Biosynthesis of Staphylococcus aureus autoinducing peptides by using the synechocystis DnaB mini-intein - PubMed (original) (raw)

Biosynthesis of Staphylococcus aureus autoinducing peptides by using the synechocystis DnaB mini-intein

Cheryl L Malone et al. Appl Environ Microbiol. 2007 Oct.

Abstract

The Agr quorum-sensing system of Staphylococcus aureus modulates the expression of virulence factors in response to autoinducing peptides (AIPs). The peptides are seven to nine residues in length and have the C-terminal five residues constrained in a thiolactone ring. We have developed a new method to generate AIP structures using an engineered DnaB mini-intein from Synechocystis sp. strain PCC6803. In the method, an oligonucleotide encoding the AIP is ligated to the intein and the fusion protein is expressed and purified by affinity chromatography. To produce the correct AIP structure, intein splicing is interrupted, allowing the cysteine side chain to catalyze thiolactone ring formation and release AIP from the resin. The technique is simple and robust, and we have successfully produced the three main classes of AIPs using the intein system. The intein-generated AIPs possessed the correct thiolactone ring modification based on biochemical analysis, and, importantly, all the samples were bioactive against S. aureus. The AIP activity was confirmed through Agr interference and activation profiling with developed S. aureus reporter strains. The simplicity of the method, benefits of DNA encoding, and scalable nature enable the production of S. aureus AIPs for many biological applications.

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Figures

FIG. 1.

FIG. 1.

The four AIP signals of S. aureus and the cross-inhibitory groups. The amino acid sequence of each of the four AIPs is shown, and the signals are boxed into three inhibitory classes. AIP-I and AIP-IV differ by only one amino acid and function interchangeably.

FIG. 2.

FIG. 2.

Schematic of intein-catalyzed protein splicing and thiolactone formation. (A) Simplified version of the protein-splicing mechanism catalyzed by an intein. X, S or O from a cysteine, serine, or threonine side chain. Details of the mechanism have been reviewed elsewhere (27). (B) Interruption of intein splicing to generate peptide thiolactones. Following the N-S acyl shift, intramolecular attack from a cysteine side chain generates the thiolactone ring.

FIG. 3.

FIG. 3.

Schematic of the method for generating the S. aureus AIP signals using the DnaB mini-intein. First, an oligonucleotide encoding the AIP peptide is ligated at the 5′ end of the DnaB intein in plasmid pDnaB8. The construct is then expressed in E. coli, cells are lysed, and the fusion protein is purified on resin. The intein performs the N-S acyl shift, creating the thioester, allowing internal attack from a cysteine side chain to release the thiolactone-containing peptide. Elution fractions are then tested for biological activity with S. aureus reporter strains.

FIG. 4.

FIG. 4.

DnaB intein activity and AIP-I purification. (A) A DHFR protein fusion was used to test DnaB activity. Plasmids pDnaB8, pDnaB8-DHFR, and pET22-bsDHFR were expressed in strain AH394 with or without IPTG induction as indicated. Overexpressed bands corresponding to intein-CBD, DHFR-intein-CBD, and DHFR are shown. (B) Samples of an iAIP-I purification were separated by SDS-PAGE and probed with CBD antibody (shown on top). Gel lanes are as follows: SM, size marker; Un, uninduced; Ind, induced; FT-1, early flowthrough sample; FT-2, late flowthrough sample; resin, chitin resin.

FIG. 5.

FIG. 5.

Verification of the iAIP-I structure. Strain AH430 (Agr-II) served as the reporter for all the tests, and GFP readings were taken 12 h after sample addition. For testing, each sample was diluted 20-fold into the AH430 culture at the beginning of logarithmic phase. As controls, TSB and supernatant from SH1000 (AIP-I) were added to AH430. To test the DnaB intein method, iAIP-I was purified from strains AH426 (shown as iAIP-I) and AH425 (cysteine mutant; shown as Mut). As indicated, the samples were left untreated or, to check for the thiolactone ring, were treated with base or hydroxylamine.

FIG. 6.

FIG. 6.

Inhibition profiling with the iAIPs. For testing, each sample was diluted 20-fold into the appropriate reporter strain at the beginning of log phase. GFP fluorescence was monitored over time and compared to that of control samples of TSB and filtered supernatants from AIP-I-, AIP-II-, and AIP-III-producing strains. (A) Strain AH429 (Agr-I reporter). (B) Strain AH430 (Agr-II reporter). (C) Strain AH431 (Agr-III reporter).

FIG. 7.

FIG. 7.

Agr activation with the iAIPs. S. aureus Agr-I (AH462), Agr-II (AH430), Agr-III (AH431) reporter strains were grown in TSB with 0.2% glucose, and 50 nM iAIP was added at the beginning of logarithmic phase. Over time, GFP fluorescence was monitored and compared to controls without additions (TSB) or with competing iAIP signals. Activation or inhibition results with control S. aureus supernatants are not shown, but all yielded the same pattern as observed with the iAIPs.

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