Thomas, P. S. | bioRxiv Science

bioRxiv2026

Chemical tools to Detect and Inhibit IgA1 Proteases in Haemophilus influenzae

Non-encapsulated ("non-typeable") Haemophilus influenzae is a major cause of mucosal infections such as otitis media, conjunctivitis, and exacerbations of chronic obstructive pulmonary disease. Rising antibiotic resistance has increased interest in anti-virulence strategies that reduce pathogenicity without exerting selective pressure for resistance. A key virulence factor of H. influenzae is immunoglobulin A1 protease (IgA1P), a secreted serine protease that promotes colonization by cleaving human IgA1 at the hinge region and enabling immune evasion. Despite its therapeutic promise, progress has been limited by the lack of chemical tools to probe IgA1P activity in its native biological environment. Here, we report the first activity-based probes that enable direct detection of active IgA1Ps in complex samples and H. influenzae clinical isolates. Competitive activity-based screening using these probes identified novel IgA1P inhibitors, and structure-activity relationship guided optimization yielded a potent lead IgA1P inhibitor. This inhibitor preserves IgA1 at the bacterial surface, demonstrating effective inhibition of IgA1P activity in its native context and chemical disruption of an IgA1P-dependent immune-evasion mechanism. Together, these chemical tools establish a versatile platform for elucidating IgA1Ps role in H. influenzae colonization and virulence, and for validating IgA1Ps as diagnostic markers and antivirulence targets.

microbiology↗

bioRxiv2024

Using BONCAT To Dissect The Proteome Of S. aureus Persisters

Bacterial persisters are a subpopulation of cells that exhibit a transient non-susceptible phenotype in the presence of bactericidal antibiotic concentrations. This phenotype can lead to the survival and regrowth of bacteria after treatment, resulting in relapse of infections. As such, it is also a contributing factor to antibacterial resistance. Multiple processes are believed to cause persister formation, yet identifying the proteins expressed during the induction of the persister state has been difficult, because the persister-state is rare, transient and does not lead to genetic changes. In this study, we used Bio-Orthogonal Non-Canonical Amino Acid Tagging (BONCAT) to label, and retrieve, the proteome expressed during the persister state for different strains of methicillin-resistant Staphylococcus aureus. After incubating antibiotic-exposed bacteria with the methionine ortholog L-azidohomoalanine to label the proteins of persister cells, we retrieved labeled proteins using click chemistry-pulldown methodology. Analysis of the retrieved proteome fraction of Methicillin resistant Staphylococcus aureus (MRSA) and Vancomycin resistant Staphylococcus aureus (VRSA) under challenge with {beta}-lactam and fluoroquinolone antibiotics with Label Free Quantification - Liquid chromatography mass spectrometry (LFQ-LCMS) based proteomics reveals the upregulation of proteins involved in stringent response, cell wall biosynthesis, purine metabolism, ppGpp biosynthesis, two component systems (TCS), lipid metabolism, ABC transporters, D-alanine biosynthesis and L-proline degradation. Conversely, we observed a decline of proteins associated with amino acid biosynthesis and degradation, protein biosynthesis, protein modification, and carbohydrate metabolism, among others. These findings indicate that modification of translational activity in persister cells enables bacterial cells to induce an active defense to survive antibiotic pressure.

microbiology↗