Neuraminidase Production by Propionibacterium acnes-Strains Isolated from Patients with Acne vulgaris, Seborrheic Eczema and Healthy Subjects

Zbl. Bakt. Hyg., I.Abt. Orig. Or ig. A 250, 122-126 (1981) Hygiene-Institut (Direktorr Prof. Dr. G.Pu/verer), H ygiene-Institut der Universitat Universirar Koln (Direkto G. Pulverer), Petzold) und Hautklinik der Universitat Heidelberg (Direktor Prof. Dr. med. D. D.Petzold) Hir Physiologische Chemie der Universitat Dr.. F. Zilliken) Institut fur Universirat Bonn (Direktor Prof. Dr

Neuraminidase Production by Propionibacterium acnes-Strains Isolated from Patients with Acne vulgaris, Seborrheic Eczema and Healthy Subjects Neuraminidase-Produktion bei Propionibacterium acnes-Stammen von Patienten mit Acne vulgaris, Seborrhoischem Ekzem und hautgesunden Vergleichspersonen U. U. HbFFLER, M. GLOOR, and H. v. NICOLAI

Rt:ceived March 12, 1981 Received

Abstract neuraminidase Among cutaneous propionibacteria, pr opion ibacteria, synthesis of neura minidase is highest in strains of thee species P. acnes. th acnes. In the present present study, neuraminidase neuraminida se activity act ivity was discovered in 90.0% of P. acnes strains stra ins isolated from from acne lesions compared with 72.7% of strains from from normal human skin. skin. Neuraminidase-positive lesions, Neuraminidase-po sitive strains from acne lesion s, moreover, produced statisti/ml bacterial suspension) than cally significant higher amounts of the enzyme (X == 727 mU mUfml isolates from from normal skin (X == 392 mU /ml). Whereas the moderate production of neuramimUfml). nidase by strains from pati patients seborrheic probably causative ents with seborr heic eczema is pr obably of no cau sati ve importance, hass to be discussed as a potential etio etiologic vulgaris. po rt ance, the enzyme ha logic factor in acne vulgar is.

Zusammenfassung Von allen Propionibakterienarten der residenten menschlichen Hautflora bilden Stamme Stamme der Spezies P. acnes acnes am haufigsten Neuraminidase. In der vorl vorliegenden iegenden Arbeit konnte bei 90,0% der aus Acne-vulgaris-Effloreszenzen Acne-vulgaris-Effloreszcnzen isolierten P. acnes-Stamme eine genuine Neuraminidaseproduktion nachgewiesen werden, wahrend 72,7% der Stamme Starnme von gesunder menschlicher Haut ddieses ieses Enzym bildeten. Neuraminidase-positive Neu ram inid ase-positive Stamme Stamrne von AkneHaut produzierten zudem statistisch signifikant hohere Enzymmengen (X = 727 mU/ml mUfml Bakteriensuspension) /ml). Wahrend die maRig Bakterien suspen sion) als Isolate von gesunder Haut (X = 392 mU mUfml). malSig hohee Neuraminidasebildung bci bei Sta Stammen Patient en mit sebo seborrhoischem hoh mmen von Parienten rrhois chem Ekzem wahrscheinlich ohne pathogenetische Bedeutung ist, mulS muR das Enzym bei der Acne vulgaris als sche inlich ohne ein atiologisch werden. atio logisch potentiell bedeutsamer bcdeut samer Faktor diskutiert werd en .

Neuraminidase production by Propionibacterium acnes-Strains

123

Introduction Two species of propionibacteria, i,i. e. P. acnes and P. granulosum, fastidious slowgrowing anaerobic coryneforms, are accused of playing a significant role in the pathogenetic process of acne vulgaris (3,9,18). P. acnes is dominant in both prevalence on different skin regions and density of population, especially in areas rich produces an extracellular lipase, cleaving about one third in sebum. The bacterium produces during their passage through the piloseof sebum triglycerides into free fatty acids during (4). In adbaceous duct. The resulting fatty acids are reported to be comedogenic (4). dition, propionibacteria are probably involved in the inflammatory reaction of acne lesions. Controversy exists over the question of whether the production of extracellular enzymes and toxins, or serological processes are of preceding importance bacteria may be involved in the etiology (5,18). Moreover, anaerobic coryneform bacteria of seborrheic eczema (dysseborrheic dermatitis). The numbers of P. acnes in the nonaffected skin of patients with seborrheic eczema are diminished, compared with (6). healthy persons of the same age and sex (6). The production of neuraminidase (acylneuraminosyl hydrolase, sialidase, EC 3.2.1.18) by strains of P. acnes, first discovered by Muller (11), (11), was proved and 3.2.1.18) bacteria using a polyacrylamide gel electrophoresis extended by us to other related bacteria (7). Recently we reported technique on neuraminidase-modified human transferrin (7). (16). Neurthe isolation, purification and properties of the P. acnes-neuraminidase (16). aminidase is widely discussed as a major etiological factor in infectional diseases (8,12,13,14) and may be involved in pathogenesis of acne vulgaris and seborrheic eczema. However, whereas a positive correlation between neuraminidase activity and virulence was clearly shown for Pasteurella multocida and Erysipelothrix rhusio(8,13), it could not yet been demonstrated for humanpathogenic pathiae in mice (8,13), microorganisms. Therefore, the aim of the present study was to determine whether differences in the neuraminidase activities of P. acnes strains isolated from there are differences different clinical specimens.

