Use of cell wall-hydrolytic enzymes in studies of the reticuloendothelial-stimulatory properties of Propionibacterium acnes

Vaccines prepared from whole cells of heat-killed Propionibacterium acnes were treated with a variety of enzymes. Only two enzymes, lysozyme and a bacteriolytic enzyme from the common European limpet, Patella vulgata, were able to abrogate the splenomegaly-inducing activity of vaccines. Inactivation of vaccine occurred without lysis of bacteria, only at high concentrations of lysozyme, and was reversed by subsequent treatment with trypsin, suggesting that lysozyme inactivation was due to a non-enzymatic adsorption of lysozyme to the bacterial surface.

The bacteriolytic enzyme from limpets was purified over 150-fold by preparative isoelectric focusing and named Patella vulgata lytic (PVL) enzyme. PVL enzyme activity in crude extracts could lyse many bacteria not lysed by lysozyme. The purified PVL enzyme had an isoelectric point of 8.3 and was a glycosidase which hydrolyzed the glycan backbone of peptidoglycan.

Treatment of vaccine with PVL enzyme abolished the splenomegaly-inducing activity of vaccine. An assay was developed to measure the ability of vaccine to inhibit the development of a transplantable tumor in BALB/c mice. Treatment of vaccine with PVL enzyme also abolished the antitumor activity of vaccine. Since PVL enzyme hydrolyzed peptidoglycan, it was concluded that intact peptidoglycan was essential to the splenomegaly-inducing and antitumor activities of P. acnes vaccine.

Formamide-extracted vaccines were as active as untreated vaccines in antitumor assays, and were also sensitive to lysis by lysozyme. Treatment of formamide-extracted vaccines with lysozyme abolished antitumor activity, indicating that peptidoglycan was responsible for the antitumor activity of formamide-extracted vaccines.

Trichloroacetic acid-extracted cell wall polysaccharide (TCA-PS) was compared with PVL enzyme-released cell wall polysaccharide (ERPS). Although antigenically similar, the ERPS had a higher molecular weight than TCA-PS, indicating that the TCA-PS had been hydrolyzed somewhat during acid-extraction and that ERPS is representative of the native cell wall polysaccharide. TCA-PS contained glucose, galactose, mannose, glucosamine, galactosamine, and small amounts of glycine and serine. ERPS contained the TCA-PS components and in addition, a small amount of lysine, and the peptidoglycan components muramic acid, alanine, glutamic acid, and diaminopimelic acid, and was therefore a complex of polysaccharide and peptidoglycan.