Surface interactome in Streptococcus pyogenes

Abstract

Very few studies have so far been dedicated to the systematic analysis of protein interactions occurring between surface and/or secreted proteins in bacteria. Such interactions are expected to play pivotal biological roles that deserve investigation. Taking advantage of the availability of a detailed map of surface and secreted proteins in Streptococcus pyogenes (group A Streptococcus (GAS)), we used protein array technology to define the "surface interactome" in this important human pathogen. Eighty-three proteins were spotted on glass slides in high density format, and each of the spotted proteins was probed for its capacity to interact with any of the immobilized proteins. A total of 146 interactions were identified, 25 of which classified as "reciprocal," namely, interactions that occur irrespective of which of the two partners was immobilized on the chip or in solution. Several of these interactions were validated by surface plasmon resonance and supported by confocal microscopy analysis of whole bacterial cells. By this approach, a number of interesting interactions have been discovered, including those occurring between OppA, DppA, PrsA, and TlpA, proteins known to be involved in protein folding and transport. These proteins, all localizing at the septum, might be part, together with HtrA, of the recently described ExPortal complex of GAS. Furthermore, SpeI was found to strongly interact with the metal transporters AdcA and Lmb. Because SpeI strictly requires zinc to exert its function, this finding provides evidence on how this superantigen, a major player in GAS pathogenesis, can acquire the metal in the host environment, where it is largely sequestered by carrier proteins. We believe that the approach proposed herein can lead to a deeper knowledge of the mechanisms underlying bacterial invasion, colonization, and pathogenesis.

Fig. 1.

Protein analysis and microarray validation. A, high throughput three-step protein purification using an AKTAxpress chromatography system yields proteins at 70–90% purity. Coomassie-stained SDS-PAGE gel of proteins was purified by AKTAxpress chromatography and used for microarray experiments. B, graphic representation of the BSA-Biotin control curve. Dots correspond to the different BSA-Biotin concentrations indicated on the x axis, whereas the continuous line corresponds to the interpolated resulting curve. MFI values are reported on the y axis. The graphic representation of the distribution of MFI values measured after incubation with Cy3-labeled streptavidin alone (negative control) is shown in the right panel. C, analysis of the biotinylated recombinant protein Spy1007 by mass spectrometry. MALDI-TOF MS spectra of the modified/unmodified protein are reported in the left panel. Spectra of unmodified (lower spectrum) and modified recombinant Spy1007 (upper spectrum) were acquired in linear mode. The singly charged monomer [MH]⁺ (1) observed from the unmodified protein is in agreement with the theoretical mass of the protein (26.8 kDa). After the biotinylation reaction, the protein was observed with singly charged monomers [MH]⁺ in agreement with the masses of the unmodified protein and with the mass of the protein increased by one, two, or three biotinylation adducts. The mass difference between the [MH]⁺ species is in agreement with the biotinylation adduct (669.75 Da). The peptide mass fingerprint of the biotinylated protein is reported in the right panel. The protein was digested with the endoprotease LysC, and the generated peptides were analyzed by MALDI-TOF MS in reflectron mode. The biotinylated peptides are assigned with a dot, the mass shift caused by the biotinylation adduct is indicated with an arrow. The three biotinylated lysines identified are indicated in red in the protein sequence reported in the lower panel. The identified peptide sequences are assigned in bold type. D, validation of protein-protein interaction experiments. A microarray was probed with biotinylated human fibronectin, and interactions were visualized by incubating the array with Cy3-labeled streptavidin and fluorescence scanning. After data processing, four proteins had signals above background.

Fig. 2.

Interactions between S. pyogenes surface-exposed or secreted proteins. The networks of interactions were visualized using Cytoscape (52). The nodes represent proteins, whereas each edge represents an interaction between the two proteins. Nodes of reciprocal interactions are indicated by blue-filled circles joined by red lines.

Fig. 3.

Networks representing all the reciprocal interactions identified. The table on the left reports the NCBI annotation for each protein involved in a reciprocal interaction.

Fig. 4.

SPR analysis of SpeI interactions with AdcA and Lmb. The SpeI, AdcA, and Lmb proteins purified in a tag-less form were immobilized on a carboxymethylated dextran-coated (CM5) sensor chip by amine coupling. Kinetics experiments were performed by injecting an increasing concentration of analyte protein in HBS-N in the presence of 5 μm Zn²⁺ over the sensor chip surface for 3 min at a flow rate of 20 μl/min. Complexes were left to dissociate for 500 s. The curves corresponding to three intermediate concentrations of analyte protein are shown. The presence of 10 mm EDTA abrogated binding for all samples. k_on, k_off, and K_D were calculated with the 1:1 Langmuir model using BiaEvaluation 4.1.

Fig. 5.

SpeI and its interactors localize to similar districts of the bacterial surface. S. pyogenes strain 3348 cells were treated with trypsin, washed, and inoculated in medium without trypsin for 0, 30, or 60 min before fixation (see “Experimental Procedures”). The cells were stained for M protein, SpeI, TlpA, or AdcA using goat anti-rabbit antibodies (green) and for cell wall peptidoglycan with biotinylated wheat germ agglutinin (red). For each sample, merging of the two images is shown in the bottom panel.

Fig. 6.

SpeI and its interactors localize in close proximity at the bacterial septum. PLA staining of complexes was performed as described under “Experimental Procedures.” A, control bacteria; B, SpeI-AdcA; C, SpeI-TlpA; D, SpeI-OppA. For each sample, merging of the two images is shown in the right panel.

Fig. 7.

Model of the acquisition of zinc ions by SpeI. a) Cartoon showing how interaction of SpeI with the substrate binding subunit of the Zn²⁺ transporter could occur at the site of SpeI export. b) Acquisition of Zn²⁺ ions by SpeI would subsequently result in SpeI dimer formation and binding to MHC II and TcR (36).