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Cyclic diGMP regulates production of sortase substrates of Clostridium difficile and their surface exposure through ZmpI protease-mediated cleavage.

Peltier J, Shaw HA, Couchman EC, Dawson LF, Yu L, Choudhary JS, Kaever V, Wren BW, Fairweather NF - J. Biol. Chem. (2015)

Bottom Line: Low c-diGMP levels induce the release of CD2831 and presumably CD3246 from the surface of cells.This regulation is mediated by proteolytic cleavage of CD2831 and CD3246 by the zinc metalloprotease ZmpI, whose expression is controlled by a type I c-diGMP riboswitch.These data reveal a novel regulatory mechanism for expression of two sortase substrates by the secondary messenger c-diGMP, on which surface anchoring is dependent.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Life Sciences, Center for Molecular Bacteriology and Infection, Imperial College London, London SW7 2AZ, United Kingdom.

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CD3246HA is anchored to the cell wall by SrtB and is released into the extracellular medium through its cleavage by ZmpI.A, schematic representation of chromosomal CD3246 showing its ribozyme-interrupted RBS and of CD3246HA encoded by pJKP070 under control of the Ptet inducible promoter and a functional RBS. The signal peptide (SP), the HA tag (HA), and the sorting signal composed of the SPKTG motif, the hydrophobic domain (in gray), and the positively charged tail (+) are represented. The SPKTG sorting motif is cleaved by srtB between the threonine (T) and the glycine (G) residues. B, supernatant (Sn), cell wall (CW), membrane (Mb), and cytosolic (Cy) compartment of wild-type (WT) and sortase mutant (ΔsrtB) strains carrying pJKP070 (expresses CD3246HA in the presence of ATc) and cultivated in the presence of 100 ng/ml ATc (+ATc) were analyzed by Western blotting with anti-HA monoclonal antibodies. Proteins were separated on 12% SDS-polyacrylamide gels. C, supernatant (Sn), cell wall (CW), membrane (Mb), and cytosolic (Cy) compartment of zmpI mutant (ΔzmpI) and srtB/zmpI double mutant (ΔsrtBΔzmpI) strains, carrying pJKP070 (expresses CD3246HA in the presence of ATc) and cultivated in the presence of 100 ng/ml ATc (+ATc), were analyzed by Western blotting with anti-HA monoclonal antibodies. Supernatant (Sn) and whole cell lysate (WL) fractions of ΔzmpI pJKP070 strain cultivated in the absence of ATc ([minus]ATc) were also analyzed as negative controls.
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Figure 10: CD3246HA is anchored to the cell wall by SrtB and is released into the extracellular medium through its cleavage by ZmpI.A, schematic representation of chromosomal CD3246 showing its ribozyme-interrupted RBS and of CD3246HA encoded by pJKP070 under control of the Ptet inducible promoter and a functional RBS. The signal peptide (SP), the HA tag (HA), and the sorting signal composed of the SPKTG motif, the hydrophobic domain (in gray), and the positively charged tail (+) are represented. The SPKTG sorting motif is cleaved by srtB between the threonine (T) and the glycine (G) residues. B, supernatant (Sn), cell wall (CW), membrane (Mb), and cytosolic (Cy) compartment of wild-type (WT) and sortase mutant (ΔsrtB) strains carrying pJKP070 (expresses CD3246HA in the presence of ATc) and cultivated in the presence of 100 ng/ml ATc (+ATc) were analyzed by Western blotting with anti-HA monoclonal antibodies. Proteins were separated on 12% SDS-polyacrylamide gels. C, supernatant (Sn), cell wall (CW), membrane (Mb), and cytosolic (Cy) compartment of zmpI mutant (ΔzmpI) and srtB/zmpI double mutant (ΔsrtBΔzmpI) strains, carrying pJKP070 (expresses CD3246HA in the presence of ATc) and cultivated in the presence of 100 ng/ml ATc (+ATc), were analyzed by Western blotting with anti-HA monoclonal antibodies. Supernatant (Sn) and whole cell lysate (WL) fractions of ΔzmpI pJKP070 strain cultivated in the absence of ATc ([minus]ATc) were also analyzed as negative controls.

