Cyclic diGMP regulates production of sortase substrates of Clostridium difficile and their surface exposure through ZmpI protease-mediated cleavage.
Bottom Line: The C-terminal PPKTG sorting motif of CD2831 is cleaved between the threonine and glycine residues, and the carboxyl group of threonine is amide-linked to the side chain amino group of diaminopimelic acid within the peptidoglycan peptide stem.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.
Affiliation: From the Department of Life Sciences, Center for Molecular Bacteriology and Infection, Imperial College London, London SW7 2AZ, United Kingdom.Show MeSH
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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).
Affiliation: From the Department of Life Sciences, Center for Molecular Bacteriology and Infection, Imperial College London, London SW7 2AZ, United Kingdom.