Materials and Methods str,lins Bacterial strains

63 P. aClles-strains Altogether 63 aCiles-strains of different clinical origin were examined. Of these, 20 strains were isolated from acne lesions, 21 strains were from the non-affected skin of patients (czema, and and further 22 strains were from from the skin and hair of healthy human with seborrheic eczema, adults. Clinical details, primary cultivation, identification and typing procedures have been The strains were selected at random, most reported in previously published studies (6,9,16). The KB and phage type I (Lit. cit. 6). of them belonging to serotype KB Bacterial cultivation cultil'atioll

All strains were held on A-agar as described earlier (6,9). Subcultivation was done for 48 h B(cton, Dickinson GmbH, Heidelon A-agar A-agar under anaerobic conditions (GasPak system; Becton, berg). For obtaining fresh fluid cultures, bacteria were inoculated into test tubes with 5 ml of a yeast extract-containing broth without addition of inductive substrates as described described (16). After incubation for 36 h at 37°C in GasPak yars, all broth cultures were previously (16). carefully shaken to establish a homogeneous suspension, and and brought to an absorbance of (measured at 540 nm against A-broth using the Beckman photometer 1211), 1211), 1.0 (measured

124

Ll.Hoffler, U.Hoffler, M.Gloor, and H. v. Nicolai

Neuraminidase assay assay Neuraminidase centrifu.ged for 15 min at 6000 x g. Sediments Samples of 2.5 ml bacterial suspension were centrifuged resuspended thoroughly in 1.0 ml 0.05 M were washed twic.e with 5 ml 0.15 M NaCI and were resuspended sodium acetate buffer pH 5.5. Neuraminidase activity was demonstrated by incubation of sodium 100 ftl1.5 mM 3'-N-acetylneuramir:osyl-Iactose 3'-N-acetylneuramir:osyl-lactose (3'-sialyllactose, II"NeuAc-Lac = NeuAca2 100ftl1.5 --+ 4Glc.) in 0.05 M acetate buffer pH 5.5 with 100 ttl bacterial suspension for 0, --+ 3Gal~1 -->(NeuAc) was determined by the 1, 2 and 4 h at 3rc. Released N-acetylneuraminic acid (NeuAc) periodic acidrrhicbarbiturate acid/thiobarbiturate method according according to Aminoff (1). (1). Protein determinations were performed by the method of Lowry et a1. (10). (10). For dry-weight Protein determinations, 0.25 ml of resuspended bacteria were transferred into pre-weighed Eppendorf tubes. The samples were centrifuged, washed twice with H 20, 2 0, and were lyophilized and weighed again.

Statistical evaluations Statistical chi 2-test with the modification of R. A. The statistical evaluations were carried out via the chi'
Results In an earlier study, different fluid media had been tried for production of neuraminidase by P. acnes, whereby high activity was observed in a yeast-extract containing broth (16). Table 1 shows the neuraminidase production of investigated bacteria in fluid cultures containing this medium. As can be seen, a total of 53/63 (~ 84.1 %) strains produced clearly measurable amounts of free, extracellular neuraminidase. The highest percentages of enzyme producing strains were found in the groups isolated from acne lesions and from the non-affected skin of patients with seborrheic eczema: 90.0% and 90.5% of strains were neuraminidase-positive, respectively, whereas strains from normal human skin were positive in only 72.7%. 2 -test with the These differences, however, could not be proved statistically (chi 2-test modification of R. A. Fisher). The results of the quantitative determinations of neuraminidase activities are shown in Table 2. P. acnes strains from normal human skin and from patients with seborrheic eczema produced neuraminidase activities of on average 392 and

