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PgaB orthologues contain a glycoside hydrolase domain that cleaves deacetylated poly-?(1,6)-N-acetylglucosamine and can disrupt bacterial biofilms.

ABSTRACT: Poly-?(1,6)-N-acetyl-D-glucosamine (PNAG) is a major biofilm component of many pathogenic bacteria. The production, modification, and export of PNAG in Escherichia coli and Bordetella species require the protein products encoded by the pgaABCD operon. PgaB is a two-domain periplasmic protein that contains an N-terminal deacetylase domain and a C-terminal PNAG binding domain that is critical for export. However, the exact function of the PgaB C-terminal domain remains unclear. Herein, we show that the C-terminal domains of Bordetella bronchiseptica PgaB (PgaBBb) and E. coli PgaB (PgaBEc) function as glycoside hydrolases. These enzymes hydrolyze purified deacetylated PNAG (dPNAG) from Staphylococcus aureus, disrupt PNAG-dependent biofilms formed by Bordetella pertussis, Staphylococcus carnosus, Staphylococcus epidermidis, and E. coli, and potentiate bacterial killing by gentamicin. Furthermore, we found that PgaBBb was only able to hydrolyze PNAG produced in situ by the E. coli PgaCD synthase complex when an active deacetylase domain was present. Mass spectrometry analysis of the PgaB-hydrolyzed dPNAG substrate showed a GlcN-GlcNAc-GlcNAc motif at the new reducing end of detected fragments. Our 1.76 Å structure of the C-terminal domain of PgaBBb reveals a central cavity within an elongated surface groove that appears ideally suited to recognize the GlcN-GlcNAc-GlcNAc motif. The structure, in conjunction with molecular modeling and site directed mutagenesis led to the identification of the dPNAG binding subsites and D474 as the probable catalytic acid. This work expands the role of PgaB within the PNAG biosynthesis machinery, defines a new glycoside hydrolase family GH153, and identifies PgaB as a possible therapeutic agent for treating PNAG-dependent biofilm infections.

SUBMITTER: Little DJ

PROVIDER: S-EPMC5933820 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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PgaB orthologues contain a glycoside hydrolase domain that cleaves deacetylated poly-β(1,6)-N-acetylglucosamine and can disrupt bacterial biofilms.

Little Dustin J DJ Pfoh Roland R Le Mauff François F Bamford Natalie C NC Notte Christina C Baker Perrin P Guragain Manita M Robinson Howard H Pier Gerald B GB Nitz Mark M Deora Rajendar R Sheppard Donald C DC Howell P Lynne PL

PLoS pathogens 20180423 4

Poly-β(1,6)-N-acetyl-D-glucosamine (PNAG) is a major biofilm component of many pathogenic bacteria. The production, modification, and export of PNAG in Escherichia coli and Bordetella species require the protein products encoded by the pgaABCD operon. PgaB is a two-domain periplasmic protein that contains an N-terminal deacetylase domain and a C-terminal PNAG binding domain that is critical for export. However, the exact function of the PgaB C-terminal domain remains unclear. Herein, we show tha ...[more]

PMID: 29684093

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Project description:The survival of commensal bacteria in the human gut partially depends on their ability to metabolize host-derived molecules. The use of the glycosidic moiety of N-glycoproteins by bacteria has been reported, but the role of N-glycopeptides or glycoamino acids as the substrates for bacterial growth has not been evaluated. We have identified in Lactobacillus casei strain BL23 a gene cluster (alf-2) involved in the catabolism of the glycoamino acid fucosyl-α-1,6-N-GlcNAc-Asn (6'FN-Asn), a constituent of the core-fucosylated structures of mammalian N-glycoproteins. The cluster consists of the genes alfHC, encoding a major facilitator superfamily (MFS) permease and the α-l-fucosidase AlfC, and the divergently oriented asdA (aspartate 4-decarboxylase), alfR2 (transcriptional regulator), pepV (peptidase), asnA2 (glycosyl-asparaginase), and sugK (sugar kinase) genes. Knockout mutants showed that alfH, alfC, asdA, asnA2, and sugK are necessary for efficient 6'FN-Asn utilization. The alf-2 genes are induced by 6'FN-Asn, but not by its glycan moiety, via the AlfR2 regulator. The constitutive expression of alf-2 genes in an alfR2 strain allowed the metabolism of a variety of 6'-fucosyl-glycans. However, GlcNAc-Asn did not support growth in this mutant background, indicating that the presence of a 6'-fucose moiety is crucial for substrate transport via AlfH. Within bacteria, 6'FN-Asn is defucosylated by AlfC, generating GlcNAc-Asn. This glycoamino acid is processed by the glycosylasparaginase AsnA2. GlcNAc-Asn hydrolysis generates aspartate and GlcNAc, which is used as a fermentable source by L.casei These data establish the existence in a commensal bacterial species of an exclusive metabolic pathway likely to scavenge human milk and mucosal fucosylated N-glycopeptides in the gastrointestinal tract.IMPORTANCE The gastrointestinal tract accommodates more than 1014 microorganisms that have an enormous impact on human health. The mechanisms enabling commensal bacteria and administered probiotics to colonize the gut remain largely unknown. The ability to utilize host-derived carbon and energy resources available at the mucosal surfaces may provide these bacteria with a competitive advantage in the gut. Here, we have identified in the commensal species Lactobacillus casei a novel metabolic pathway for the utilization of the glycoamino acid fucosyl-α-1,6-N-GlcNAc-Asn, which is present in the core-fucosylated N-glycoproteins from mammalians. These results give insight into the molecular interactions between the host and commensal/probiotic bacteria and may help to devise new strategies to restore gut microbiota homeostasis in diseases associated with dysbiotic microbiota.

Dataset Information

PgaB orthologues contain a glycoside hydrolase domain that cleaves deacetylated poly-?(1,6)-N-acetylglucosamine and can disrupt bacterial biofilms.

Publications

PgaB orthologues contain a glycoside hydrolase domain that cleaves deacetylated poly-β(1,6)-N-acetylglucosamine and can disrupt bacterial biofilms.

Similar Datasets

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