Active immunization of NMRI mice against Serratia marcescens i. phenol-water lipopolysaccharide fractions and purified metalloproteases

The minimal intraperitoneal (i.p.) immunogenic doses of phenol-hot water lipopolysaccharide (P-W- LPS) preparations from three strains of Serratia marcescens for juvenile NMRI mice ranged from 3.2 to 16 ng following i.p. challenge infection with homologous strains. Dual autoclaving (121°C, 15 min) abolished the cross-immunogenicity of two selected P-W LPS extracts. Four purified metalloproteases from S. marcescens strains SF 178, SH 186, SV 01, and SE 182 shared the following properties: a) inhibition of proteolytic (azocasein hydrolysis) activity by 50 mM of EDTA; b) heat-lability (60°C, 15 min); c) identical molecular weights (54000 Daltons = 54 K) as documented with the SDS-PAGE procedure; d) close serologic relatedness (ELISA technique, polyclonal rabbit immune sera); and e) uniform reactivity of the 54 K polypeptide bands with polyclonal rabbit anti-metal-loprotease immune sera (Western blots). The minimal immunogenic dose of metalloprotease SF 178, the sole significantly murine immunogenic enzyme, was 1000 ng = 1 μg. Die minimale intraperitoneale (i.p.) immunogene Dosis von 3 Serratia marcescens Phenol-Heißwasser-Lipopolysaccharidfraktionen (P-W LPS) für juvenile NMRI Mäuse betrug 3,2 bis 16 ng. Zweimal autoklavierte (121 °C, 15 min) P-W LPS Extrakte waren nicht mehr kreuzimmunogen. Vier gereinigte Metalloproteasen der S. marcescens Stämme SF 178, SH 186, SV Ol und SE 182 zeigten folgende gemeinsame Eigenschaften: a) Inhibition der proteolytischen Aktivität durch 50 mM EDTA; b) Hitze-Labilität (60 °C, 15 min); c) identisches Molekulargewicht (54000 Daltons = 54 K) in SDS-PAGE Elektropherogrammen; d) enge serologische Verwandtschaft (ELISA-Verfahren, polyklonale anti-Metalloprotease Kaninchenimmunseren); e) einheitliche Reaktivität der 54 K Polypeptide mit polyklonalen Kaninchenimmunseren mittels Western blots. Die minimale murine immunogene Dosis der Metalloprotease SF 178 betrug 1000 ng = 1 μg.