Propionibacterium acnes-strains from different cliTable 1. Neuraminidase production of Propionibacterium nical origins Neuraminidase production negative positive

Origin

No. of strains

Normal human skin Acne lesions a SE b Patients with SE

22 20 21 21

6 2 2

16 (72,7%) 18 (90,0%) 19 (90,5%)

total

63 63

10

(84,1 %) 53 (84,1

a b

(chi 2 test, R.A.Fisher) not significant (chi? not significant (chi (chi>2 test, R.A.Fisher)

Neuraminidase Production Production by Propionibacterium acnes-Strains Neuraminidase

125 125

Neuraminidase activities of Propionibacterium aClles-strains from differentclinical different clinical Table 2. Neuraminidase origins origins

Origin

No. of positive positive strains (total) (toral) strains

Neuraminidase activities activities Neuraminidase X [mU/ml] a [mU/ml] lmU/mll lmU/mll

Normal human skin Acne lesions lesions a Acne Patients with SE SE b Patients

16 16 18 18 19 19

392

a b

p

728

383

57

174 174 90

< 0.01 (Wilcoxon-test)

not significant (Wilcoxon-test)

383 mUlml bacterial suspension (~ 6.54 and 6.39 pkat/ml), pkat/rnl), respectively, whereas from acne lesions showed activities of on average 727 mU/ml (~12.1 (~12.1 pkat/ pkatl strains from from persons with seborrheic eczema and and with ml). The difference between strains from not significant, but strains isolated from acne lesions produced normal skin is not significant higher amounts (Wilcoxon-test) than neuraminidase in statistically significant from normal human skin (Table 2). The same tendency was observed with strains from regard to values referred to mg dry weight as to mg bacterial protein.

Discussion Propionibacteria are part of the normal resident bacterial flora of healthy human units (18). (18). Some extracellular adults, normally being situated in the pilosebaceous units enzymes and toxins produced by these coryneforms have been discussed as possible pathogenetic factors in etiology of acne vulgaris (4,5, 18). Among them, neuraminihighest in strains of the species P. acnes (83% positive), whereas dase synthesis is highest and P. granustrains of the other cutaneous Propionibacterium-species, P. avidum and seldom produce measurable amounts of this enzyme (20% (20% and 6% positive, losum, seldom (7)). We also observe more frequent neuraminidase production from respectively (7)). from from acne lesions, compared with strains from normal P. acnes-strains isolated from human skin. Additionally neuraminidase-positive strains produce statistically significant higher amounts of the enzyme than strains from normal skin. Whereas the low number of propionibacteria on the skin of patients with seborrheic eczema (6) (6) low and the average production of neuraminidase by these strains is probably of no causative importance to this disease, it seems probable that the enzyme acts as one of several etiological factors in acne vulgaris. between N -acetylneuraminic acid The P. acnes-enzyme cleaves oc-ketosidic linkages between and remaining carbohydrate chains of oligosaccharides and glycoproteins, and its substrate specificity pattern is similar to that of neuraminidases from other bacteria of low pathogenicity towards man (2, (2, 14-17). The high susceptibility of the NeuAcoc2 ---7> 6GaltJl---7> 4GlcNAc-structure and of glycoprotein type substrates to the P. acnes (16) demonstrates the possible responsibility of the enzyme for malneuraminidase (16) surface glycocalyx structures in the epithelial wall of the sebaceous function of cell surface follicle. Thus there is reason to believe that the neuraminidase activity of P. acnes within the acne microcomedo contributes to the incitation of its rupture.

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U.Hoffler, M.Gloor, and H. v. Nicolai

Acknowledgements. This study was supported by Research Grants from the Deutsche The authors wish to thank Mrs. Mrs. Forschungsgemeinschaft (Ho 757/2, Ni 147/6, GI 91/4). The D.Stefanik, Mrs. M.Franke-Hoffmann, Mrs. C.Mohring and Mrs. N.Silberstein for helpful technical assistance.

