Structure-function analysis of peptide signaling in the Clostridium perfringens Agr-like quorum sensing system

Abstract

The accessory growth regulator (Agr)-like quorum sensing (QS) system of Clostridium perfringens controls the production of many toxins, including beta toxin (CPB). We previously showed (J. E. Vidal, M. Ma, J. Saputo, J. Garcia, F. A. Uzal, and B. A. McClane, Mol Microbiol 83:179-194, 2012, http://dx.doi.org/10.1111/j.1365-2958.2011.07925.x) that an 8-amino-acid, AgrD-derived peptide named 8-R upregulates CPB production by this QS system. The current study synthesized a series of small signaling peptides corresponding to sequences within the C. perfringens AgrD polypeptide to investigate the C. perfringens autoinducing peptide (AIP) structure-function relationship. When both linear and cyclic ring forms of these peptides were added to agrB null mutants of type B strain CN1795 or type C strain CN3685, the 5-amino-acid peptides, whether in a linear or ring (thiolactone or lactone) form, induced better signaling (more CPB production) than peptide 8-R for both C. perfringens strains. The 5-mer thiolactone ring peptide induced faster signaling than the 5-mer linear peptide. Strain-related variations in sensing these peptides were detected, with CN3685 sensing the synthetic peptides more strongly than CN1795. Consistent with those synthetic peptide results, Transwell coculture experiments showed that CN3685 exquisitely senses native AIP signals from other isolates (types A, B, C, and D), while CN1795 barely senses even its own AIP. Finally, a C. perfringens AgrD sequence-based peptide with a 6-amino-acid thiolactone ring interfered with CPB production by several C. perfringens strains, suggesting potential therapeutic applications. These results indicate that AIP signaling sensitivity and responsiveness vary among C. perfringens strains and suggest C. perfringens prefers a 5-mer AIP to initiate Agr signaling.

Importance: Clostridium perfringens possesses an Agr-like quorum sensing (QS) system that regulates virulence, sporulation, and toxin production. The current study used synthetic peptides to identify the structure-function relationship for the signaling peptide that activates this QS system. We found that a 5-mer peptide induces optimal signaling. Unlike other Agr systems, a linear version of this peptide (in addition to thiolactone and lactone versions) could induce signaling. Two C. perfringens strains were found to vary in sensitivity to these peptides. We also found that a 6-mer peptide can inhibit toxin production by some strains, suggesting therapeutic applications.

FIG 1

Peptides used in this study.

FIG 2

CPB production by CN3685 or a CN3685 agrB null mutant (top of panel A) versus CN1795 or a CN1795 agrB null mutant (bottom of panel A) in the presence of 100 μM concentrations of different peptides. (A) Wild-type CN3685, the CN3685agrBko null mutant, wild-type CN1795, and the CN1795agrBko null mutant were used to compare natural CPB production levels by overnight TGY cultures. The last lanes of both panels show purified CPB, with a molecular mass of 35 kDa, as a positive control. In all other lanes, the indicated peptides were added overnight to TGY broth inoculated with CN3685agrBko (top) or CN1795agrBko (bottom). The image shows representative results that were reproducible over three repetitions. (B) Quantitative analysis of CPB production using ImageJ software, from panel A CPB Western blots. Points without error bars had errors too small to depict.

FIG 3

Restoration of CPB production to agrB null mutants using different doses of peptides (5-R, 5-L, and 8-R). (A) Western blot showing CPB production by CN3685agrBko inoculated into TGY-containing peptides at a final concentration of 5 to 100 μM, followed by overnight incubation at 37°C. (B) Quantitative Western blot of CPB production, using ImageJ software, by overnight cultures supplemented with different concentrations of signaling peptides (5-R, 5-L, and 8-R). Asterisks indicate a statistically significant difference (P < 0.05) by a treatment compared to wild-type CPB production. Results are the means plus standard deviation (SD) from three repetitions. Points without error bars had errors too small to depict. (C) Western blot detection of CPB production by the CN3685 agrB null mutant or CN1795 agrB null mutant after overnight TGY culture in the presence of a final concentration of 25 μM signaling peptides. Purified CPB was used as a positive control.

FIG 4

Time course study of CPB production by CN1795agrBko and CN3685agrBko in the presence of peptides 5-R and 5-L. Signaling peptide (100 μM) was added to the TGY broth followed by the inoculation of bacteria at 37°C. Samples were collected at 3 h, 6 h, and 16 h (overnight), and those supernatants were then used for Western blot detection for CPB production. The kinetics of natural CPB production by TGY cultures of wild-type CN3685 or CN1795, without added peptide, are shown for comparison. All experiments were repeated three times.

