Streptolytic Activities of a Lytic Enzyme from Staphylococcus hyicus

Zbl. Bakt. 271, 54-60 (1989)

Streptolytic Activities of a Lytic Enzyme from Staphylococcus hyicus * CH. FREDE, D. CHRIST, and CH. LAMMLER Institut fur Bakteriologie und Immunologie, Justus-Liebig-Universitiit, D-6300 Giei~en

With 5 Figures· Received September 8, 1988 . Accepted October 27, 1988

Summary The LE IIIb fraction of the bacteriolytic enzyme from Staphylococcus hyicus could be isolated by ionic exchange chromatography and subsequent gel filtration. Isoelectric focussing of the highly purified enzyme preparation revealed an isoelectric point at pH 10.3. The lytic activity of LE IIIb on streptococci of various serogroups could be effectively analyzed with the help of an aggregometer and expressed as increase of transmittance at 546 nm. The streptococci differed in their LE IIIb lysis pattern, which was more pronounced with those of serological groups C and D and Streptococcus uberis. The lytic effect of the LE IIIb fraction was optimal at low molarity (0.01 mol/I) of the lysis buffer and reduced in the presence of HgCl z and EDTA. Zusammenfassung Die LE IIIb-Fraktion des bakteriolytischen Enzyms von Staphylococcus hyicus konnte durch Ionenaustauschchromatographie und anschlielSende Gelfiltration isoliert werden. Die hochgereinigten Enzympriiparationen hatten einen isoelektrischen Punkt bei pH 10,3. Die lytische Aktivitiit von LE IIIb auf Streptokokken unteschiedlicher serologischer Gruppen war mit Hilfe eines Aggregometers durch Anstieg der Transmission bei OD 546 nm nachweisbar. Die Streptokokkenkulturen zeigten ein unterschiedliches Lyseverhalten. Streptokokken der serologischen Gruppen C, D und Streptococcus uberis wurden dabei deutlich Iysiert. Die Iytische Wirkung der LE IIIb-Fraktion war besonders ausgepriigt bei niedriger Molaritiit (0,01 molll) des Lysepuffers, wurde aber in Gegenwart von HgClz oder EDTA gehemmt.

Introduction Bacteriolytic enzymes proved to be a powerful tool for solubilization of bacterial surface antigens (14,4), for bacterial plasmid isolation (15) and for protoplast prepara-

* Supported by the Deutsche Forschungsgemeinschaft, Bonn, FRG.

Streptolytic Activities of a Lytic Enzyme from S. hyicus

55

tion (12). Among these, great interest has been focussed on lytic enzymes from staphylococcal species (9). Lysostaphin, the lytic enzyme from Staphylococcus staphylolyticus could be separated into three enzyme fractions L I to L III (3). These enzyme fractions, characterized as endo-N-acetyl-glucosidase (L I), endopeptidase (L II), and N-acetyl-muramyl-Lalanin amidase (L III), differed in their lysis pattern to various micrococcal and staphylococcal species, (18, 16,8). Further bacteriolytic as well as serological tests indicated, that the principal activity of lysostaphin was associated with the staphylococcal endopeptidase (16,2). The endopeptidase cleaved the interpeptidic pentaglycin bridges of the peptidoglycan (1). Lytic enzymes from other staphylococcal species (S. aureus, S. simulans and S. saprophyticus) were characterized as endo-N-acetyl-glucosaminidase and allowed a separation of staphylococci in various lyogroups (17). More recently, Muller and Blobel (7) described two lytic enzymes from S. hyicus (LE IlIa and LE IIIb), which effectively lyzed micrococcal and streptococcal cell walls. The purpose of the present study was to examine the lytic activity of a highly purified LE IlIb fraction on streptoccci of various serogroups. The lytic effects were determined with the help of an aggregometer.

