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Studies on the Specific Soluble Protein of Pasteurella Pestis and Allied Organisms¹

I. Isolation, Fractionation and Certain Physical, Chemical and Serological Properties

From the Department of Epidemiology, All India Institute of Hygiene and Public Health, Calcutta

Abstract

1. The specific soluble proteins of virulent and avirulent plague and pseudotuberculosis bacilli were isolated from the filtrates of their growth in casein hydrolysate broth at 28 C for 24 days. The sodium or ammonium sulphate precipitate was found serologically more active than the acetic acid precipitate.

2. Five fractions were isolated from each filtrate by precipitating at one-third, half, half minus one one third, and full minus half saturation of sodium sulphate, the residue being taken as the last fraction. The fraction obtained by half saturation (P fraction) was serologically active and formed the bulk (75%) of the precipitate. The rest, including the residue, was serologically inactive. About 90 per cent of the active P fraction was constituted by P fraction (i.e. obtained by one-third saturation with Na₂SO₄) and the rest by P(–) fraction, both of which are serologically active.

3. Similar active protein fractions were also isolated from the water soluble extracts of the virulent plague organisms grown on casein hydrolysate agar at 37 C for 72 hours. The P fraction (called here L_auto P) formed the major bulk (80–90%) of the precipitate reacting with an antiplague horse serum in as high a dilution as 1,280,000. This fraction was found practically absent in the avirulent non-protective strains and completely absent in the pseudotuberculosis strains.

4. Certain chemical physical and serological properties of these protein fractions were studied. Their solubility was found low. The water extractable protein fraction were more soluble and contained higher nitrogen values than those obtained from the broth filtrates. They resembled nucleoprotein in character, but a difference was noted in the tryptophane content of the protein fractions recovered from different organisms. The protein solutions did not give any specific optical rotation but slight differences were noted between virulent and avirulent protective plague strains in the ultraviolet absorption curves.

5. The P fraction of the virulent and avirulent protective plague strains (called Antigen A) differed serologically from their P(–) fraction (called Antigen B) as well as from the P fractions of the non-protective avirulent plague and pseudotuberculosis strains, the latter two fractions behaving almost identically amongst themselves and cross-reacting with the antisera against antigen B and boiled virulent plague organism. It is considered that antigen A is the specific antigen of virulent plague bacillus corresponding to the envelope antigen of Schütze, and that antigen B acts as a common somatic link between the plague and pseudotuberculosis organisms.

6. The antigens A and B are both extractable from both filtrates whereas only antigen A is obtainable from water soluble extracts, the latter providing the most specific soluble antigen of the virulent plague bacillus. An antiserum prepared against this fraction (L_auto P) agglutinates only the protective plague strains to the complete exclusion of the pseudotuberculosis organisms.

7. The results also indicate that there is a progressive deterioration of the virulent plague strains due to subculture until they completely lose the specific antigen A, for the isolation of which a fully nutritive media as provided by the enriched casein hydrolysate broth or agar is necessary.

8. Further work on these protein fractions will be reported later.

Footnotes

¹ Most of this work was carried out in 1941–42 at the Haffkine Institute, Bombay and was presented before the 37th session of the Indian Science Congress Association held at Poona in January, 1950.