Understanding protease-mediated host-pathogen interactions in Proteus mirabilis
ZapA is a zinc-dependent metalloprotease secreted by Proteus mirabilis, a Gram-negative opportunistic pathogen that is primarily implicated in catheter-associated urinary tract infections (CAUTIs), which account for 80 % of all nosocomial infections. ZapA is an important virulence factor that contributes to the evasion of P. mirabilis from innate immunity via the hydrolysis of host structural proteins such as cytoskeletal and extracellular matrix components, and immunoproteins such as immunoglobulins, complement and antimicrobial peptides. The work in this thesis has further elucidated the immunomodulatory role of ZapA. Purification of native, proteolytically active ZapA from P. mirabilis strain BB2000-conditioned media via hydrophobic interaction chromatography (HIC) and subsequent coincubation with novel host substrates or with prostate and bladder cell lines in vitro shows that it degrades interleukins (ILs)-6 and -8 and putatively disarms protease-activated receptors (PARs) 1 and 2 (via enzyme-linked immunosorbent and calcium mobilisation assays, respectively). These cytokines and receptors are heavily involved in pro-inflammatory responses and therefore these data corroborate previous literature that demonstrate the immuno-evasive properties of ZapA. However, it was also found that ZapA proteolytically processes pro-IL-1ß into its mature active form, IL-1ß, another pro-inflammatory cytokine. Furthermore, it was found that ZapA degrades flagellin monomers; this may be particularly useful during swarming motility, in which P. mirabilis cells become hyperflagellated, to avoid Toll-like receptor 5 (TLR5)-mediated recognition. The N-alpha mercaptoamide dipeptide inhibitor, ME-YV, was included in these experiments and shown to prevent ZapA-mediated effects, validating the efficacy of ZapA as a target for the action of anti-virulence agents. Multiplex substrate profiling by mass spectrometry (MSP-MS) of ZapA and other Proteus spp. proteases has potentially facilitated the development of unique, highly specific substrates which could be applied as part of a point-of-care (POC) assay to rapidly diagnose urinary tract infections, demonstrating the efficacy of proteases as biomarkers.
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