Cleavage of bacterial flagellin with proteolytic enzymes: II. Induction of enhanced delayed-type hypersensitivity to flagellin by peptic fragments of the protein

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Digestion of Salmonella adelaide flagellin (mol wt 40,000) with pepsin at suboptimal pH results in the release of 3–4 large, “pepsin-resistant peptides” (mol wt 13,000–15,000), which carry most of the antigenic determinants of the protein. These large peptides are slowly degraded by prolonged pepsin treatment into low molecular weight peptic peptides (mol wt < 4,000) which have little or no antigenic activity. A single injection into rats of the peptic digests of flagellin failed to produce detectable anti-flagellin antibodies, but was still capable of priming rats for a secondary antibody response to flagellin. This priming effect was mediated by the “pepsin-resistant peptides” present in the peptic digests, but not by the low molecular weight peptic peptides. In contrast to the antibody response, preparations of flagellin that had been subjected to prolonged peptic digestion induced higher levels of flagellin-specific delayed-type hypersensitivity than undigested flagellin. Furthermore, on fractionation of such digests, it was found that both the “pepsin-resistant peptides” and the low molecular weight peptic peptides induced delayed-type hypersensitivity to flagellin. A partial purification of the small peptic peptides which provoked delayed hypersensitivity was achieved by high voltage paper electrophoresis. The hypersensitivity inducing peptides were shown to be derived from regions of the flagellin molecule which carried antibody forming determinants. From the observation that degraded antigen preferentially induces delayed hypersensitivity, it is proposed that macrophage-degraded antigen plays an important role in regulating humoral and cell-mediated immunity in vivo. It is also suggested that degraded antigen favours the induction of delayed hypersensitivity due to its inability to promote cell-cell interaction between immunocompetent cells.