References

1. Aminoff, D.: Methods for the quantitative estimation of N-acetyl-neuraminic acid and J. 81 (1961) (1961) 384-392 their application to hydrolysates of sialomucoids. Biochem. J. 2. Crampen, M., M., H. v. Nicolai, and F. Zilliken: Properties and substrate specificities of two two neuraminidases from Trichomonas fetus. Hoppe-Seyler's Z. Physiol. Chern. 360 (1979) 1703-1712 (1979) ducts of un3. Gloor, M. and M. Franke: On the propionibacteria in the pilosebaceous ducts Derm. Res. 262 (1978) (1978) 125-129 involved skin of acne patients. Arch. Derrn. und J.Hordcek: 4. Gloor, M. und j.Horaiek: Uber die Hautoberf1achenlipide. Hautoberflachenlipide. In: Handbuch der HautTeil 41, Seite 263-348, G.]adasG.Jadasund und Geschlechtskrankheiten, Erganzungswerk, Bd. 1, Tei141, (1979) sohn (ed.). Springer-Verlag, Berlin (1979) 5. Hoffler, U.: Ober Uber die pathogenetische Bedeutung der Propionibaktericn Acne vulgaPropionibakterien bei Acne Fette, Seifen, Anstrichm. 82 (1980) (1980) 510-513 ris. Fette, ris. 6. Hoffler, U., M.Gloor, G.Peters, H.L.Ko, A.Briiutigam, A. Thurn, and G.Pulverer: Qualitative and quantitative investigations on the resident bacterial skin flora in healthy persons and in the non-affected skin of patieLts patier.rs with seborrheic eczema. Arch. Derm. Derm. Res. 268 (1980) (1980) 297-312 Pro7. Hoffler, U., H.-Ch. Kohler, R. Adam, and G. Mauff: Neuraminidase production of Pro(1978) 177-179 pionibacterium acnes and related bacteria. FEMS Microbiol. Lett. 4 (1978) und H. E. Muller: Die Virulenz von Erysipelothrix rhusiopatbiaerhusiopathiae8. Krasemann, Chr. und Abt, Orig. A 231 Stammen und und ihrc Neuraminidase-Produkticn. Neuraminidase-Produktion. Zbl. Bakt. Hyg., I. 1. Abt. (1975) 206-213 206-213 (1975) 9. Lentze, I., H. L. Ko und U. U. Hoffler: Differenzierung unterschiedlicher PropionibakterienSpezies aus Acne Acne vulgaris-Effloreszenzen. Hautarzt 30 (1979) (1979) 242-247 10. Lowry, O.H., N.].Rosebrough, R.j.Randall: Protein measurement with N.J. Rosebrough, A.L.Farr, and R.J.Randall: the folin phenol reagent. ]. J. BioI. Bio!. Chern. 193 (1951) (1951) 265-275 265-275 Ubcr das Vorkommen von Neuraminidase bei Corynebacterium acnes. 11. Miiller, H. E.: Obcr Med. Microbiol. Microbio!' Immunol. Immuno!. 156 (1971) (1971) 240-249 240-249 Z. Med. Pathoger.itatsfaktor bei mikrobiellen Zb!. 12. Muller, H. E.: Neuraminidase als Parhogcr.itatsfaktor rnikrobiellen Infektionen. Zbl. 1. Abt. Orig. A 235 (1976) (1976) 105-110 Bakt. Hyg., I. H.E. und Chr.Krasemann: Die Virulcnz Viruknz von Pasteurella multocida-Stamrnen multocida-Stammen 13. Muller, H.E. Neuraminidase-Produktion. Zbl. Zb!. Bakt. Hyg., 1. Abt. Orig. A 229 (1974) (1974) 391-400 undihre Neurarninidase-Produktion. Hyg., I. Miiller, H.E., H. v. Nicolai und F.Zilliken: Michaelis-Konstanten von Neuraminidasen 14. Muller, (1975) 417-419 bei pathogenen und apathogenen Mikroorganismen. Z. Naturforsch. 30c (1975) 15. Nicolai, H. v., P. Esser, and E. Lauer: Partial purification and properties of neuramini(1981) 153 Physiol. Chern. 362 (1981) dase from Bifidobacterium lactentis. Hoppe-Seyler's Z. Physiol. -162 U. Hoffler, and F. Zilliken: Isolation, purification and properties of neur16. Nicolai, H. v., U. 1. Abt. Orig. A 247 (1980) (1980) aminidase from Propionibacterium acnes. Zbl. Bakt. Hyg. I. 84-94 acd properties of two two neur17. Nicolai, H. v., H.E.Muller, and F.Zilliken: Purification ar.d FEBS Lett. 117 (1980) (1980) 107-110 aminidases from Streptococcus viridans II. FEBS Treatmer"t. Springer-Verlag, 18. Plewig, G. and A.M.Kligman: Acne, Morphogenesis and Treatment. (1975) Berlin-Heidelberg-New York (1975)

U. Hoffler, Hygiene-Institut der Universitat, Universitiit, Goldenfelsstr. 21, D-5000 Koln 41 Dr. U.