Development of a High-Throughput Screen and Its Use in the Discovery of Streptococcus pneumoniae Immunoglobulin A1 Protease Inhibitors

Abstract

Streptococcus pneumoniae relies on a number of virulence factors, including immunoglobulin A1 protease (IgA1P), a Zn2+ metalloprotease produced on the extracellular surface of the bacteria, to promote pathogenic colonization. IgA1P exhibits a unique function, in that it catalyzes the proteolysis of human IgA1 at its hinge region to leave the bacterial cell surface masked by IgA1 Fab, enabling the bacteria to evade the host's immune system and adhere to host epithelial cells to promote colonization. Thus, S. pneumoniae IgA1P has emerged as a promising antibacterial target; however, the lack of an appropriate screening assay has limited the investigation of this metalloprotease virulence factor. Relying on electrostatics-mediated AuNP aggregation, we have designed a promising high-throughput colorimetric assay for IgA1P. By using this assay, we have uncovered inhibitors of the enzyme that should be useful in deciphering its role in pneumococcal colonization and virulence.

Gold Nanoparticles for In Vitro Diagnostics

“…76 Using a similar principle, AuNPs were used to detect Streptococcus pneumoniae immunoglobulin A1 protease (IgA1P) based on the electrostatics-mediated AuNP assembly. 77 IgA1P catalyzes the proteolysis of human IgA1 to yield IgA1 Fc α and positively charged lgA1 Fab α . The negatively charged AuNPs selectively bind to the positively charged lgA1 Fab α to produce a visual AuNPs aggregation.…”

LSPR‐based colorimetric biosensing for food quality and safety

“…The same assay has been utilized for the detection of class A β-lactamases, which are made by E. coli and Bacillus cereus (Jiang et al, 2009). The detection of enzyme immunoglobulin A1 (IgA1) protease made by Streptococcus pneumonia has been achieved using 20-nm citrateadsorbed AuNPs aggregated by the positively charged Ab-antigen fragment regions (Garner et al, 2013). As nonfunctionalized AuNPs are used, this assay is easy to perform.…”

How to Translate a Bioassay Into a Screening Assay for Natural Products: General Considerations and Implementation of Antimicrobial Screens

“…Another virulence factor targeted by recent screening campaigns is bacterial motility, for which a screening assay utilizing a miniaturized version of the classical soft agar method in combination with viability staining to distinguish growth inhibitors from motility inhibitors has been described [112]. In addition, bacteria-specific exoenzymes have inspired the development of target-based screening assays based on the detection of the cleavage products [113].…”