Surface Interactome in Streptococcus pyogenes

Surface proteins play a fundamental role in bacterial adaptation, survival, and proliferation. They sense the chemical and physical conditions of the external environment and send appropriate signals to the cytoplasmic compartment, control the in/out trafficking of a plethora of molecules and ions, serve as anchoring tools for adhesion, and promote biofilm formation. In pathogenic bacteria, they also participate in tissue colonization and invasion and contribute considerably to counteract the defense mechanisms of the host. As a consequence of their role in key biological processes, surface protein identification has become instrumental not only for the definition of the mechanisms underlying bacterial physiology and pathogenesis but also for the development of new antimicrobial therapeutic and prophylactic products. The precise elucidation of bacterial surface proteomes (“surfome”) is experimentally challenging. Contamination of the surface/membrane protein preparations with cytoplasmic proteins can prevent accurate proteome characterization both in qualitative and quantitative terms. Furthermore, functionally important complexes that may form on the bacterial surface can remain largely undetected because the experimental conditions used for sample preparation and analysis usually destroy noncovalent protein-protein interactions. In the last few years, new effective protocols for the identification and quantification of surface-associated proteins have been developed. They include in silico analysis of genome sequences combined with the use of antibodies specific for each predicted surface protein to confirm its surface exposure on whole bacterial cells (1–4), in vivo labeling of surface proteins coupled to mass spectrometry (5), protease “shaving” of bacterial surfaces and analysis of proteolytic peptides by mass spectrometry (6–9), and mass spectrometry analysis of outer membrane vesicles (10, 11). In regard to the identification of surface protein complexes, several experimental methods exist (12), the most commonly used being the yeast two-hybrid system (13, 14), the tandem affinity purification (tagging) approach combined with protein identification using mass spectrometry (15, 16) and protein microarray (17, 18). However, none of them have so far been exploited to decipher the interactions taking place at the bacterial surface. Therefore, whereas the number of bacterial “surfomes” determined with a sufficiently high degree of accuracy is growing, the characterization of “surface interactomes” remains a field almost completely unexplored. The aim of this study is to further our understanding of this field. Using Streptococcus pyogenes (group A Streptococcus (GAS))1 as a model system and taking advantage of the availability of its surfome at a good level of resolution (6),2 protein arrays of 83 surface-exposed proteins have been produced. The ability of these proteins to form complexes has then been investigated by probing the array with biotin-labeled derivatives of each of the spotted proteins. Some of the identified interactions have been validated by determining the kinetic and thermodynamic constants of interactions using surface plasmon resonance and by demonstrating co-localization of some the interacting proteins on the bacterial surface by confocal microscopy. Overall, the approach has unraveled a network of interactions taking place at the surface of GAS, interactions that might explain some fundamental mechanisms of the biology and virulence of this important human pathogen.