The Virulence-Enhancing Factor of Mucins 4. THE ROLE OF PARTICULATE INSOLUBLE MATTER AND A VISCOUS MEDIUM IN VIRULENCE ENHANCEMENT, WITH A REVISED ASSAY OF THE THIRD FACTOR INVOLVED BY H. SMITH,

In Part 3 (Smith, 1951) of this series a virulenceenhancing fraction of hog gastric mucin was separated from the insoluble particulate residue. This residue had an adjuvant action on the enhancement ofthe virulence by the activefactor comparable with that already demonstrated (Smith, 1950b) for the viscosity of the original mucin. However, using a biological assay involving a medium of constant viscosity for injection, no residue was obtained completelyfree from virulence-enhancing activity, even after autoclaving and washing with hot water. The suggestion was made, therefore, that a viscous medium, together with the particulate residue, could impede the host defence mechanisms by themselves, quite apart from their probable action in delaying the absorption by the host of the active factor. This suggestion is supported by the work of Olitzki & Koch (1945), who reported the adjuvant action of kaolin, fuller's earth, Norit A and talcum on the small virulence-enhancing activity of 0-3% agar for Shigella dysenteriae, Escherichia coli and Bacterium typhosum (Salmonella typhosa), and of Olitzki, Shelubsky & Hestrin (1946), who noted a similar action of kaolin on the activity of a number of carbohydrates, although the viscosities of the carbohydrate preparations used were not noted or measured. The work described in this paper shows that the suggestion is in fact true. Thus one can no longer refer to the virulenceenhancing factor ofhog gastric mucin; the activity is due to a combination ofthree factors, all ofwhich are slightly active alone, and none of which can be singled out as having a much greater share in the combined activity than the other two. Nev&rtheless, in contrast with the particulate insoluble matter and the viscous medium, the third factor is more specific. Knowledge of its chemical nature would be extremely interesting, especially for comparison with that of various bacterial products which have been shown to have virulence-enhancing activity (Olitzki, 1948). It was essential for further chemical fractionation to obtain a reliable assessment of this third factor, samples of which are still called (somewhat inaccurately) virulence-enhancing samples. Although an assay involving a simple injection of the sample, together with Bact. typhosum, would be desirable, this was found to be impossible; too large a quantity is needed to inject adequate batches ofmice with the relatively high concentrations required (approximately 1 %, w/v). A constant amount ofparticulate residue in a medium of constant viscosity is, therefore, combined with the third factor, which then need be used in concentrations of the order of only 0-025% (w/v). The necessary alterations in the technique of the assay are described in this paper.