Multiplexed on-yeast serological assay for immune escape screening of SARS-CoV-2 variants.

The emergence of the SARS-CoV-2 Omicron variant altered patient risk profiles and shifted the trajectory of the COVID-19 pandemic. Therefore, sensitive serological tests capable of analyzing patient IgG responses to multiple variants in parallel are highly desirable. Here, we present an adaptable serological test based on yeast surface display and serum biopanning that characterizes immune profiles against SARS-CoV-2 Wuhan (B lineage), Delta (B.1.617.2 lineage), and Omicron (B.1.1.529 lineage) receptor-binding domain (RBD) variants. We examined IgG titers from 30 serum samples from COVID-19-convalescent and vaccinated cohorts in Switzerland, and assessed the relative affinity of polyclonal serum IgG for RBD domains. We demonstrate that serum IgGs from patients recovered from severe COVID-19 between March-June 2021 bound tightly to both original Wuhan and Delta RBD variants, but failed to recognize Omicron RBDs, representing an affinity loss of >10- to 20-fold. Our yeast immunoassay is easily tailored, expandable and parallelized with newly emerging RBD variants.

Titrating Avidity of Yeast-Displayed Proteins Using a Transcriptional Regulator.

Yeast surface display is a valuable tool for protein engineering and directed evolution; however, significant variability in the copy number (i.e., avidity) of displayed variants on the yeast cell wall complicates screening and selection campaigns. Here, we report an engineered titratable display platform that modulates the avidity of Aga2-fusion proteins on the yeast cell wall dependent on the concentration of the anhydrotetracycline (aTc) inducer. Our design is based on a genomic Aga1 gene copy and an episomal Aga2-fusion construct both under the control of an aTc-dependent transcriptional regulator that enables stoichiometric and titratable expression, secretion, and display of Aga2-fusion proteins. We demonstrate tunable display levels over 2-3 orders of magnitude for various model proteins, including glucose oxidase enzyme variants, mechanostable dockerin-binding domains, and anti-PDL1 affibody domains. By regulating the copy number of displayed proteins, we demonstrate the effects of titratable avidity levels on several specific phenotypic activities, including enzyme activity and cell adhesion to surfaces under shear flow. Finally, we show that titrating down the display level allows yeast-based binding affinity measurements to be performed in a regime that avoids ligand depletion effects while maintaining small sample volumes, avoiding a well-known artifact in yeast-based binding assays. The ability to titrate the multivalency of proteins on the yeast cell wall through simple inducer control will benefit protein engineering and directed evolution methodology relying on yeast display for broad classes of therapeutic and diagnostic proteins of interest.

Adhesive Virulence Factors of Staphylococcus aureus Resist Digestion by Coagulation Proteases Thrombin and Plasmin.

Staphylococcus aureus (S. aureus) is an invasive and life-threatening pathogen that has undergone extensive coevolution with its mammalian hosts. Its molecular adaptations include elaborate mechanisms for immune escape and hijacking of the coagulation and fibrinolytic pathways. These capabilities are enacted by virulence factors including microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) and the plasminogen-activating enzyme staphylokinase (SAK). Despite the ability of S. aureus to modulate coagulation, until now the sensitivity of S. aureus virulence factors to digestion by proteases of the coagulation system was unknown. Here, we used protein engineering, biophysical assays, and mass spectrometry to study the susceptibility of S. aureus MSCRAMMs to proteolytic digestion by human thrombin, plasmin, and plasmin/SAK complexes. We found that MSCRAMMs were highly resistant to proteolysis, and that SAK binding to plasmin enhanced this resistance. We mapped thrombin, plasmin, and plasmin/SAK cleavage sites of nine MSCRAMMs and performed biophysical, bioinformatic, and stability analysis to understand structural and sequence features common to protease-susceptible sites. Overall, our study offers comprehensive digestion patterns of S. aureus MSCRAMMs by thrombin, plasmin, and plasmin/SAK complexes and paves the way for new studies into this resistance and virulence mechanism.

Optimal Sacrificial Domains in Mechanical Polyproteins: S. epidermidis Adhesins Are Tuned for Work Dissipation.

The opportunistic pathogen Staphylococcus epidermidis utilizes a multidomain surface adhesin protein to bind host components and adhere to tissues. While it is known that the interaction between the SdrG receptor and its fibrinopeptide target (FgB) is exceptionally mechanostable (∼2 nN), the influence of downstream B domains (B1 and B2) is unclear. Here, we studied the mechanical relationships between folded B domains and the SdrG receptor bound to FgB. We used protein engineering, single-molecule force spectroscopy (SMFS) with an atomic force microscope (AFM), and Monte Carlo simulations to understand how the mechanical properties of folded sacrificial domains, in general, can be optimally tuned to match the stability of a receptor-ligand complex. Analogous to macroscopic suspension systems, sacrificial shock absorber domains should neither be too weak nor too strong to optimally dissipate mechanical energy. We built artificial molecular shock absorber systems based on the nanobody (VHH) scaffold and studied the competition between domain unfolding and receptor unbinding. We quantitatively determined the optimal stability of shock absorbers that maximizes work dissipation on average for a given receptor and found that natural sacrificial domains from pathogenic S. epidermidis and Clostridium perfringens adhesins exhibit stabilities at or near this optimum within a specific range of loading rates. These findings demonstrate how tuning the stability of sacrificial domains in adhesive polyproteins can be used to maximize mechanical work dissipation and serve as an adhesion strategy by bacteria.

Engineered Molecular Therapeutics Targeting Fibrin and the Coagulation System: a Biophysical Perspective.

The coagulation cascade represents a sophisticated and highly choreographed series of molecular events taking place in the blood with important clinical implications. One key player in coagulation is fibrinogen, a highly abundant soluble blood protein that is processed by thrombin proteases at wound sites, triggering self-assembly of an insoluble protein hydrogel known as a fibrin clot. By forming the key protein component of blood clots, fibrin acts as a structural biomaterial with biophysical properties well suited to its role inhibiting fluid flow and maintaining hemostasis. Based on its clinical importance, fibrin is being investigated as a potentially valuable molecular target in the development of coagulation therapies. In this topical review, we summarize our current understanding of the coagulation cascade from a molecular, structural and biophysical perspective. We highlight single-molecule studies on proteins involved in blood coagulation and report on the current state of the art in directed evolution and molecular engineering of fibrin-targeted proteins and polymers for modulating coagulation. This biophysical overview will help acclimatize newcomers to the field and catalyze interdisciplinary work in biomolecular engineering toward the development of new therapies targeting fibrin and the coagulation system.