Materials and Methods

Bacterial cultures. A total of 65 streptococcal cultures of serological groups A, B, C, D, G and Streptococcus uberis were used in this study. The cultures had been isolated from infections of humans, bovines, equines and canines. Most of the cultures were kindly obtained from Dr. R. Weiss (Institut fur Hygiene und Infektionskrankheiten der Tiere der Justus-Liebig-Universitat, Giegen) and from Dr. G. Hahn (Institut fur Hygiene der Bundesanstalt fur Milchforschung, Kiel). Isolation of the murolytic enzyme. For isolation of the lytic enzyme, S. hyicus NCTC 10350 was cultivated as described by Muller and Blobel (7). After centrifugation, the culture supernatant (2 1) was dialyzed against 0.01 molll phosphate buffer, pH 6.3, for 24 hand incubated with 7.5 g amberlite (CG 50 II; Serva, Heidelberg, FRG) for 3 hand 4°C on a rotary shaker. The amberlite had been equilibrated in 0.01 molll phosphate buffer, pH 6.3. After repeated washings the elution was performed with a 0.01 molll phosphate buffer, pH 6.3, to 0.5 molll phosphate buffer, pH 6.3, gradient at a flow rate of 40 mllh. Fractions with lytic activity were pooled, concentrated with polyethylene glycol (PEG 35000; Serva, Heidelberg, FRG) and further purified by gel filtration (G75-sepharose; PharmaciaiLKB, Freiburg FRG). Proteolytic activities of the fractions were determined with azocasein (Sigma, Deisenhofen, FRG), according to Scharmann and Balke (11). Isoelectric focussing. Isoelectric focussing was carried out with 100 ml culture supernatant as well as with the purified enzyme preparation and carrier ampholytes (Pharmacial LKB, Freiburg, FRG), pH 5-7 and pH 8-11 (1: 1) (10). Standardization of the lytic enzyme. The lytic activities of the enzyme preparations were standardized with the Micrococcus luteus reference culture CCM169 by the help of an aggregometer (Universal-Aggregometer, Braun, Melsungen, FRG). Lysis of the cells was expressed by the increase of transmittance (1:; Tr) of the bacterial suspension at 546 nm and 37°C after addition of the lytic enzyme from S. hyicus (5). One unit of lytic activity (1 U) was defined as increase of 0.01 per min. Lysis assay. For this, the streptococci were cultivated in Todd Hewitt broth (THB, Difco Laboratories, DetroitlMichigan, USA) for 24 h at 3rC, washed in 0.01 mol/l phosphate buffer, pH 7.2, and finally adjusted photometerically to 20% transmission (546 nm) in the same buffer. 36 U of the purified enzyme were added to 2 ml of the bacterial suspension. Changes in transmission, induced by the lytic enzyme, were continuously registered using a

56

Ch. Frede, D. Christ, and Ch. Liimmler

chart recorder (Kompensationsschreiber Servogor RE 511; U1trawest, Niirnberg, FRG). For further characterization of the lytic enzyme activities the lytic assay was performed with 0.01 to 0.1 molJllysis buffer and in the presence of increasing concentrations of HgCI 2, EDTA, NaNJ , CoCi 2, BaCI2> and CaCho Results The bacteriolytic enzyme from S. hyicus could be isolated from culture supernatant by ionic exchange chromatography on amberlite CG 50 II and further purified by gel filtration using G75-sepharose. The elution profiles and the lytic activity of the fractions are shown in Fig. 1 and Fig. 2. Fractions containing the lytic enzyme without proteolytic activities were used for further studies. Preparative isoelectric focussing of the culture supernatant revealed rwo major points with bacteriolytic activities at pH 9.8 and pH 10.3 (Fig. 3). Isoelectric focussing of the highly purified enzyme prepara. tion from S. hyicus demonstrated enhanced bacteriolytic activity at pH 10.3 (Fig. 4). The bacteriolytic activity of the isolated enzyme preparations on 65 streptococcal cultures was recorded as increase of transmittance at 546 nm after 20 min incubation time. The results are presented in Table 1 and Fig. 5. The lytic activity was more pronounced with streptococci of serological groups C, D and Streptococcus uberis, moderate with streptococci of serological groups Band G and weak for streptococci of serological group A. Further characterization of the bacteriolytic activity was performed with respectively one culture of group D and group G streptococci. An increase of molarity of the lysis buffer over 0.01 molll as well as the presence of HgCh and EDTA reduced the lytic effects. The addition of CaClz slightly increased lysis of both cultures, whereas CoCh, NaN3, and BaClz even in concentrations of 1 mmolJl had no measurable effects (Table 2).

.o.Tr 80

00280

70 60

2,0

50 1,5 40 30 20

,

:

1,0

qs

10 00

.....'

.~

0,..0

,

............

Fig. 1. Elution pattern of the lytic enzyme from S. hyicus NCTC 10350 after ionic exchange chromatography on amberlite CG 50 II. The bacteriolytic activity of the fractions (100 fJl) was expressed as increase of transmittance (546 nm) of Micrococcus luteus cells after 20 min at 37°C (.-.). The fractions were monitored for protein at 280 nm (e__e).

