G. Gowland

Partial Purification and Characterization of Lipase (EC 3.1.1.3) from Propionibacterium acnes

Lipase from Propionibacterium acnes has been purified 4800-fold from crude culture supernatant. The purified enzyme preparation had no assayable protease, hyaluronate lyase or acid phosphatase activities. The molecular weight of the lipase was 46,770 as determined by gel filtration. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed a major protein component (mol. wt 41,190) together with two minor protein components (mol. wt 67,000 and 125,900). The lipase had a pH optimum of 6.8, was most stable in the pH range 5.0 to 6.0 and was completely inactivated after 30 min at 60 degrees C. The lipase hydrolysed trilaurin, triolein, trimyristin and tripalmitin at decreasing rates and did not exhibit phospholipase activity. Analysis of the reaction products from the hydrolysis of triolein by P. acnes lipase did not demonstrate an accumulation of 2-monoolein which suggested that the enzyme did not exhibit a positional specificity for the 1-position of the triacylglycerol. Crude lipase preparations contained an aggregated high molecular weight form of the enzyme which was eluted with the void volume from Sephadex G-200. This aggregated form was dissociated to produce the lower molecular weight lipase species by subsequent dialysis and elution from Sephadex G-200 using buffer with a higher ionic strength.

Cellular immunity to P. acnes in the normal population and patients with acne vulgaris

Patients with varying degrees of acne, acne‐free adult controls and samples of cord blood were investigated for cell mediated immunity to P. acnes using a leukocyte migration inhibition test. Despite the fact that the mean migration index tended to decrease with acne severity, only the patients with severe acne showed cell‐mediated immunity. It is suggested that when cellular immunity arises it is a late event which may contribute to inflammation but is probably not a factor in its initiation.

Antibodies to P. acnes and P. acnes exocellular enzymes in the normal population at various ages and in patients with acne vulgaris

Total serum IgM and IgG agglutinins to P. acnes and neutralizing antibodies to P. acnes lipase, hyaluronate lyase and acid phosphatase were measured in normal individuals of different age groups. Agglutinins to P. acnes were detected in infants at 4 months of age and were present at a high level throughout life. A switch from predominantly IgM agglutinins in children, to IgG agglutinins in adults, occurred during adolescence. Anti‐P. acnes lipase antibodies were present in 20% of teenagers and 17–42% of adults. Anti‐P. acnes hyaluronate lyase antibodies were found in adults only (4–17%). Antibodies to acid phosphatase were not detected.

Purification and partial characterization of hyaluronate lyase (EC 4.2.2.1) from Propionibacterium acnes.

Hyaluronidase from Propionibacterium acnes has been purified 13,000-fold from the culture supernatant to homogeneity (as determined by polyacrylamide disc gel electrophoresis). The molecular weight of the purified enzyme was 85,110 as determined by gel filtration. The purified enzyme had a pH optimum at 6.4, was stable between pH 5 and 5.8 and was completely inactivated after 15 min at 50 degrees C. Preliminary studies suggested that the enzyme is active against chondroitin 4- and 6-sulphates, but not against dermatan sulphate. Analysis by paper chromatography of the reaction products from the degradation of hyaluronic acid by bacterial, testicular and P. acnes enzymes suggested that the P. acnes enzyme is similar in its mode of action to other bacterial hyaluronate lyases. The enzyme from P. acnes may thus be tentatively classified as a hyaluronate lyase.

Purification and Partial Characterization of an Acid Phosphatase (EC 3.1.3.2) Produced by Propionibacterium acnes

A strain of Propionibacterium acnes (type I; Marples & McGinley, 1974), isolated from a blackhead acne lesion, produced an acid phosphatase which was present in the culture supernatant in the late-exponential and early-stationary phases of growth. This acid phosphatase was purified more than 45 000-fold (4.5% yeild). The purified enzyme gave two protein bands on sodium dodecyl sulphate-polyacrylamide gel electrophoresis corresponding to molecular weights of 155 000 and 87 100. The enzyme had a single peak of activity on Sephadex G-100, with a molecular weight corresponding to 93 000. The highly purified acid phosphatase had an optimum activity at pH 5.8, was stable from pH 4.0 to 5.5 and was totally inactivated after 30 min at 55 degrees C. The enzyme did not show an absolute requirement for metal ions, but was stimulated by Mg2+, Ca2+, Zn2+ and K+ at concentrations between 0.1 and 1 mM. The acid phosphatase was active against a number of monophosphate esters.

Humoral responses to Malassezia furfur serovars A, B and C in normal individuals of various ages

A transferable solid‐phase (TSP) ELISA was developed for the determination of antibody titres specific to Malassezia furfur serovars A, B and C in human sera. A survey of levels of class‐specific antibodies (IgM, IgG and IgA) to M. furfur serovars A, B and C in relation to age (2–64 years: 60 individuals) demonstrated that individuals had immunity to M. furfur by the age of 2–3 years. There was no difference in either IgM or IgG levels into adulthood. The only age‐related differences were lower IgM titres to the three serovars in the 60–64 year age‐group compared with younger individuals. There was, however, a difference between titres of antibody specific to the three serovars. The mean reciprocal log2 IgM titre to serovar A (6·9) was significantly higher (P < 0·05) than that to serovar B (mean reciprocal log2 titre of 5·8), but not to serovar C (6·1). In contrast, the mean reciprocal log2 IgG titre to serovar A (6·5) was significantly lower (P <0 ·05) than those to serovars B and C (mean reciprocal log2 titre of 8·9 in both cases).

IgG subclasses in acne vulgaris

IgG subclasses were measured in male patients with very low grade or severe acne. No IgG subclass deficiencies were found. Patients with severe acne had a significant increase in total IgG attributable to their exposure to antigens which stimulate the production of antibodies in the IgG2 and IgG3 subclasses.

Difficulties in producing antibodies to purified Propionibacterium acnes exocellular enzymes

Attempts were made to produce antisera to purified preparations of Propionibacterium acnes lipase, hyaluronate lyase and acid phosphatase in rabbits. Antiserum to lipase (neutralizing titre 1:32) was produced using conventional methods. Lipase (30 μg) in Freunds complete adjuvant (FCA) was injected into multiple sites thrice at weekly intervals. Antibody levels were boosted by i.v. injections of 30 μg in saline at 2‐weekly intervals for 2 months. Such regimes failed to raise antibodies to hyaluronate lyase and acid phosphatase.