Molecular analysis of a metalloprotease from Proteus mirabilis.
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.
Wassif
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Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore 21202, USA.
Cheek
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Belas
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002413:Cations, Divalent / Q000494:pharmacology; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D006801:Humans; D007132:Immunoglobulin Isotypes / Q000378:metabolism; D008666:Metalloendopeptidases / Q000145:classification / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D011513:Proteus mirabilis / Q000201:enzymology / Q000235:genetics*; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D002413:Cations, Divalent; D007132:Immunoglobulin Isotypes; D011994:Recombinant Proteins; D008666:Metalloendopeptidases; C095666:serralysin