Molecular characterization of the interaction between human IgG and the M-related proteins from Streptococcus pyogenes.

Group A Streptococcal M-related proteins (Mrps) are dimeric α-helical-coiled-coil cell membrane-bound surface proteins. During infection, Mrp recruit the fragment crystallizable region of human immunoglobulin G via their A-repeat regions to the bacterial surface, conferring upon the bacteria enhanced phagocytosis resistance and augmented growth in human blood. However, Mrps show a high degree of sequence diversity, and it is currently not known whether this diversity affects the Mrp-IgG interaction. Herein, we report that diverse Mrps all bind human IgG subclasses with nanomolar affinity, with differences in affinity which ranged from 3.7 to 11.1 nM for mixed IgG. Using surface plasmon resonance, we confirmed Mrps display preferential IgG-subclass binding. All Mrps were found to have a significantly weaker affinity for IgG3 (p < 0.05) compared to all other IgG subclasses. Furthermore, plasma pulldown assays analyzed via Western blotting revealed that all Mrp were able to bind IgG in the presence of other serum proteins at both 25 °C and 37 °C. Finally, we report that dimeric Mrps bind to IgG with a 1:1 stoichiometry, enhancing our understanding of this important host-pathogen interaction.

Elucidating the Stoichiometries of Host-Pathogen Protein Interactions with Mass Photometry.

Mass photometry (MP) is a single molecule technique that enables the characterization of individual proteins. Here we show a detailed workflow using the Refeyn OneMP to investigate molecular complexes, using the M53 protein, a plasminogen-binding group A streptococcal M-like protein (PAM), and human plasminogen as exemplar proteins. The methodology described herein confirmed a 1:1 binding stoichiometry for the M53-plasminogen complex. Additionally, MP was used to identify the oligomerization state, homogeneity, purity, and approximate molecular weights of each protein.

Neuroserpin and transthyretin are extracellular chaperones that preferentially inhibit amyloid formation.

Neuroserpin is a secreted protease inhibitor known to inhibit amyloid formation by the Alzheimer's beta peptide (Aß). To test whether this effect was constrained to Aß, we used a range of in vitro assays to demonstrate that neuroserpin inhibits amyloid formation by several different proteins and protects against the associated cytotoxicity but, unlike other known chaperones, has a poor ability to inhibit amorphous protein aggregation. Collectively, these results suggest that neuroserpin has an unusual chaperone selectivity for intermediates on the amyloid-forming pathway. Bioinformatics analyses identified a highly conserved 14-residue region containing an α helix shared between neuroserpin and the thyroxine-transport protein transthyretin, and we subsequently demonstrated that transthyretin also preferentially inhibits amyloid formation. Last, we used rationally designed neuroserpin mutants to demonstrate a direct involvement of the conserved 14-mer region in its chaperone activity. Identification of this conserved region may prove useful in the future design of anti-amyloid reagents.

Current Understanding of Group A Streptococcal Biofilms.

BACKGROUND: It has been proposed that GAS may form biofilms. Biofilms are microbial communities that aggregate on a surface, and exist within a self-produced matrix of extracellular polymeric substances. Biofilms offer bacteria an increased survival advantage, in which bacteria persist, and resist host immunity and antimicrobial treatment. The biofilm phenotype has long been recognized as a virulence mechanism for many Gram-positive and Gram-negative bacteria, however very little is known about the role of biofilms in GAS pathogenesis. OBJECTIVE: This review provides an overview of the current knowledge of biofilms in GAS pathogenesis. This review assesses the evidence of GAS biofilm formation, the role of GAS virulence factors in GAS biofilm formation, modelling GAS biofilms, and discusses the polymicrobial nature of biofilms in the oropharynx in relation to GAS. CONCLUSION: Further study is needed to improve the current understanding of GAS as both a monospecies biofilm, and as a member of a polymicrobial biofilm. Improved modelling of GAS biofilm formation in settings closely mimicking in vivo conditions will ensure that biofilms generated in the lab closely reflect those occurring during clinical infection.