Proteus mirabilis ZapA Metalloprotease Degrades a Broad Spectrum of Substrates, Including Antimicrobial Peptides

Abstract

The 54-kDa extracellular metalloprotease ZapA is an important virulence factor of uropathogenic Proteus mirabilis. While ZapA has the ability to degrade host immunoglobulins (Igs), the dramatic attenuation of virulence in ZapA mutants suggests that this enzyme may have a broader spectrum of activity. This hypothesis was tested by in vitro assays with purified ZapA and an array of purified protein or peptide substrates. The data reveal that many proteins found in the urinary tract are substrates of ZapA proteolysis, including complement (C1q and C3), cell matrix (collagen, fibronectin, and laminin), and cytoskeletal proteins (actin and tubulin). Proteolysis of IgA and IgG was significantly enhanced by conditions that denatured the Igs. It was discovered that the antimicrobial peptides human ␤-defensin 1 (hBD1) and LL-37 are readily cleaved by the enzyme. To the best of our knowledge, this is the first report of a bacterial protease capable of cleaving hBD1, a component of the human renal tubule innate immune response. Proteolysis of hBD1 resulted in ca. six peptides, while proteolysis of LL-37 resulted in at least nine products. Matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis of the molecular masses of the reaction products indicated that ZapA preferred no distinct peptide bond. The antimicrobial activity of hBD1 and LL-37 was significantly reduced following ZapA treatment, suggesting that proteolysis results in inactivation of these peptides. The data suggest that a function of ZapA during urinary tract infections is the proteolysis of antimicrobial peptides associated with the innate immune response.

Burkholderia cenocepacia zinc metalloproteases influence resistance to antimicrobial peptides

“…Fewer studies have examined the activity of bacterial proteases against other antimicrobial peptides but Pr. mirabilis ZapA has been shown to degrade hbD-1 (Belas et al, 2004) and LasB has been shown to cleave SLPI (Sponer et al, 1991). Our studies have shown that at least one of the B. cenocepacia ZmpA and ZmpB proteases had activity against hbD-1, LL-37, elafin and SLPI.…”

“…Many proteases have been reported to degrade LL-37, including Proteus mirabilis ZapA (Belas et al, 2004), Staphlylococcus aureus aureolysin (SieprawskaLupa et al, 2004), Ps. aeruginosa LasB, Enterococcus faecalis gelatinase, Streptococcus pyogenes SpeB (Schmidtchen et al, 2002), and a Bacillus anthracis metalloprotease (Thwaite et al, 2006).…”

Cloning, characterization and molecular analysis of a metalloprotease from Proteus mirabilis

“…The substrate specificity of ZapA had been characterized using fluorescent peptides derived from bioactive peptides and the oxidized β-chain of insulin. As one of the most important virulence factors of P. mirabilis, the role of ZapA in pathogenesis was probably due to the destruction of IgA, antimicrobial peptides, bioactive molecules, and structural components of the host cells (Fernandes et al 2000;Anéas et al 2001;Belas et al 2004).…”

“…Metalloprotease ZapA from P. mirabilis degrades a broad spectrum of substrates, including IgA and antimicrobial peptides (Loomes et al 1990;Almogren et al 2003;Belas et al 2004). The substrate specificity of ZapA had been characterized using fluorescent peptides derived from bioactive peptides and the oxidized β-chain of insulin.…”

Comparison of the binding specificity of two bacterial metalloproteases, LasB of Pseudomonas aeruginosa and ZapA of Proteus mirabilis, using N-alpha mercaptoamide template-based inhibitor analogues

“…These metalloproteases are members of the thermolysin and serralysin families respectively. It has been demonstrated that each of these bacterial proteases functions as a virulence factor during the process of infection, each having a deleterious effect on the host including the degradation of a broad range of host tissue proteins, and biomolecules involved in innate immunity such as immunoglobulins, complement factors, antimicrobial peptides and cytokines [7][8][9][10][11][12]. In addition, LasB acts within the bacterial cell as a regulator of polysaccharide (alginate) secretion [13].…”