Mentions: CD3246, identified as another putative SrtB substrate, harbors a SPKTG sorting motif and is annotated as a putative adhesin. CD3246 is not conserved across all C. difficile lineages and is notably absent from ribotype 027 strains. CD3246 shares similarities with CD2831. First, a c-diGMP-II riboswitch (Cdi2_1) is present upstream of CD3246 (20). This riboswitch controls transcription and also initiation of translation through the splicing of an allosteric self-splicing ribozyme, located between the riboswitch and the CD3246-coding sequence (Fig. 10A) (35, 36). Second, CD3246 contains seven consecutive ZmpI cleavage sites gathered in the C-terminal extremity of the protein (30). We therefore investigated the subcellular localization of CD3246 to determine whether surface attachment of CD3246 is controlled by the same mechanism as that of CD2831. We constructed a derivative of CD3246 with an HA tag inserted five residues downstream of the signal peptide. A functional RBS was also inserted upstream of the CD3246-coding sequence because the native CD3246 RBS is interrupted by the ribozyme (Fig. 10A). This construct was then placed under the control of Ptet, yielding pJKP070, and introduced into C. difficile 630 and ΔsrtB strains. Upon induction, anti-HA antibodies detected the majority of the CD3246HA in the culture supernatant of 630 (pJKP070) and a small proportion in the cell wall fraction. A similar profile was obtained with the ΔsrtB (pJKP070) strain grown in the presence of ATc, indicating that the low level of cell wall associated CD3246HA is not mediated by SrtB (Fig. 10B). Plasmid pJKP070 was then introduced into the ΔzmpI mutant. As expected, subcellular localization of CD3246HA was affected by the zmpI deletion with the majority of the protein now found associated to the cell wall (Fig. 10C). Also, the apparent molecular weight of CD3246HA was significantly larger in the absence of ZmpI, in agreement with the proteolytic activity of ZmpI on CD3246 (Fig. 10, B and C).


Cyclic diGMP regulates production of sortase substrates of Clostridium difficile and their surface exposure through ZmpI protease-mediated cleavage.

Peltier J, Shaw HA, Couchman EC, Dawson LF, Yu L, Choudhary JS, Kaever V, Wren BW, Fairweather NF - J. Biol. Chem. (2015)

