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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

Elevated c-diGMP levels induce formation of long chain of cells in C. difficile 630.A, phase contrast (left and center panels) and fluorescence (right panel) microscopy images C. difficile wild-type (WT) strain carrying an empty plasmid (vector) or pECC12 (constitutively expresses DccA). For the fluorescence microscopy, membranes were stained with MitoTracker Green. White triangles indicate positions of septa. Scale bars, 20 μm. B, phase contrast microscopy views of C. difficile wild-type (WT) strain carrying pECC17 (expresses DccA in the presence of ATc). Expression of DccA from pECC17 was induced with the indicated concentrations of ATc. Black bars, 20 μm.
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Figure 9: Elevated c-diGMP levels induce formation of long chain of cells in C. difficile 630.A, phase contrast (left and center panels) and fluorescence (right panel) microscopy images C. difficile wild-type (WT) strain carrying an empty plasmid (vector) or pECC12 (constitutively expresses DccA). For the fluorescence microscopy, membranes were stained with MitoTracker Green. White triangles indicate positions of septa. Scale bars, 20 μm. B, phase contrast microscopy views of C. difficile wild-type (WT) strain carrying pECC17 (expresses DccA in the presence of ATc). Expression of DccA from pECC17 was induced with the indicated concentrations of ATc. Black bars, 20 μm.

Mentions: Immunofluorescence microscopy analysis was performed to detect CD2831. In the wild-type strain carrying pECC12 (constitutive expression of dccA) or pECC17 and grown in the presence of 100 ng/ml ATc (inducible expression of dccA), fluorescence was not detected (data not shown). Phase contrast microscopy revealed that cells carrying pECC12 grew as chains compared with the wild-type strain carrying an empty vector (Fig. 9A). These chains contained incomplete cell septa as visualized by staining with MitoTracker Green, suggesting that increased intracellular levels of c-diGMP result in aberrant cell division. Chains of cells were also observed in the strain carrying pECC17 upon induction with 100 ng/ml ATc, but to a much lesser extent (Fig. 9B). Indeed, in the strain carrying pECC17, chain length was found to correlate to the inducer concentration. Thus in C. difficile 630, formation of long chains of cells is likely the direct consequence of the elevation of c-diGMP levels. The absence of CD2831 immunolabeling at the surface of cells expressing high levels of c-diGMP might be related to the formation of chains or to insufficient levels of surface-localized CD2831. To explore this second hypothesis, the levels of CD2831 in strains 630 (pECC12) and 630 Δzmp1 (pJKP041) were compared. The levels of CD2831 were extremely low in 630 (pECC12) compared with 630 Δzmp1 (pJKP041), after induction with ATc, and were therefore probably too low to be detected by immunofluorescence (Fig. 8D).


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)

Elevated c-diGMP levels induce formation of long chain of cells in C. difficile 630.A, phase contrast (left and center panels) and fluorescence (right panel) microscopy images C. difficile wild-type (WT) strain carrying an empty plasmid (vector) or pECC12 (constitutively expresses DccA). For the fluorescence microscopy, membranes were stained with MitoTracker Green. White triangles indicate positions of septa. Scale bars, 20 μm. B, phase contrast microscopy views of C. difficile wild-type (WT) strain carrying pECC17 (expresses DccA in the presence of ATc). Expression of DccA from pECC17 was induced with the indicated concentrations of ATc. Black bars, 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 9: Elevated c-diGMP levels induce formation of long chain of cells in C. difficile 630.A, phase contrast (left and center panels) and fluorescence (right panel) microscopy images C. difficile wild-type (WT) strain carrying an empty plasmid (vector) or pECC12 (constitutively expresses DccA). For the fluorescence microscopy, membranes were stained with MitoTracker Green. White triangles indicate positions of septa. Scale bars, 20 μm. B, phase contrast microscopy views of C. difficile wild-type (WT) strain carrying pECC17 (expresses DccA in the presence of ATc). Expression of DccA from pECC17 was induced with the indicated concentrations of ATc. Black bars, 20 μm.
Mentions: Immunofluorescence microscopy analysis was performed to detect CD2831. In the wild-type strain carrying pECC12 (constitutive expression of dccA) or pECC17 and grown in the presence of 100 ng/ml ATc (inducible expression of dccA), fluorescence was not detected (data not shown). Phase contrast microscopy revealed that cells carrying pECC12 grew as chains compared with the wild-type strain carrying an empty vector (Fig. 9A). These chains contained incomplete cell septa as visualized by staining with MitoTracker Green, suggesting that increased intracellular levels of c-diGMP result in aberrant cell division. Chains of cells were also observed in the strain carrying pECC17 upon induction with 100 ng/ml ATc, but to a much lesser extent (Fig. 9B). Indeed, in the strain carrying pECC17, chain length was found to correlate to the inducer concentration. Thus in C. difficile 630, formation of long chains of cells is likely the direct consequence of the elevation of c-diGMP levels. The absence of CD2831 immunolabeling at the surface of cells expressing high levels of c-diGMP might be related to the formation of chains or to insufficient levels of surface-localized CD2831. To explore this second hypothesis, the levels of CD2831 in strains 630 (pECC12) and 630 Δzmp1 (pJKP041) were compared. The levels of CD2831 were extremely low in 630 (pECC12) compared with 630 Δzmp1 (pJKP041), after induction with ATc, and were therefore probably too low to be detected by immunofluorescence (Fig. 8D).

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