Streptolytic Activities of a Lytic Enzyme from S. hyicus

lI.Tr 40

30

57

00280 0,15

0,1

20

.............

'"..._......'

0,05 10

....

,•.e.'

'

o ~~1--3~O------~40-------5~O------~~c------7~O-------~~i

Fig. 2. Gelfiltration of the lytic enzyme on G75-sepharosej bacteriolytic activity of the fractions (10 fAll (.-.), optical density at 280 nm ( _ . ) .

ATr 70

ATr pH

80

pH

70

60

9,5 50

60 9,5

50

40

40

30

10,0

20

30

lOP

20

10 ~

__~~__~__~__~___ T 5 10 15 20 2S

10,5

10 10

15

20

25

T

10,5

Fig. 3. Isoelectric focussing of the culture supernatant from S. hyicus; bacteriolytic activity of the fractions (100 fAll (.-.), pH of the fractions (.). Fig. 4. Isoelectric focussing of the purified enzyme preparationj bacteriolytic activity of the fraction (100 Ill) (.-.), pH of the fractions (.).

58

Ch. Frede, D. Christ, and Ch. Liimmler 80

E c

...'"

U'l

z

60

0

iii til

~

z 40

Ul <[ 0::

>-

Fig. 5. Typical lysis pattern of streptococci of serological groups D (I), G (II), and s. uberis (III) in the presence of the lytic enzyme from

s. hyicus (36 U).

Table 1. Lysis of streptococci of serological groups A, B, C, D, G, and presence of LE IIIb from s. hyicus

n

Streptococci of serological groups

Origin

A

human human, bovine human, bovine, equine bovine human, bovine, equine bovine

B

C" D G S. uberis

s.

uberis in the ~Tr

5 9

16 14 9 8

mean

range

1.8 9.5 10.8 17.6 7 10.9

0-5 6-14 4-19 7-39 3-11 4-21

" including cultures of S. equisimilis (3), S. equi (3), S. zooepidemicus (6), and S. dysgalac-

tiae (4)

n = number of cultures ~ Tr = increase of transmittance of the bacterial suspension after addition of 36 U of the LE I1Ib fraction of the lytic enzyme from Staphylococcus hyicus and 20 min incubation time.

Table 2. Lytic effect of LE IIIb on a group D (I) and group G (II) streptococcus in the presence of various additives

II

0.01 molll phosphate buffer + 0.001 mmolll HgCl 2 + 0.01 mmolll HgCl 2 + 0.1 mmolll HgCl 2

+ 1.0 + 1.0

+ 1.0 + 1.0 + 1.0

mmolll mmol/l mmolll mmol/l mmolll

CaCl1 EDTA BaCl2 NaNJ CoCl1

100%" 20%

100%"

110% 70% 90% 100% 100%

111% 33% 78% 67% 89%

33%

22%

" Lysis of the streptococci expressed as increase of transmittance after 20 min incubation time; lysis in 0.01 molll phosphate buffer, pH 7.2, was regarded as 100%.

Streptolytic Activities of a Lytic Enzyme from S. hyicus

59

Discussion

In the present study a lytic enzyme from S. hyicus could be highly purified by ionic exchange chromatography and subsequent gel filtration. The lytic enzyme had an isoelectric point at pH 10.3 and was therefore assigned to the LE II1b fraction, described by Milller and Blobel (7). The lytic effects of the LE IIIb fraction on streptococci of various serogroups and on Streptococcus uberis were recorded as increase of transmittance at 546 nm and could be effectively analyzed with the help of an aggregometer. This method had already been used for the demonstration of mutanolysin susceptibility of bovine pathogenic Actinomyces pyogenes (5). The streptococci used in this study differed in their LE IIIb lysis pattern. Lysis had optimal activity at low molarity of lysis buffer and the activity was reduced in the presence of HgCb and EDTA. The latter indicated some Ca ++ -ion dependency. The LE IIIb fraction of the lytic enzyme from S. hyicus might be a useful tool, which can be used in parallel with the murolytic enzyme from Streptomyces globisporus for solubilization of streptococcal group and type specific antigens (4, 6), for extraction of plasmaprotein receptors from streptococcal surface (14) or DNA fingerprinting (13).