CD3246HA is anchored to the cell wall by SrtB and is released into the extracellular medium through its cleavage by ZmpI.A, schematic representation of chromosomal CD3246 showing its ribozyme-interrupted RBS and of CD3246HA encoded by pJKP070 under control of the Ptet inducible promoter and a functional RBS. The signal peptide (SP), the HA tag (HA), and the sorting signal composed of the SPKTG motif, the hydrophobic domain (in gray), and the positively charged tail (+) are represented. The SPKTG sorting motif is cleaved by srtB between the threonine (T) and the glycine (G) residues. B, supernatant (Sn), cell wall (CW), membrane (Mb), and cytosolic (Cy) compartment of wild-type (WT) and sortase mutant (ΔsrtB) strains carrying pJKP070 (expresses CD3246HA in the presence of ATc) and cultivated in the presence of 100 ng/ml ATc (+ATc) were analyzed by Western blotting with anti-HA monoclonal antibodies. Proteins were separated on 12% SDS-polyacrylamide gels. C, supernatant (Sn), cell wall (CW), membrane (Mb), and cytosolic (Cy) compartment of zmpI mutant (ΔzmpI) and srtB/zmpI double mutant (ΔsrtBΔzmpI) strains, carrying pJKP070 (expresses CD3246HA in the presence of ATc) and cultivated in the presence of 100 ng/ml ATc (+ATc), were analyzed by Western blotting with anti-HA monoclonal antibodies. Supernatant (Sn) and whole cell lysate (WL) fractions of ΔzmpI pJKP070 strain cultivated in the absence of ATc ([minus]ATc) were also analyzed as negative controls.
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Figure 10: CD3246HA is anchored to the cell wall by SrtB and is released into the extracellular medium through its cleavage by ZmpI.A, schematic representation of chromosomal CD3246 showing its ribozyme-interrupted RBS and of CD3246HA encoded by pJKP070 under control of the Ptet inducible promoter and a functional RBS. The signal peptide (SP), the HA tag (HA), and the sorting signal composed of the SPKTG motif, the hydrophobic domain (in gray), and the positively charged tail (+) are represented. The SPKTG sorting motif is cleaved by srtB between the threonine (T) and the glycine (G) residues. B, supernatant (Sn), cell wall (CW), membrane (Mb), and cytosolic (Cy) compartment of wild-type (WT) and sortase mutant (ΔsrtB) strains carrying pJKP070 (expresses CD3246HA in the presence of ATc) and cultivated in the presence of 100 ng/ml ATc (+ATc) were analyzed by Western blotting with anti-HA monoclonal antibodies. Proteins were separated on 12% SDS-polyacrylamide gels. C, supernatant (Sn), cell wall (CW), membrane (Mb), and cytosolic (Cy) compartment of zmpI mutant (ΔzmpI) and srtB/zmpI double mutant (ΔsrtBΔzmpI) strains, carrying pJKP070 (expresses CD3246HA in the presence of ATc) and cultivated in the presence of 100 ng/ml ATc (+ATc), were analyzed by Western blotting with anti-HA monoclonal antibodies. Supernatant (Sn) and whole cell lysate (WL) fractions of ΔzmpI pJKP070 strain cultivated in the absence of ATc ([minus]ATc) were also analyzed as negative controls.
Mentions: CD3246, identified as another putative SrtB substrate, harbors a SPKTG sorting motif and is annotated as a putative adhesin. CD3246 is not conserved across all C. difficile lineages and is notably absent from ribotype 027 strains. CD3246 shares similarities with CD2831. First, a c-diGMP-II riboswitch (Cdi2_1) is present upstream of CD3246 (20). This riboswitch controls transcription and also initiation of translation through the splicing of an allosteric self-splicing ribozyme, located between the riboswitch and the CD3246-coding sequence (Fig. 10A) (35, 36). Second, CD3246 contains seven consecutive ZmpI cleavage sites gathered in the C-terminal extremity of the protein (30). We therefore investigated the subcellular localization of CD3246 to determine whether surface attachment of CD3246 is controlled by the same mechanism as that of CD2831. We constructed a derivative of CD3246 with an HA tag inserted five residues downstream of the signal peptide. A functional RBS was also inserted upstream of the CD3246-coding sequence because the native CD3246 RBS is interrupted by the ribozyme (Fig. 10A). This construct was then placed under the control of Ptet, yielding pJKP070, and introduced into C. difficile 630 and ΔsrtB strains. Upon induction, anti-HA antibodies detected the majority of the CD3246HA in the culture supernatant of 630 (pJKP070) and a small proportion in the cell wall fraction. A similar profile was obtained with the ΔsrtB (pJKP070) strain grown in the presence of ATc, indicating that the low level of cell wall associated CD3246HA is not mediated by SrtB (Fig. 10B). Plasmid pJKP070 was then introduced into the ΔzmpI mutant. As expected, subcellular localization of CD3246HA was affected by the zmpI deletion with the majority of the protein now found associated to the cell wall (Fig. 10C). Also, the apparent molecular weight of CD3246HA was significantly larger in the absence of ZmpI, in agreement with the proteolytic activity of ZmpI on CD3246 (Fig. 10, B and C).

Bottom Line: Low c-diGMP levels induce the release of CD2831 and presumably CD3246 from the surface of cells.This regulation is mediated by proteolytic cleavage of CD2831 and CD3246 by the zinc metalloprotease ZmpI, whose expression is controlled by a type I c-diGMP riboswitch.These data reveal a novel regulatory mechanism for expression of two sortase substrates by the secondary messenger c-diGMP, on which surface anchoring is dependent.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Life Sciences, Center for Molecular Bacteriology and Infection, Imperial College London, London SW7 2AZ, United Kingdom.

Show MeSH
Related in: MedlinePlus