Versatile Substrates and Probes for IgA1 Protease Activity

Abstract

Bacterial meningitis is a severe infectious disease with high mortality. Gram-positive and Gram-negative bacteria that cause meningitis secrete immunoglobulin A1 (IgA1) proteases to assist in mucosal colonization, invasion, and immune evasion. IgA1 proteases have unique selectivity, with few reported substrates other than IgA1 from human tissue. Here we describe the design, characterization, and application of peptide substrates for diverse IgA1 proteases from Neisseria, Haemophilus, and Streptococcus bacteria. IgA1 proteases from diverse strains showed unexpected selectivity profiles among peptide substrates derived from autoproteolytic sites. A fluorescence probe derived from one of these peptides was used to quantitate IgA1 protease activity in buffer and in human cerebrospinal fluid; it was able to detect recombinant Haemophilus influenzae type 1 IgA1 protease at less than 1 μg mL(-1) . We also used the probe to establish the first high-throughput screen for IgA1 protease inhibitors. This work provides tools that will help investigate the roles of IgA1 proteases in bacterial colonization, immune evasion, and infection.

A basis for vaccine development: Comparative characterization of Haemophilus influenzae outer membrane vesicles

“…Amongst others, we have identified the serine protease HtrA, an important virulence factor (Loosmore et al, 1998), as well as the vaccine candidates OMP 26 and protein D (Pichichero et al, 2010) to be present in OMVs derived from all strains tested. In contrast, other proteins like the protective surface antigen D15, a vaccine candidate (Loosmore et al, 1997), as well as the IgA1 protease, an important virulence factor (Choudary et al, 2013), were exclusively found in NTHi 2019-R OMVs.…”

Small-Molecule Inhibitors of Haemophilus influenzae IgA1 Protease

“…The limitations of existing drug discovery efforts targeting IgA proteases led us to explore alternative substrates as potential starting points for HTS. In prior work, we developed the first synthetic Förster resonance energy transfer (FRET) probe for monitoring IgA1P activity, molecule F2.2 (Figure c) …”

“…F2.2 incorporates a peptide sequence from an autoproteolytic site of Neisseria gonorrheae type 2 IgA1P. F2.2 is cleaved by a variety of IgA1Ps from Gram-negative pathogens, including H. influenzae . We previously demonstrated the compatibility of F2.2 with a 384-well HTS format using Neisseria meningitidis IgA1P.…”

“…In prior work, we developed the first synthetic Forster resonance energy transfer (FRET) probe for monitoring IgA1P activity, molecule F2.2 (Figure 1c). 30 F2.2 incorporates a peptide sequence from an autoproteolytic site of Neisseria gonorrheae type 2 IgA1P. F2.2 is cleaved by a variety of IgA1Ps from Gram-negative pathogens, including H. influenzae.…”

Microbial Evasion of IgA Functions

“…Studies using various hybrids of IgA as substrate show that most but not all IgA1 proteases will cleave a postproline peptide bond in one half of the IgA1 hinge, even when seen in the context of the IgA2 Fc (Senior et al, 2000). Fluorescent peptide probes for the rapid, sensitive, and quantitative detection of IgA1 protease activity were reported (Choudary et al, 2013).…”