Streptococcus pyogenes Forms Serotype- and Local Environment-Dependent Interspecies Protein Complexes.

Streptococcus pyogenes is known to cause both mucosal and systemic infections in humans. In this study, we used a combination of quantitative and structural mass spectrometry techniques to determine the composition and structure of the interaction network formed between human plasma proteins and the surfaces of different S. pyogenes serotypes. Quantitative network analysis revealed that S. pyogenes forms serotype-specific interaction networks that are highly dependent on the domain arrangement of the surface-attached M protein. Subsequent structural mass spectrometry analysis and computational modeling of one of the M proteins, M28, revealed that the network structure changes across different host microenvironments. We report that M28 binds secretory IgA via two separate binding sites with high affinity in saliva. During vascular leakage mimicked by increasing plasma concentrations in saliva, the binding of secretory IgA was replaced by the binding of monomeric IgA and C4b-binding protein (C4BP). This indicates that an upsurge of C4BP in the local microenvironment due to damage to the mucosal membrane drives the binding of C4BP and monomeric IgA to M28. These results suggest that S. pyogenes has evolved to form microenvironment-dependent host-pathogen protein complexes to combat human immune surveillance during both mucosal and systemic infections. IMPORTANCE Streptococcus pyogenes (group A Streptococcus [GAS]), is a human-specific Gram-positive bacterium. Each year, the bacterium affects 700 million people globally, leading to 160,000 deaths. The clinical manifestations of S. pyogenes are diverse, ranging from mild and common infections like tonsillitis and impetigo to life-threatening systemic conditions such as sepsis and necrotizing fasciitis. S. pyogenes expresses multiple virulence factors on its surface to localize and initiate infections in humans. Among all these expressed virulence factors, the M protein is the most important antigen. In this study, we perform an in-depth characterization of the human protein interactions formed around one of the foremost human pathogens. This strategy allowed us to decipher the protein interaction networks around different S. pyogenes strains on a global scale and to compare and visualize how such interactions are mediated by M proteins.

Structural determination of Streptococcus pyogenes M1 protein interactions with human immunoglobulin G using integrative structural biology.

Streptococcus pyogenes (Group A streptococcus; GAS) is an important human pathogen responsible for mild to severe, life-threatening infections. GAS expresses a wide range of virulence factors, including the M family proteins. The M proteins allow the bacteria to evade parts of the human immune defenses by triggering the formation of a dense coat of plasma proteins surrounding the bacteria, including IgGs. However, the molecular level details of the M1-IgG interaction have remained unclear. Here, we characterized the structure and dynamics of this interaction interface in human plasma on the surface of live bacteria using integrative structural biology, combining cross-linking mass spectrometry and molecular dynamics (MD) simulations. We show that the primary interaction is formed between the S-domain of M1 and the conserved IgG Fc-domain. In addition, we show evidence for a so far uncharacterized interaction between the A-domain and the IgG Fc-domain. Both these interactions mimic the protein G-IgG interface of group C and G streptococcus. These findings underline a conserved scavenging mechanism used by GAS surface proteins that block the IgG-receptor (FcγR) to inhibit phagocytic killing. We additionally show that we can capture Fab-bound IgGs in a complex background and identify XLs between the constant region of the Fab-domain and certain regions of the M1 protein engaged in the Fab-mediated binding. Our results elucidate the M1-IgG interaction network involved in inhibition of phagocytosis and reveal important M1 peptides that can be further investigated as future vaccine targets.

Capacitive Sensor to Monitor Enzyme Activity by Following Degradation of Macromolecules in Real Time.

A capacitive sensor was developed to analyze the presence and enzymatic activity of a model protease from standard solutions by following the degradation of the substrate in real time. The enzyme was chosen based on its specific digestion of the hinge region of immunoglobulin G (IgG). Real-time enzyme activity was monitored by measuring the change in capacitance (∆C) based on the release of IgG fragments after enzymatic digestion by the enzyme. The results indicated that the developed capacitive system might be used successfully for label-free and real-time monitoring of enzymatic activity of different enzymes in a sensitive, rapid, and inexpensive manner in biotechnological, environmental, and clinical applications.

Capacitive Saccharide Sensor Based on Immobilized Phenylboronic Acid with Diol Specificity.

A capacitive sensor for saccharide detection is described in this study. The detection is based on selective interaction between diols and aminophenylboronic acid (APBA) immobilized on a gold electrode. Glucose, fructose, and dextran (MW: 40 kDa) were tested with the system over wide concentration ranges (1.0 x 10-8 M - 1.0 x 10-3 M for glucose, 1.0 x 10-8 M - 1.0 x 10-2 M for fructose and 1.0 x 10-10 M - 1.0 x 10-5 M for dextran). The limits of detection (LODs) were 0.8 nM for glucose, 0.6 nM for fructose, and 13 pM for dextran. These data were comparable to the others reported previously. In order to demonstrate glycoprotein detection with the same sensor, human immunoglobulin G (IgG) as well as horseradish peroxidase were used as model analytes. The sensor responded to IgG in the concentration range of 1.0 x 10-13 M - 1.0 x 10-7 M with a LOD value of 16 fM. The performance of the assay of peroxidase was compared to a spectrophotometric assay by determining the enzymatic activity of a captured analyte. The results showed that the method might be useful for label-free, fast, and sensitive detection of saccharides as well as glycoproteins over a wide concentration range.

A novel capacitive sensor based on molecularly imprinted nanoparticles as recognition elements.

Molecularly Imprinted Polymers (MIPs) are synthetic receptors capable of selective binding to their target (template) molecules and, hence, are used as recognition elements in assays and sensors as a replacement for relatively unstable enzymes and antibodies. Herein, we describe a manufacturing-friendly protocol for integration of MIP nanoparticles (nanoMIPs) with a (label-free) capacitive sensor. The nanoMIPs were produced by solid-phase synthesis for two templates with different sizes and properties, including a small molecule tetrahydrocannabinol (THC) and a protein (trypsin). NanoMIPs were deposited on the surface of the sensor and the change in capacitance (ΔC) upon binding of the target was measured. The significant improvement in the selectivity and limit of detection (one order of magnitude compared to previously used MIP microparticles) can be attributed to their increased surface-to-volume ratio and higher specificity of the nanoMIPs produced by the solid-phase method. The methodology described is also compatible with common sensor fabrication approaches, as opposed to methods involving in situ MIP polymerisation. The proposed sensor shows high selectivity, fast sensor response (45 min including injection, regeneration and re-equilibration with running buffer), and straightforward data analysis, which makes it viable for label-free monitoring in real-time. The set of targets assessed in this manuscript shows the general applicability of the biosensor platform.