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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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Related in: MedlinePlus

Display of CD2831 on the clostridial cell surface. The wild-type (WT), zmpI mutant (ΔzmpI), and srtB/zmpI double mutant (ΔsrtBΔzmpI) strains carrying pJKP041 (overexpresses CD2831 in the presence of ATc) were grown in BHIS broth in the presence of 100 ng/ml ATc (+ATc) and labeled with rabbit anti-LMW-SlpA (green, left panels) to label the surface of all bacteria and mouse anti-CD2831 (red, middle panels) to assess CD2831 localization. Overlays of those images are also showed (merge, right panels). Labeling was visualized using Cy2 anti-rabbit conjugate and rhodamine red-X anti-mouse conjugate. The ΔzmpI strain carrying pJKP041 and grown in the absence of the inducer (−ATc) was used as a negative control. White bars represent 10 μm.
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Figure 3: Display of CD2831 on the clostridial cell surface. The wild-type (WT), zmpI mutant (ΔzmpI), and srtB/zmpI double mutant (ΔsrtBΔzmpI) strains carrying pJKP041 (overexpresses CD2831 in the presence of ATc) were grown in BHIS broth in the presence of 100 ng/ml ATc (+ATc) and labeled with rabbit anti-LMW-SlpA (green, left panels) to label the surface of all bacteria and mouse anti-CD2831 (red, middle panels) to assess CD2831 localization. Overlays of those images are also showed (merge, right panels). Labeling was visualized using Cy2 anti-rabbit conjugate and rhodamine red-X anti-mouse conjugate. The ΔzmpI strain carrying pJKP041 and grown in the absence of the inducer (−ATc) was used as a negative control. White bars represent 10 μm.

Mentions: To assess CD2831 localization at the individual cell level, immunofluorescence microscopy analysis was performed on intact bacteria. All bacteria were uniformly labeled with anti-LMW SLP, which detects the S-layer that completely coats C. difficile (Fig. 3). CD2831 could be detected at the surface of the induced ΔzmpI pJKP041 strain (Fig. 3), but the intensity of labeling by the anti-CD2831 antibodies appeared to be heterogeneous in the bacterial population. Interestingly, CD2831 labeling was not distributed along the entire bacterial cell but was concentrated mostly at one cell pole. Similar features have been previously reported for SrtB substrates in Listeria monocytogenes (see below) (32). Most cells from the induced wild-type pJKP041 culture were not stained with anti-CD2831, with the exception of polar staining in a few cells. This residual fluorescence was not observed in the uninduced ΔzmpI pJKP041 strain, indicating that a small fraction of CD2831 remains at the cell surface in the induced wild-type pJKP041 strain. It is likely that the level of ZmpI in the extracellular medium is insufficient to cleave efficiently the large amount of CD2831 protein produced under inducing conditions.


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)

Display of CD2831 on the clostridial cell surface. The wild-type (WT), zmpI mutant (ΔzmpI), and srtB/zmpI double mutant (ΔsrtBΔzmpI) strains carrying pJKP041 (overexpresses CD2831 in the presence of ATc) were grown in BHIS broth in the presence of 100 ng/ml ATc (+ATc) and labeled with rabbit anti-LMW-SlpA (green, left panels) to label the surface of all bacteria and mouse anti-CD2831 (red, middle panels) to assess CD2831 localization. Overlays of those images are also showed (merge, right panels). Labeling was visualized using Cy2 anti-rabbit conjugate and rhodamine red-X anti-mouse conjugate. The ΔzmpI strain carrying pJKP041 and grown in the absence of the inducer (−ATc) was used as a negative control. White bars represent 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4591827&req=5

Figure 3: Display of CD2831 on the clostridial cell surface. The wild-type (WT), zmpI mutant (ΔzmpI), and srtB/zmpI double mutant (ΔsrtBΔzmpI) strains carrying pJKP041 (overexpresses CD2831 in the presence of ATc) were grown in BHIS broth in the presence of 100 ng/ml ATc (+ATc) and labeled with rabbit anti-LMW-SlpA (green, left panels) to label the surface of all bacteria and mouse anti-CD2831 (red, middle panels) to assess CD2831 localization. Overlays of those images are also showed (merge, right panels). Labeling was visualized using Cy2 anti-rabbit conjugate and rhodamine red-X anti-mouse conjugate. The ΔzmpI strain carrying pJKP041 and grown in the absence of the inducer (−ATc) was used as a negative control. White bars represent 10 μm.
Mentions: To assess CD2831 localization at the individual cell level, immunofluorescence microscopy analysis was performed on intact bacteria. All bacteria were uniformly labeled with anti-LMW SLP, which detects the S-layer that completely coats C. difficile (Fig. 3). CD2831 could be detected at the surface of the induced ΔzmpI pJKP041 strain (Fig. 3), but the intensity of labeling by the anti-CD2831 antibodies appeared to be heterogeneous in the bacterial population. Interestingly, CD2831 labeling was not distributed along the entire bacterial cell but was concentrated mostly at one cell pole. Similar features have been previously reported for SrtB substrates in Listeria monocytogenes (see below) (32). Most cells from the induced wild-type pJKP041 culture were not stained with anti-CD2831, with the exception of polar staining in a few cells. This residual fluorescence was not observed in the uninduced ΔzmpI pJKP041 strain, indicating that a small fraction of CD2831 remains at the cell surface in the induced wild-type pJKP041 strain. It is likely that the level of ZmpI in the extracellular medium is insufficient to cleave efficiently the large amount of CD2831 protein produced under inducing conditions.

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