Abstract
Bacterial secreted proteins constitute a biologically important subset of proteins involved in key processes related to infection such as adhesion, colonization, and dissemination. Bacterial extracellular proteases, in particular, have attracted considerable attention, as they have been shown to be indispensable for bacterial virulence. Here, we analyzed the extracellular subproteome of Clostridium difficile and identified a hypothetical protein, CD2830, as a novel secreted metalloprotease. Following the identification of a CD2830 cleavage site in human HSP90β, a series of synthetic peptide substrates was used to identify the favorable CD2830 cleavage motif. This motif was characterized by a high prevalence of proline residues. Intriguingly, CD2830 has a preference for cleaving Pro-Pro bonds, unique among all hitherto described proteases. Strikingly, within the C. difficile proteome two putative adhesion molecules, CD2831 and CD3246, were identified that contain multiple CD2830 cleavage sites (13 in total). We subsequently found that CD2830 efficiently cleaves CD2831 between two prolines at all predicted cleavage sites. Moreover, native CD2830, secreted by live cells, cleaves endogenous CD2831 and CD3246. These findings highlight CD2830 as a highly specific endoproteinase with a preference for proline residues surrounding the scissile bond. Moreover, the efficient cleavage of two putative surface adhesion proteins points to a possible role of CD2830 in the regulation of C. difficile adhesion.
Highlights
We analyzed the extracellular subproteome of Clostridium difficile and identified a hypothetical protein, CD2830, as a novel secreted metalloprotease
After the initial identification of HSP90 as a substrate for CD2830, we identified a CD2830 cleavage motif using a synthetic peptide library screen
The identified CD2830 cleavage motif is unique and has hitherto not been described for any other protease [24, 25]
Summary
Chemicals Used—IgA was purchased from VWR International (Amsterdam, the Netherlands) (401098), and recombinant HSP90 (SPR102A) and recombinant HSP90␣ (SPR-101A) were obtained from Sanbio (Uden, the Netherlands). The PCR product was digested with NdeI and BamHI and ligated into vector pET16b (Novagen, Darmstadt, Germany), digested with NdeI and BamHI This resulted in the construction of a CD2830 expression vector containing 10 consecutive histidines at its N-terminus, replacing the signal sequence (supplemental Fig. S1B). After desalting on a reverse phase cartridge as described above, peptides were eluted using 50% acetonitrile, 0.1% formic acid and directly infused into the Q-TOF mass spectrometer (maXis, Bruker) with a syringe pump (flow rate ϭ 30 l/h). Identification of CD2830 Cleavage Products of CD2831 and CD3246 in Conditioned Medium—The conditioned minimal growth medium of C. difficile cells (10 ml of the supernatant after 30 min at 30,000g) was desalted on a reverse phase cartridge (C-18 Oasis HLB, 1 cc, 30 mg, Waters, Etten-Leur, the Netherlands), and peptides were eluted stepwise using 150 l of 20% and 50% acetonitrile in 0.1% formic acid. MS/MS data acquisition was continuously performed on m/z values corresponding to the expected peptides (m/z 680.8 (CD2831 peptide) and m/z 638.8 (CD3246 peptide))
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