References

J. M.: Use of bacteriolytic enzymes in determination of wall structure and their role in cell metabolism. Bact. Rev. 32 (1968) 425-464 2. Grov, A., O. J. Iversen, and C. Endresen: Studies on lysostaphin. Serological crossreactions between three isolated enzymes. Eur. ]. Biochem. 48 (1974) 193-198 3. Iversen, O. J. and A. Grov: Studies on lysostaphin. Separation and characterization of three enzymes. Eur. ]. Biochem. 38 (1973) 293-300 4. Liimmler, Ch., Ch. Frede, and H. Blobel: Effective murolytic solubilization of streptococcal group-specific antigen. ]. Clin. Microbiol. 24 (1986) 903-904 5. Liimmler, Ch. and Ch. Frede: Mutanolysin-induced lysis of Actinomyces pyogenes determined by aggregometry. Zbl. Bakt. Hyg. A 269 (1988) 447-453 6. Liimmler, Ch., K. Gurturk, and H. Blobel: Streptococcal group B type antigen X in group L streptococci. ]. Clin. Microbiol. 25 (1987) 1803-1804 7. Muller, H. P. and H. Blobel: Isolation and characterization of a staphylococcal enzyme bacteriolytic on streptococci. Zbl. Bakt. Hyg. A 264 (1987) 41-46 8. Poutrel, B. and J. B. Caffin: Lysostaphin disk test for routine presumptive identification of staphylococci. ]. Clin. Microbiol. 13 (1981) 1023-1025 9. Satta, G., P. E. Varaldo, G. Grazi, and R. Fontana: Bacteriolytic activity in staphylococci. Infect. Immun. 16 (1977) 37-42 10. Schaeg, W., R. Bingol, and H. Blobel: Purification of penicillinase (~-Iactamase) and acid phosphatase from Staphylococcus au reus in one procedure. Biochim. Biophys. Acta 169 (1972) 542-549 11. Scharmann, W. and E. Balke: Untersuchungen tiber eine Protease (Elastase) von Pseudomonas aeruginosa. Bildung und Reinigung der Protease. Hoppe-Seyler's Z. Physiol. Chern. 365 (1974) 443-450 12. Siegel, J. L., S. F. Hurst, E. S. Liberman, S. E. Coleman, and A. S. Bleiweis: Mutanolysin induced spheroplasts of Streptococcus mutans are true protoplasts. Infect. Immun. 31 (1981) 808-815 13. Skjold, S. A., P. G. Quie, L. A. Fries, M. Barnham, and P. P. Cleary: DNA fingerprinting of Streptococcus zooepidemicus (Lancefield group C) as an aid to epidemiological study.]. Infect. Dis. 155 (1987) 1145-1150 1. Ghuysen,

60

Ch. Frede, D. Christ, and Ch. Liimmler

14. Tille, D., G. S. Chhatwal, and H. Blobel: Release of Fc-receptors after streptococcal lysis induced by a lytic enzyme from Streptomyces globisporus. Med. Microbiol. Immunol. 175 (1986) 35-41

15. Tompkins, L. S.: DNA methods in clinical microbiology. In: Manual of Clinical Microbiology (Lennette, E. H., A. Balows, W. J. Hausler, and H. J. Shadomy, eds.), pp. 1023-1028. ASM, WashingtonlDC (1985)

16. Tuncan, E. U. and S. E. Martin: Lysostaphin lysis procedure for detection of Staphylococcus aureus by the firely bioluminescent ATP method. Appl. Environ. Microbiol. 53 (1987) 88-91

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17. Varaldo, P. E., G. Sattaa, G. Grazi,and C. A. Romanzi: Grouping of staphylococci on the basis of their bacteriolytic activity pattern: a new approach to the taxonomy of the Micrococcaceae. I. Identification of six different "lyogroups". Int. J. System. Bact. 28 (1978) 141-147 18. Wadstrom, T. and o. Vesterberg: Studies on endo-~-N-acetyl-glucosaminidase, staphylolytic peptidase, and N-acetyl-muramyl-L-alanin amidase in lysostaphin and from Staphylococcus aureus. Acta Path. Microbiol. Scand. Sect. B 769 (1971) 248-264

Dr. Christoph Liimmler, Inst. f. Bakteriologie und Immunologie, Fachbereich Veteriniirmedizin der Justus Liebig-Universitiit, Frankfurter Str. 107, D-6300 Gief~en