PgaB orthologues contain a glycoside hydrolase domain that cleaves deacetylated poly-β(1,6)-N-acetylglucosamine and can disrupt bacterial biofilms.
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 co...
Main Authors: | , , , , , , , , , , , , |
---|---|
Format: | Article |
Language: | English |
Published: |
Public Library of Science (PLoS)
2018-04-01
|
Series: | PLoS Pathogens |
Online Access: | https://doi.org/10.1371/journal.ppat.1006998 |
id |
doaj-eb91361e84b14e24a860218a28db4e5a |
---|---|
record_format |
Article |
spelling |
doaj-eb91361e84b14e24a860218a28db4e5a2021-04-21T17:43:50ZengPublic Library of Science (PLoS)PLoS Pathogens1553-73661553-73742018-04-01144e100699810.1371/journal.ppat.1006998PgaB orthologues contain a glycoside hydrolase domain that cleaves deacetylated poly-β(1,6)-N-acetylglucosamine and can disrupt bacterial biofilms.Dustin J LittleRoland PfohFrançois Le MauffNatalie C BamfordChristina NottePerrin BakerManita GuragainHoward RobinsonGerald B PierMark NitzRajendar DeoraDonald C SheppardP Lynne HowellPoly-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 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.https://doi.org/10.1371/journal.ppat.1006998 |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Dustin J Little Roland Pfoh François Le Mauff Natalie C Bamford Christina Notte Perrin Baker Manita Guragain Howard Robinson Gerald B Pier Mark Nitz Rajendar Deora Donald C Sheppard P Lynne Howell |
spellingShingle |
Dustin J Little Roland Pfoh François Le Mauff Natalie C Bamford Christina Notte Perrin Baker Manita Guragain Howard Robinson Gerald B Pier Mark Nitz Rajendar Deora Donald C Sheppard P Lynne Howell PgaB orthologues contain a glycoside hydrolase domain that cleaves deacetylated poly-β(1,6)-N-acetylglucosamine and can disrupt bacterial biofilms. PLoS Pathogens |
author_facet |
Dustin J Little Roland Pfoh François Le Mauff Natalie C Bamford Christina Notte Perrin Baker Manita Guragain Howard Robinson Gerald B Pier Mark Nitz Rajendar Deora Donald C Sheppard P Lynne Howell |
author_sort |
Dustin J Little |
title |
PgaB orthologues contain a glycoside hydrolase domain that cleaves deacetylated poly-β(1,6)-N-acetylglucosamine and can disrupt bacterial biofilms. |
title_short |
PgaB orthologues contain a glycoside hydrolase domain that cleaves deacetylated poly-β(1,6)-N-acetylglucosamine and can disrupt bacterial biofilms. |
title_full |
PgaB orthologues contain a glycoside hydrolase domain that cleaves deacetylated poly-β(1,6)-N-acetylglucosamine and can disrupt bacterial biofilms. |
title_fullStr |
PgaB orthologues contain a glycoside hydrolase domain that cleaves deacetylated poly-β(1,6)-N-acetylglucosamine and can disrupt bacterial biofilms. |
title_full_unstemmed |
PgaB orthologues contain a glycoside hydrolase domain that cleaves deacetylated poly-β(1,6)-N-acetylglucosamine and can disrupt bacterial biofilms. |
title_sort |
pgab orthologues contain a glycoside hydrolase domain that cleaves deacetylated poly-β(1,6)-n-acetylglucosamine and can disrupt bacterial biofilms. |
publisher |
Public Library of Science (PLoS) |
series |
PLoS Pathogens |
issn |
1553-7366 1553-7374 |
publishDate |
2018-04-01 |
description |
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 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. |
url |
https://doi.org/10.1371/journal.ppat.1006998 |
work_keys_str_mv |
AT dustinjlittle pgaborthologuescontainaglycosidehydrolasedomainthatcleavesdeacetylatedpolyb16nacetylglucosamineandcandisruptbacterialbiofilms AT rolandpfoh pgaborthologuescontainaglycosidehydrolasedomainthatcleavesdeacetylatedpolyb16nacetylglucosamineandcandisruptbacterialbiofilms AT francoislemauff pgaborthologuescontainaglycosidehydrolasedomainthatcleavesdeacetylatedpolyb16nacetylglucosamineandcandisruptbacterialbiofilms AT nataliecbamford pgaborthologuescontainaglycosidehydrolasedomainthatcleavesdeacetylatedpolyb16nacetylglucosamineandcandisruptbacterialbiofilms AT christinanotte pgaborthologuescontainaglycosidehydrolasedomainthatcleavesdeacetylatedpolyb16nacetylglucosamineandcandisruptbacterialbiofilms AT perrinbaker pgaborthologuescontainaglycosidehydrolasedomainthatcleavesdeacetylatedpolyb16nacetylglucosamineandcandisruptbacterialbiofilms AT manitaguragain pgaborthologuescontainaglycosidehydrolasedomainthatcleavesdeacetylatedpolyb16nacetylglucosamineandcandisruptbacterialbiofilms AT howardrobinson pgaborthologuescontainaglycosidehydrolasedomainthatcleavesdeacetylatedpolyb16nacetylglucosamineandcandisruptbacterialbiofilms AT geraldbpier pgaborthologuescontainaglycosidehydrolasedomainthatcleavesdeacetylatedpolyb16nacetylglucosamineandcandisruptbacterialbiofilms AT marknitz pgaborthologuescontainaglycosidehydrolasedomainthatcleavesdeacetylatedpolyb16nacetylglucosamineandcandisruptbacterialbiofilms AT rajendardeora pgaborthologuescontainaglycosidehydrolasedomainthatcleavesdeacetylatedpolyb16nacetylglucosamineandcandisruptbacterialbiofilms AT donaldcsheppard pgaborthologuescontainaglycosidehydrolasedomainthatcleavesdeacetylatedpolyb16nacetylglucosamineandcandisruptbacterialbiofilms AT plynnehowell pgaborthologuescontainaglycosidehydrolasedomainthatcleavesdeacetylatedpolyb16nacetylglucosamineandcandisruptbacterialbiofilms |
_version_ |
1714665864392867840 |