FIG 5

Structure-function analysis of the signaling peptides. (A) Western blot detection of overnight CPB production by TGY cultures of the CN3685 and CN1795 agrB gene null mutants after activation by 100 μM concentrations of AgrD sequence-based peptides (5-R and 5-L) or variant peptides (5-RCS, 5-LCV, Ca5c, 6-L, and 6-R). A 6-mer random sequence peptide (6Ctrl) was used as a negative control. (B) CPB production by overnight TGY cultures of the CN3685 agrB null mutant and the CN1795 agrB null mutant after treatment with 100 μM concentrations of 5-R, 5-L, 5-RCS, 5-LCS, 5-LCV, Ca5c. A similar concentration of a 5-mer random sequence peptide (5Ctrl) was used as a negative control. These experiments were repeated twice.

FIG 6

Inactivation of the cpb gene in C. perfringens type B strain CN1795 using the TargeTron gene-knockout system. (A) PCR confirmation of the isogenic cpb gene null mutant CN1795cpbko. Using primers (cpbF/cpbR) (18) that target the specific cpb gene in C. perfringens, a PCR assay using primers to an internal cpb sequence amplified an ∼900-bp product from the isolated DNA of wild-type CN1795. However, PCR using the same primers amplified an ∼1.8-kb product from the isolated DNA of CN1795cpbko, which indicated the insertion of the ∼900-bp group II intron Ltr. Lane M, 1-kb molecular mass marker. (B) Southern blot hybridization of a DIG-labeled intron-specific probe with DNA purified from wild-type CN1795 and cpb null mutant CN1795cpbko. DNA from each strain was digested with EcoRI and electrophoresed on a 1% agarose gel prior to blotting and hybridization with the intron probe. (C) Western blot for CPB production by overnight TGY cultures of wild-type CN1795 and cpb null mutant CN1795cpbko using an anti-CPB antibody.

FIG 7

Transwell assay to determine if the natural signaling peptide produced by other C. perfringens strains can also activate the Agr-like system in CN3685agrBko and CN1795agrBko. The agrB null mutants (CN3685agrBko [A] and CN1795agrBko [B]) were inoculated into fresh TGY broth in the bottom chamber of Transwells as a recipient, while the donor strain was inoculated into fresh TGY broth in the top chamber. A membrane filter with 0.45-μm pores separated the two chambers, preventing bacterial passage between chambers but allowing the exchange of polypeptides. Donor strains included type A strain 13, SM101, type B strain CN1795cpbko, type C strain CN3685cpbko, and type D strains CN2068 and CN3718. The Transwell plate was anaerobically incubated overnight at 37°C, and culture supernatant was then collected for Western blot detection of CPB production. Wild-type strains CN3685 and CN1795 were used to compare CPB production in overnight TGY cultures. The image shows representative results that were reproducible over three repetitions.

FIG 8

Investigation of the possible inactivation of signaling peptides by CN1795 or CN3685. A 100 μM concentration of peptides 5-R or 5-L was incubated for 2 h with 100 μl of supernatant from TGY broth cultures of CN1795cpbko or CN3685cpbko, followed by adding 900 μl of fresh TGY broth. CN1795agrBko or CN3685agrBko was then inoculated into this mixture. After a 5-h anaerobic culture at 37°C, the culture supernatants were collected for CPB detection using Western blot analysis. (A) CPB production by CN1795agrBko. Wild-type strain CN1795 was used as a positive control, while CN1795agrBko was used as a negative control. (B) CPB production by CN3685agrBko. Wild-type strain CN3685 was used as a positive control, while CN3685agrBko was used as a negative control.

FIG 9

Peptide 6-R shows an inhibitory effect on the native signaling peptide produced by some C. perfringens strains. (A) Competition between native AIP and the synthetic signaling peptide 6-R to inhibit overnight CPB production by agrB null mutants of CN1795 (top) or CN3685 (bottom). (B) Effects of 6-R on CPB toxin production by other strains, including type B PS49, CN1793, Bar2, and CN2003 and type C Bar3, CN5383, CN3955, and CN3763. A final concentration of 100 μM 6-R was added into fresh TGY broth, followed by inoculation of these wild-type strains and overnight anaerobic incubation at 37°C. Bacterial culture in the absence of 6-R was used as a control. The presence of CPB in the overnight culture was detected by CPB Western blotting. (C) CPB production was upregulated after 2 h in the presence of Caco-2 host cells, and the 6-R peptide could inhibit this toxin upregulation.

FIG 10

Peptide 6-R can compete with 5-R in the agrB-agrD system. Final 100 or 250 μM concentrations of peptide 6-R, along with a 100 μM concentration of peptide 5-R, were added into fresh TGY broth, followed by inoculation of that broth with CN1795agrBko. The culture was anaerobically incubated overnight at 37°C. Culture supernatant was collected for detection of CPB using a Western blot assay. Cultures of wild-type CN1795, CN1795agrBko, CN1795agrBko with the addition of 100 μM 5-R, and CN1795agrBko with the presence of 100 μM 6-R were tested similarly as controls.