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

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.

Enantioselective high-performance liquid chromatography of therapeutically relevant aminoalcohols as their fluorescent 1-naphthyl isocyanate derivatives.

A method for determining the enantiomers of 10 therapeutically relevant aminoalcohols using HPLC and precolumn derivatization was developed. Naphthyl isocyanate reacted with racemic aminoalcohols to form urea derivatives which were separated isocratically on a cellulose tris(3,5-dimethylphenylcarbamate) coated silica gel column, and detected fluorometrically in the lower ng mL(-1) range. The effluents can also be monitored at lower sensitivity, using an ultraviolet detector operated at 220 nm.

Characterization and purification of an outer membrane metalloproteinase from Pseudomonas aeruginosa with fibrinogenolytic activity.

A membrane proteinase from Pseudomonas aeruginosa, called insulin-cleaving membrane proteinase (ICMP), was located in the outer membrane leaflet of the cell envelope. The enzyme is expressed early in the logarithmic phase parallel to the bacterial growth during growth on peptide rich media. It is located with its active center facing to the outermost side of the cell, because its whole activity could be measured in intact cells. The very labile membrane proteinase was solubilized by non-ionic detergents (Nonidet P-40, Triton X-100) and purified in its amphiphilic form to apparent homogeneity in SDS-PAGE by copper chelate chromatography and two subsequent chromatographic steps on Red-Sepharose CL-4B (yield 58.3%, purification factor 776.3). It consisted of a single polypeptide chain with a molecular mass of 44.6 kDa, determined by mass spectrometry. ICMP was characterized to be a metalloprotease with pH-optimum in the neutral range. The ICMP readily hydrolyzed Glu(13)-Ala(14) and Tyr(16)-Leu(17) bonds in the insulin B-chain. Phe(25)-Tyr(26) and His(10)-Leu(11) were secondary cleavage sites suggesting a primary specificity of the enzyme for hydrophobic or aromatic residues at P'(1)-position. The ICMP differed from elastase, alkaline protease and LasA in its cleavage specificity, inhibition behavior and was immunologically diverse from elastase. The amino acid sequence of internal peptides showed no homologies with the known proteinases. This outer membrane proteinase was capable of specific cleavage of alpha and beta fibrinogen chains. Among the p-nitroanilide substrates tested, substrates of plasminogen activator, complement convertase and kallikrein with arginine residues in the P(1)-subsite were the substrates best accepted, but they were only cleaved at a very low rate.

Fusobacterium nucleatum pericarditis.

A pericardial effusion was diagnosed by echocardiography in a 49 year old man who suffered acute cough, orthopnea, and chest pain. Because of a positive tuberculin skin test, mycobacteria were initially suspected as the cause of the pericarditis. The patient was therefore treated with antituberculosis drugs. The pericardial effusion failed to resolve, however, and pericardiectomy was performed. Culture of the pericardial fluid yielded pure Fusobacterium nucleatum growth. The patient responded to antibiotic therapy and was in good health 3 weeks after being discharged from the hospital. This represents the first report of F. nucleatum pericarditis.

Identification of toxigenic Clostridium difficile by counterimmunoelectrophoresis.

A counterimmunoelectrophoresis (CIE) technique which reacted positively with culture filtrates of Clostridium difficile was developed and compared with a cytotoxicity assay in human embryonic lung cell cultures. CIE, employing C. sordellii antitoxin, detected 17 of 17 C. difficile strains. Of those positive by CIE, 13 were cytotoxic in cell culture. Fourteen Clostridium species other than C. difficile, C. sordellii, and C. bifermentans were negative by CIE. C. sordellii and C. bifermentans gave positive CIE results but were not cytotoxic. Similar sensitivity of toxin detection was observed for both methods. Optimal conditions for performing CIE included use of 48-h chopped meat-glucose broth cultures as the antigen source, use of a 10x-concentrated U.S. Standard C. sordillii antitoxin, and electrophoresis for 1.5 h in 0.05 M tris(hydroxymethyl)aminomethane-barbital-sodium barbital, pH 8.8, at a constant current of 6 mA/slide. CIE appears to be a suitable alternative to the cytotoxicity assay and may serve as a means for presumptive identification of C. difficile.

Rapid identification of group B streptococci by counterimmunoelectrophoresis.

Beta-hemolytic streptococcal isolates have been examined by counterimmunoelectrophoresis (CIE) with group B antiserum to determine whether this techinque is of value in the rapid identification of group B strains. Ninety stock cultures and 100 clinical isolates of beta-hemolytic streptococci including representatives of groups A, D, C, G, and B were inoculated into Todd-Hewitt broth; after incubation at 37 C for 1, 2, 3, and 4 h, aliquots of the whole broth cultures were removed and tested by CIE. Antigen was not regularly detected in the 1-, 2-, and 3-h samples, but after 4 h all 126 group B streptococcal strains identified by the capillary precipitin reaction gave CIE precipitin bands with group B antiserum. None of the 58 non-group B strains gave precipitin reactions with this antiserum. Cerebrospinal fluid from an infant with group B streptococcal meningitis and peritoneal fluid from a patient with group B streptococcal peritonitis had free group B antigen detected by the CIE technique. CIE of broth cultures and direct body fluids appears to be a rapid and sensitive method for the identification of group B streptococcal strains.