Cwp2 from Clostridium difficile exhibits an extended three domain fold and cell adhesion in vitro

William J. Bradshaw; Jonathan M. Kirby; April K. Roberts; Clifford Shone; K. Ravi Acharya

doi:10.1111/febs.14157

Cwp2 from Clostridium difficile exhibits an extended three domain fold and cell adhesion in vitro

William J. Bradshaw, Jonathan M. Kirby, April K. Roberts, Clifford Shone^*, K. Ravi Acharya

^*Corresponding author for this work

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Abstract

Colonization of the gut by Clostridium difficile requires the adhesion of the bacterium to host cells. A range of cell surface located factors have been linked to adhesion including the S-layer protein LMW SLP and the related protein Cwp66. As well as these proteins, the S-layer of C. difficile may contain many others. One such protein is Cwp2. Here, we demonstrate the production of a C. difficile strain 630 cwp2 knockout mutant and assess the effect on the bacterium. The mutant results in increased TcdA (toxin A) release and impaired cellular adherence in vitro. We also present the extended three domain structure of the ‘functional’ region of Cwp2, consisting of residues 29–318 at 1.9 Å, which is compared to that of LMW SLP and Cwp8. The adhesive properties of Cwp2 and LMW SLP, which are likely to be shared by Cwp8, are predicted to be mediated by the variable loop regions in domain 2. Databases: Structural data are available in the PDB under the accession number 5NJL.

Original language	English
Pages (from-to)	2886-2898
Number of pages	13
Journal	FEBS Journal
Volume	284
Issue number	17
DOIs	https://doi.org/10.1111/febs.14157
Publication status	Published - Sept 2017

Bibliographical note

Funding Information:
The authors would like to thank Diamond Light Source for beamtime (proposal mx8922), and the staff of beamline I04 for assistance with crystal testing and X-ray data collection. This work was supported by postgraduate studentships from Public Health England (PHE, Porton Down, England) and the University of Bath to WJB and from PHE to JMK, and a Medical Research Council (UK) project grant (MK/K027123/ 1) to KRA and CCS. KRA wishes to thank University of Bath for a 6-month academic sabbatical leave. This research made use of the Balena High Performance Computing (HPC) Service at the University of Bath. The authors would like to thank Lucy Blandford for assistance with the adhesion assay.

Publisher Copyright:
© 2017 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

Keywords

Clostridium difficile
bacterial adhesion
cell wall
colitis
crystal structure

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10.1111/febs.14157

Cite this

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title = "Cwp2 from Clostridium difficile exhibits an extended three domain fold and cell adhesion in vitro",

abstract = "Colonization of the gut by Clostridium difficile requires the adhesion of the bacterium to host cells. A range of cell surface located factors have been linked to adhesion including the S-layer protein LMW SLP and the related protein Cwp66. As well as these proteins, the S-layer of C. difficile may contain many others. One such protein is Cwp2. Here, we demonstrate the production of a C. difficile strain 630 cwp2 knockout mutant and assess the effect on the bacterium. The mutant results in increased TcdA (toxin A) release and impaired cellular adherence in vitro. We also present the extended three domain structure of the {\textquoteleft}functional{\textquoteright} region of Cwp2, consisting of residues 29–318 at 1.9 {\AA}, which is compared to that of LMW SLP and Cwp8. The adhesive properties of Cwp2 and LMW SLP, which are likely to be shared by Cwp8, are predicted to be mediated by the variable loop regions in domain 2. Databases: Structural data are available in the PDB under the accession number 5NJL.",

keywords = "Clostridium difficile, bacterial adhesion, cell wall, colitis, crystal structure",

author = "Bradshaw, {William J.} and Kirby, {Jonathan M.} and Roberts, {April K.} and Clifford Shone and Acharya, {K. Ravi}",

note = "Funding Information: The authors would like to thank Diamond Light Source for beamtime (proposal mx8922), and the staff of beamline I04 for assistance with crystal testing and X-ray data collection. This work was supported by postgraduate studentships from Public Health England (PHE, Porton Down, England) and the University of Bath to WJB and from PHE to JMK, and a Medical Research Council (UK) project grant (MK/K027123/ 1) to KRA and CCS. KRA wishes to thank University of Bath for a 6-month academic sabbatical leave. This research made use of the Balena High Performance Computing (HPC) Service at the University of Bath. The authors would like to thank Lucy Blandford for assistance with the adhesion assay. Publisher Copyright: {\textcopyright} 2017 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.",

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AU - Bradshaw, William J.

AU - Kirby, Jonathan M.

AU - Roberts, April K.

AU - Shone, Clifford

AU - Acharya, K. Ravi

N1 - Funding Information: The authors would like to thank Diamond Light Source for beamtime (proposal mx8922), and the staff of beamline I04 for assistance with crystal testing and X-ray data collection. This work was supported by postgraduate studentships from Public Health England (PHE, Porton Down, England) and the University of Bath to WJB and from PHE to JMK, and a Medical Research Council (UK) project grant (MK/K027123/ 1) to KRA and CCS. KRA wishes to thank University of Bath for a 6-month academic sabbatical leave. This research made use of the Balena High Performance Computing (HPC) Service at the University of Bath. The authors would like to thank Lucy Blandford for assistance with the adhesion assay. Publisher Copyright: © 2017 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

PY - 2017/9

Y1 - 2017/9

N2 - Colonization of the gut by Clostridium difficile requires the adhesion of the bacterium to host cells. A range of cell surface located factors have been linked to adhesion including the S-layer protein LMW SLP and the related protein Cwp66. As well as these proteins, the S-layer of C. difficile may contain many others. One such protein is Cwp2. Here, we demonstrate the production of a C. difficile strain 630 cwp2 knockout mutant and assess the effect on the bacterium. The mutant results in increased TcdA (toxin A) release and impaired cellular adherence in vitro. We also present the extended three domain structure of the ‘functional’ region of Cwp2, consisting of residues 29–318 at 1.9 Å, which is compared to that of LMW SLP and Cwp8. The adhesive properties of Cwp2 and LMW SLP, which are likely to be shared by Cwp8, are predicted to be mediated by the variable loop regions in domain 2. Databases: Structural data are available in the PDB under the accession number 5NJL.

AB - Colonization of the gut by Clostridium difficile requires the adhesion of the bacterium to host cells. A range of cell surface located factors have been linked to adhesion including the S-layer protein LMW SLP and the related protein Cwp66. As well as these proteins, the S-layer of C. difficile may contain many others. One such protein is Cwp2. Here, we demonstrate the production of a C. difficile strain 630 cwp2 knockout mutant and assess the effect on the bacterium. The mutant results in increased TcdA (toxin A) release and impaired cellular adherence in vitro. We also present the extended three domain structure of the ‘functional’ region of Cwp2, consisting of residues 29–318 at 1.9 Å, which is compared to that of LMW SLP and Cwp8. The adhesive properties of Cwp2 and LMW SLP, which are likely to be shared by Cwp8, are predicted to be mediated by the variable loop regions in domain 2. Databases: Structural data are available in the PDB under the accession number 5NJL.

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KW - cell wall

KW - colitis

KW - crystal structure

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JO - FEBS Journal

JF - FEBS Journal

IS - 17

ER -

Cwp2 from Clostridium difficile exhibits an extended three domain fold and cell adhesion in vitro

Abstract

Bibliographical note

Keywords

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