Adhesive polypeptides of Staphylococcus aureus identified using a novel secretion library technique in Escherichia coli

Abstract

Background: Bacterial adhesive proteins, called adhesins, are frequently the decisive factor in initiation of a bacterial infection. Characterization of such molecules is crucial for the understanding of bacterial pathogenesis, design of vaccines and development of antibacterial drugs. Because adhesins are frequently difficult to express, their characterization has often been hampered. Alternative expression methods developed for the analysis of adhesins, e.g. surface display techniques, suffer from various drawbacks and reports on high-level extracellular secretion of heterologous proteins in Gram-negative bacteria are scarce. These expression techniques are currently a field of active research. The purpose of the current study was to construct a convenient, new technique for identification of unknown bacterial adhesive polypeptides directly from the growth medium of the Escherichia coli host and to identify novel proteinaceous adhesins of the model organism Staphylococcus aureus.

Results: Randomly fragmented chromosomal DNA of S. aureus was cloned into a unique restriction site of our expression vector, which facilitates secretion of foreign FLAG-tagged polypeptides into the growth medium of E. coli ΔfliCΔfliD, to generate a library of 1663 clones expressing FLAG-tagged polypeptides. Sequence and bioinformatics analyses showed that in our example, the library covered approximately 32% of the S. aureus proteome. Polypeptides from the growth medium of the library clones were screened for binding to a selection of S. aureus target molecules and adhesive fragments of known staphylococcal adhesins (e.g coagulase and fibronectin-binding protein A) as well as polypeptides of novel function (e.g. a universal stress protein and phosphoribosylamino-imidazole carboxylase ATPase subunit) were detected. The results were further validated using purified His-tagged recombinant proteins of the corresponding fragments in enzyme-linked immunoassay and surface plasmon resonance analysis.

Conclusions: A new technique for identification of unknown bacterial adhesive polypeptides was constructed. Application of the method on S. aureus allowed us to identify three known adhesins and in addition, five new polypeptides binding to human plasma and extracellular matrix proteins. The method, here used on S. aureus, is convenient due to the use of soluble proteins from the growth medium and can in principle be applied to any bacterial species of interest.

Figure 1

Elements used in construction of the polypeptide secretion library of S. aureus in E. coli. A. Expression vector pSRP18/0 contains an expression cassette comprised of a 5' untranslated sequence upstream of the flagellin gene of E. coli MG1655 (fliC_MG1655) here indicated fliC5'UTR, a DNA fragment encoding the N-terminal 20 amino acids fliC_MG1655 (fliC1-60), a synthetic FLAG tag encoding sequence (flag) and a 3' untranslated region downstream of fliC_MG1655 (fliC3'UTR). EcoRV indicates the unique cloning site for foreign DNA fragments, horizontal arrows indicate the oligonucleotides used as primers for PCR (017F, 025F and 028R) and sequencing (017F and 071R) of the cloned inserts and black lines indicate sequences of the plasmid pBR322. SalI and BamHI indicate the restriction sites created during cloning of the expression cassette into pBR322. B. Agarose gel electrophoretic analysis of the chromosomal DNA isolated from S. aureus NCTC 8325-4 and used in generation of the library. The purified DNA is shown in the left lane, randomly fragmented and blunted DNA in the right lane. Ns indicate not sonicated and s stands for sonicated and polished DNA. The positions of molecular weight markers in base pairs are shown to the right.

Figure 2

Distribution of DNA fragments of the Flag-tag positive library clones on the S. aureus chromosome. The height of the bars represents the density of matches in windows of 4 kbp for the first sequence batch obtained with primer 017F (innermost circle) and the second sequence batch obtained using primer 071R (middle circle). The size of the chromosome is 2.82 Mbp (outermost circle); coordinates of the chromosome are indicated in Mbp.

Figure 3

Properties of polypeptides secreted into the growth medium by the Ftp library clones and purified His-recombinant polypeptides. A. Upper panel shows the binding of cell-free growth media from the library clones to ECM proteins and the control protein fetuin immobilized in polystyrene microtitre wells as analyzed by ELISA. Lower panel shows Western blot analysis with monoclonal anti-FLAG antibodies of bacterial cells (C) and TCA-precipitated cell-free growth media (S) of the corresponding clones. Vector indicates growth medium from MKS12 (pSRP18/0), D1-D3 denotes polypeptides secreted by MKS12 (pSRP18/0D1-D3), and the names indicate individual library clones. The cell samples correspond to 50 μl and the supernatants to 500 μl bacterial culture, except in the case of clones ΔPBP, ΔUsp, ΔIspD, ΔEbh where supernatant samples corresponding only to 100 μl of culture were loaded due to the high expression level of the polypeptide. B. Upper panel presents the binding of His-tagged recombinant polypeptides to ECM proteins immobilized in polystyrene microtiter wells as analyzed by ELISA and the lower panel shows SDS-PAGE analysis of affinity-purified recombinant polypeptides. The names following His-indicate polypeptides encoded by gene fragments subcloned from corresponding individual library clones. The values are averages of 2 to 3 parallels from 2 to 4 individual experiments, showing the standard deviation as error bars. CI, type I collagen; CIV, type IV collagen; Fn, fibronectin; Fg, fibrinogen; Fet, control protein fetuin. Molecular masses in kDa are indicated to the left.