Low molecular mass protein patterns in mycobacterial culture filtrates and purified protein derivatives.

Mycobacterial polypeptides from 2 kDa to 14 kDa may be involved in host response to infection and be useful for new diagnostics and vaccines. Tris-tricine SDS-polyacrylamide gel electrophoresis separation of proteins in tuberculin purified protein derivative (PPD) and in 6-8-week culture filtrates of Mycobacterium tuberculosis, Mycobacterium bovis BCG, and other Mycobacterium species demonstrated as many as 10 low molecular mass bands. Common and distinct bands were observed among different species and PPD. These low molecular mass culture filtrate proteins may represent potential diagnostic reagents and vaccines for Mycobacterium tuberculosis.

Identification of an elastolytic protease in stationary phase culture filtrates of M. tuberculosis.

Culture filtrate from M. tuberculosis grown in Sauton broth was screened at weekly intervals for elastase and caseinase activity. Both activities were detected concomitantly at 4 weeks and had similar inhibitor and pH profiles. Highest activity occurred between pH 6.5 and pH 7.5. Azocaseinase activity was linear with time between 12 and 28 h at 37 degrees C. Enzymatic activity was inhibited by EDTA, EGTA, dithiothreitol, ethylmaleimide, 1,10-phenanthroline, Zincov, N-tosyl-L-phenylalanine chloromethyl ketone, and N-methoxysuccinyl-Ala-Ala-Pro-Val chloromethyl ketone. SDS-PAGE analysis revealed breakdown of casein after incubation with culture filtrate. These results indicate the presence of a calcium-dependent, elastolytic metalloprotease in stationary phase cultures of M. tuberculosis.

Cross-reactivity of Shigella flexneri serotype 2a O antigen antibodies following immunization or infection.

To study the cross-reactivity pattern of Shigella flexneri 2a O-antigen antibodies, sera from humans and monkeys challenged with S. flexneri 2a, and from humans and guinea pigs immunized with a recombinant vaccine expressing serotype 2a O-antigen, were tested against a panel of lipopolysaccharide extracted from heterologous S. flexneri. Sera from the two groups of humans, who were volunteers in either a clinical challenge or vaccination study, showed similar patterns: cross-reactivity was more often seen with IgA antibodies, and these were mostly cross-reactive with serotype 2b, which shares the type II antigen, and serotypes 1a, 5a, and Y, which share 4 or 3, 4 group antigen, with 2a. The majority of sera from immunized guinea pigs showed both IgG and IgA cross-reactivity with 1a, 5a, and Y, but not 2b. The majority of sera from challenged monkeys showed cross-reactivity with almost all flexneri serotypes tested, with 1a, 2b, and Y being recognized most often, and the cross-reactive antibodies were more often IgG than IgA. These results show that either immunization or challenge with the 2a serotype induces cross-reactive antibodies which recognize similar subsets of heterologous serotypes, and suggest that it may be possible to design multivalent vaccines against S. flexneri.