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Molecular analysis of a metalloprotease from Proteus mirabilis
1Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore 21202, USA.
Abstract:
Proteus mirabilis is known for its ability to differentiate from swimmer to swarmer cells, a process crucial for the pathogenesis of these bacteria during urinary tract infections. Among the many virulence factors produced during swarmer cell differentiation is an extracellular metalloprotease. A cosmid containing a large fragment of P. mirabilis chromosomal DNA was obtained by measuring protease expression in recombinant Escherichia coli. The recombinant and native enzymes were purified to over 95% homogeneity from culture supernatants by use of phenyl-Sepharose affinity chromatography and found to be identical. The activity of the 55-kDa enzyme was stimulated by divalent cations (Ca2+ > Mg2+) and inhibited by a chelator of these cations. The enzyme possesses substrate specificity for both serum and secretory forms of immunoglobulin A1 (IgA1) and IgA2 as well as IgG and, unlike classic IgA proteases, digested to completion both human and mouse IgA. Following subcloning, a 5-kb DNA fragment encoding recombinant protease activity was identified by insertional mutagenesis with Tn5. Four open reading frames were identified within this 5-kb region by limited nucleotide sequence analysis of DNA flanking the transposon. The nucleotide and deduced amino acid sequences of the metalloprotease structural gene (zapA) were obtained. Computerized homology studies revealed that the P. mirabilis metalloprotein is a member of the serralysin family of proteases and may be part of an operon comprising genes encoding an ATP-dependent ABC transporter in addition to the metalloprotease. The relevance of the metalloprotease to swarmer cell differentiation and pathogenicity is discussed.
Insights
Proteus mirabilis produces a metalloprotease essential for swarmer cell differentiation and urinary tract infections. This enzyme degrades immunoglobulin A (IgA) and immunoglobulin G (IgG), contributing to bacterial virulence.
Area of Science:
- Microbiology
- Bacterial Pathogenesis
- Enzymology
Background:
- Proteus mirabilis exhibits cell differentiation crucial for urinary tract infection (UTI) pathogenesis.
- Extracellular metalloproteases are key virulence factors during swarmer cell differentiation.
Purpose of the Study:
- To identify and characterize the extracellular metalloprotease involved in Proteus mirabilis swarmer cell differentiation.
- To elucidate the genetic basis and substrate specificity of this metalloprotease.
Main Methods:
- Cosmid cloning and expression in Escherichia coli for protease isolation.
- Purification using phenyl-Sepharose affinity chromatography.
- Enzyme activity assays, substrate specificity determination (IgA1, IgA2, IgG), and genetic analysis (Tn5 mutagenesis, DNA sequencing).
Main Results:
- A 55-kDa extracellular metalloprotease was purified and characterized.
- The enzyme's activity is dependent on divalent cations (Ca2+, Mg2+).
- It exhibits broad substrate specificity, degrading human and mouse IgA and IgG.
- The metalloprotease gene (zapA) was identified and sequenced, revealing homology to serralysin family proteases.
- The gene may be part of an operon including an ABC transporter.
Conclusions:
- The identified metalloprotease (ZapA) is a significant virulence factor in Proteus mirabilis.
- Its ability to degrade immunoglobulins contributes to bacterial survival and pathogenesis in UTIs.
- The enzyme's genetic organization suggests a coordinated system for its secretion and function.