ZapA, a possible virulence factor from Proteus mirabilis exhibits broad protease substrate specificity

Abstract

The opportunistic bacterium Proteus mirabilis secretes a metalloprotease, ZapA, considered to be one of its virulence factors due to its IgA-degrading activity. However, the substrate specificity of this enzyme has not yet been fully characterized. In the present study we used fluorescent peptides derived from bioactive peptides and the oxidized ß-chain of insulin to determine the enzyme specificity. The bradykinin-and dynorphin-derived peptides were cleaved at the single bonds Phe-Ser and Phe-Leu, with catalytic efficiencies of 291 and 13 mM/s, respectively. Besides confirming already published cleavage sites, a novel cleavage site was determined for the ß-chain of insulin (Val-Asn). Both the natural and the recombinant enzyme displayed the same broad specificity, demonstrated by the presence of hydrophobic, hydrophilic, charged and uncharged amino acid residues at the scissile bonds. Native IgA, however, was resistant to hydrolysis by ZapA.

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

“…Many strains of P. mirabilis from a variety of sources have been reported to secrete EDTA-sensitive metalloproteases (Loomes et al 1992;Wassif et al 1995;Anéas et al 2001). In this study, ZapA from Pm7-a strain isolated from marine environments-was reconstituted and purified from BL21(DE3) for the first time.…”

“…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).…”

Identification of Natural Target Proteins Indicates Functions of a Serralysin-Type Metalloprotease, PrtA, in Anti-Immune Mechanisms

“…We found altogether 16 PAT proteins, most of which are minor components of the hemolymph. PrtA readily and selectively cleaved these and, as judged by the high substrate/proteinase ratios and the relatively short reaction times, their sensitivity to PrtA was substantially higher than those of several, partly immune-related proteins to other serralysin-type enzymes, ZapA of Proteus mirabilis (1,2,35,38) and serralysin of S. marcescens (28,29). These features and the fact that M. sexta is a potential host for the deadly insect pathogen Photorhabdus luminescens, which secrets PrtA early during infection, suggest that the cleavage of PAT proteins might have a role in virulence.…”

“…There are a number of enzymes in M10B, also called serralysins, which are secreted by a wide range of microorganisms, including plant and human pathogens. Serralysin-type proteases are generally supposed to be nonspecific enzymes, involved in, e.g., bioconversion of the host tissues, because they cleave with a relaxed residue preference in a variety of synthetic peptide substrates and denatured oligopeptides of biological origin (1,2,9,22,23,25,30). However, serralysins, like most of the proteases, encounter substrate proteins under physiological conditions, where they are in native conformation.…”

“…Due to this, the generally supposed virulence factor role of serralysins has still not been confirmed experimentally. The only observation in this regard is the in vitro cleavage of immunoglobulin A and G proteins and several human defensins, as well as some cell matrix and interconnecting filament proteins, by ZapA of Proteus mirabilis (1,2,35,38) and serralysin of Serratia marcescens (28,29). However, the in vivo significance of these reactions, too, remains to be established because the applied conditions, a large enzyme/substrate molar ratio and the very long incubation time (1:10 to 1:6,000 and 3 to 24 h, respectively), do not indicate the sensitive cleavage which might be expected in the case of specific target proteins.…”