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 Zn(2+) 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.

Figure 1

IgA1 cleavage by IgA1Ps. (a) IgA1P-catalyzed degradation of IgA1. Black = heavy chain, grey = light chain. (b) Sequence of amino acids in the hinge region of the α chain of IgA1 Fab and proposed cleavage site by S. pneumoniae IgA1P.

Figure 2

AuNP-based colorimetric IgA1P assay. (a) Assay design. Black = heavy chain, grey = light chain. (b) Proof-of- concept data. wt+ = D39 strain S. pneumoniae supernatant treated with secretory IgA. wt- = D39 strain S. pneumoniae supernatant without secretory IgA. wt/IgA2 = D39 strain S. pneumoniae supernatant treated with IgA2. KO/IgA1 = P1285 strain S. pneumoniae supernatant treated with secretory IgA. A₆₅₀/A₅₂₀ = raw ratiometric absorbance signal.

Figure 3

Characterization of AuNP assay for IgA1P-mediated IgA1 cleavage. A₆₅₀/A₅₂₀ = normalized ratiometric absorbance signal with respect to the negative control (wt-). (a) Time dependence. (b) Dependence on the concentration of secretory IgA. (c) Dependence on the volume of D39 strain S. pneumoniae supernatant containing IgA1P. (d) Z′ factor.

Figure 4

Identified inhibitors of S. pneumoniae IgA1P. (a) Natural product hits. (b) 3,4-HOPTO-based hits.