faecalis</i> in diluted THB medium (•) and in diluted THB medium supplemented with 2 mg/ml hLF (▪) or isolated N-linked glycans from hLF (▴). Optical density (OD) at 620 nm was determined at indicated time points. Error bars indicate the standard deviation from the mean of three independent experiments.</p

Influence of human lactoferrin on biofilm formation of <i>Enterococcus faecalis</i>.

<p>Biofilm formation of <i>E. faecalis</i> was measured using the crystal violet assay and is expressed as OD₅₅₀. Human lactoferrin (hLF) was added either fully glycosylated (hLF) or deglycosylated (de-hLF) due to the treatment with EndoE. Error bars indicate the standard deviation from the mean of three independent experiments with three replicates. w/o: no hLF added.</p

Binding of human lactoferrin to the surface of <i>Enterococcus faecalis</i> and recombinant enolase.

<p>A. Western blot analysis, using anti-lactoferrin antibodies, of human lactoferrin (hLF) bound to <i>E. faecalis</i> OG1. <i>E. faecalis</i> was incubated with indicated concentrations of hLF or EndoE treated hLF (de-hLF). <i>E. faecalis</i> cell extract was separated on 10% SDS-PAGE and electro-blotted onto PVDF membranes. Control: 1 µg hLF. B. Binding of different hLF concentrations to recombinant enolase immobilized to a microtiter plate. Anti-hLF antibodies were used to detect the binding of hLF to the enolase. Error bars indicate the standard deviation from the mean of three independent experiments.</p

PNGaseF released N-glycans identified from human lactoferrin.

<p>The depicted glycan structure is based on the Oxford glycan nomenclature <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091035#pone.0091035-Harvey1" target="_blank">[56]</a>. Glycans were detected as [M-2H]²⁻ ions. GU values were generated as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091035#pone.0091035-Guile1" target="_blank">[54]</a>. In situations where chromatographic peaks containing multiple structures, the associated peak area was divided equally among the structures for simplicity.</p

Activity on and binding of EndoE to human lactoferrin.

<p>A. Human lactoferrin (hLF) was incubated with EndoE, EndoE(E186Q) and EndoE(E662Q), separated on 10% SDS-PAGE and stained with Coomassie (upper panel), or electro-blotted onto PVDF membranes and was analyzed with ConA lectin (lower panel). Incubation of hLF with PBS was used as a negative control. B. Plasmon surface resonance assay to analyze binding of EndoE to hLF. The plots show binding of EndoE(E186Q) and EndoE(E662Q) to hLF.</p

Glycan analysis of lactoferrin.

<p>Hydrophilic interaction liquid chromatography (HILIC)-fluorescence chromatogram of 2-AB labeled glycans released from human lactoferrin by the endoglycosidase EndoE (A) and the endoglycosidase PNGaseF, respectively (B). Identified glycans are separated into peaks. The numbers correspond to the glycan structures depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091035#pone-0091035-g003" target="_blank">Figure 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091035#pone-0091035-g004" target="_blank">4</a>.</p

EndoE released N-glycans identified from human lactoferrin.

<p>The depicted glycan structure is based on the Oxford glycan nomenclature <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091035#pone.0091035-Harvey1" target="_blank">[56]</a>. Glycans were detected as [M-H] and [M-2H]²⁻ ions. * denotes single charged ions. Glycan names denoted with a subscript E refer to glycans released using EndoE. GU values were generated as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091035#pone.0091035-Guile1" target="_blank">[54]</a>. In situations where chromatographic peaks containing multiple structures, the associated peak area was divided equally among the structures for simplicity.</p

Uncovering the Role of <i>N</i>‑Glycan Occupancy on the Cooperative Assembly of Spike and Angiotensin Converting Enzyme 2 Complexes: Insights from Glycoengineering and Native Mass Spectrometry

Interactions between the SARS-CoV-2 Spike protein and ACE2 are one of the most scrutinized reactions of our time. Yet, questions remain as to the impact of glycans on mediating ACE2 dimerization and downstream interactions with Spike. Here, we address these unanswered questions by combining a glycoengineering strategy with high-resolution native mass spectrometry (MS) to investigate the impact of N-glycan occupancy on the assembly of multiple Spike-ACE2 complexes. We confirmed that intact Spike trimers have all 66 N-linked sites occupied. For monomeric ACE2, all seven N-linked glycan sites are occupied to various degrees; six sites have >90% occupancy, while the seventh site (Asn690) is only partially occupied (∼30%). By resolving the glycoforms on ACE2, we deciphered the influence of each N-glycan on ACE2 dimerization. Unexpectedly, we found that Asn432 plays a role in mediating dimerization, a result confirmed by site-directed mutagenesis. We also found that glycosylated dimeric ACE2 and Spike trimers form complexes with multiple stoichiometries (Spike-ACE2 and Spike2-ACE2) with dissociation constants (Kds) of ∼500 and <100 nM, respectively. Comparing these values indicates that positive cooperativity may drive ACE2 dimers to complex with multiple Spike trimers. Overall, our results show that occupancy has a key regulatory role in mediating interactions between ACE2 dimers and Spike trimers. More generally, since soluble ACE2 (sACE2) retains an intact SARS-CoV-2 interaction site, the importance of glycosylation in ACE2 dimerization and the propensity for Spike and ACE2 to assemble into higher oligomers are molecular details important for developing strategies for neutralizing the virus