The cell envelope-bound metalloprotease (camelysin) from Bacillus cereus is a possible pathogenic factor

Abstract

A novel membrane proteinase of the nosocomial important bacteria species Bacillus cereus (synonyms: camelysin, CCMP) was purified up to homogeneity as was shown by mass spectrometry in its amphiphilic form. Camelysin is a neutral metalloprotease with a molecular mass of 19 kDa. Its unique N-terminus Phe-Phe-Ser-Asp-Lys-Glu-Val-Ser-Asn-Asn-Thr-Phe-Ala-Ala-Gly-Thr-Leu-Asp-Leu-Thr-Leu-Asn-Pro-Lys-Thr-Leu-Val-Asp-(Ile-Lys-Asp)- was not detected in the protein data bases during BLAST searches, but in the partially sequenced genome of Bacillus anthracis, coding for an unknown protein. Cleavage sites of the membrane proteinase for the insulin A- and B-chains were determined by mass spectrometry and N-terminal sequencing. Camelysin prefers cleavage sites in front of aliphatic and hydrophilic amino acid residues (-OH, -SO3H, amido group), avoiding bulky aromatic residues. The internally quenched fluorogenic substrates of the matrix metalloproteases 2 and 7 were cleaved with the highest efficiency at the Leu-decrease-Gly or Leu-decrease-Ala bond with the smaller residue in the P1' position. The protein specificity is broad--all various kinds of casein were cleaved as well as acid-soluble collagen, globin and ovalbumin; intact insulin was destroyed only to a low extent. Actin, collagen type I, fibrinogen, fibrin, alpha2-antiplasmin and alpha1-antitrypsin were cleaved. The protease formed SDS-stable complexes with Glu-plasminogen and antithrombin III, visible after SDS electrophoresis by gold staining and Western blot. The CCMP-plasminogen complex caused a partial activation of plasminogen to plasmin. Camelysin interacts with proteins of the blood coagulation cascade and could facilitate the penetration of fibrin clots and of the extracellular matrix during bacterial invasion.

Purification of Bovine Bone Oligophosphopeptide with High Calcium-binding Activity by <i>Bacillus cereus </i>MBL13 Collagenolytic Protease

“…Bacterial collagenolytic proteases in various forms have found practical applications worldwide, such as additives to laundry detergents and experimental reagents in laboratory work [21,22]. Several B. cereus species have been previously reported to produce collagenolytic proteases [23,24]. However, information on the specificity of collagenolytic proteases toward animal bones is limited.…”

Camelysin Is a Novel Surface Metalloproteinase from Bacillus cereus

“…The protease was purified in its detergent form. It was homogeneous in mass spectrometry (MS), with a molecular mass of 19,073.1 Ϯ 15 Da, and was called caseincleaving metalloproteinase or camelysin (16,17). The protein differs from known extra-and intracellular Bacillus proteinases in its N-terminal sequence, substrate specificity, and inhibition pattern.…”

“…The protein differs from known extra-and intracellular Bacillus proteinases in its N-terminal sequence, substrate specificity, and inhibition pattern. Camelysin preferentially cleaves peptide bonds in front of aliphatic hydrophobic amino acids and hydrophilic amino acid residues, avoiding bulky aromatic residues in the P 1 Ј position, and is therefore almost completely unable to release chromogenic and fluorogenic groups from this position (17). Camelysin belongs to the neutral metalloproteases, showing their typical strong inhibition by metal chelators (16), but it is insensitive to phosphoramidon or zincov, which are the strongest inhibitors of neutral metalloproteinases of the thermolysin-type (clan MA) (47).…”

“…Significant examples are the cell wall-anchored, complement C5a-cleaving peptidase from group B streptococci (8), the plasminogen-activating outer membrane proteases (OmpT) (54), and some members of the serine protease autotransporters (26,58) in gramnegative bacteria. Camelysin from B. cereus could have a similar significant role in host-pathogen interactions, because it cleaves serum protease inhibitors, collagen type I, fibrin, and fibrinogen and causes a partial activation of human plasminogen (17). Extension fragments with known DNA sequences were obtained by PCR.…”

Interactions of Francisella Tularensis With Components of the Host Fibrinolytic System

“…(77)(78), staphylococcal spp. (79)(80)(81)(82), and Bacillus anthracis (83)(84), as well as Gram-negative bacteria such as Borrelia spp. (85)(86)(87) have been shown the potential to augment their invasive capacity by interacting with such plasma proteins as fibrinogen, fibronectin, and/or PLG.…”