5ied. Microbiol. Immunol. 161,127--132 (1975) 9 by Springer-Verlag 1975

Proteolytic Activity of Mycoplasma salivarium and Mycoplasma orale 1 Tsuguo W a t a n a b e Department of Bacteriology, School of Dental Medicine, Tsurumi University, Yokohama Received December 5, 1974

Abstract. Proteolytic activity was found in whole ceils and cultural supernatants of Mycoplasma salivarium (PG20 und 8 isolates) and Mycoplasma orale I (CH 19299 and 8 isolates). Additionally, the activity was examined in cell membrane and soluble fractions of PG20 and CH19299, and detected in them. The level of the activity appeared higher in M. salivarium than M. orale 1. And some differences were found between these mycoplasmas in the affinity for substrates as a result of examination of the activity in cultural supernatants using horse serum, casein and bovine albumin as substrates. That is, PG20 had a higher affinity for horse serum proteins than casein, while CH19299 the latter than the former, although the lowest affinity for bovine albumin was common to these two strains. P r o t e o l y t i c a c t i v i t y as m e a s u r e d b y l i q u e f a c t i o n o f h e a t - c o a g u l a t e d a n i m a l s e r u m or g e l a t i n has been d e m o n s t r a t e d in M. mycoides, A. laidlawii ( F r e u n d t , 1958; R a z i n a n d Oliver, 1961), M. canis ( F e u n d t , 1958) a n d M. arthritidis ( A l u o t t o et al., 1970; Czekalowski, 1973). H o w e v e r , t h e presence of the a c t i v i t y in h u m a n m y c o p l a s m a s h a d been u n k n o w n u n t i l t h e a u t h o r a n d his colleagues ( W a t a n a b e et al., 1973 ; W a t a n a b e , 1974) d e s c r i b e d t h a t M. salivarium, M. orale 2, M. hominis, M. ]ermentans, M. pneumoniae, a n d oral, p h a r y n g e a l a n d u r o g e n i t a l T - m y c o p l a s m a s d e c o m p o s e d horse s e r u m proteins, t h o u g h M. orale 1 d i d not. This finding will p a r t l y evidence t h e presence o f p r o t e o l y t i c a c t i v i t y in h u m a n m y c o p l a s m a s . B u t s e r u m of c o m p l e x c o m p o s i t i o n is p r e s u m e d to h a v e a v a r i e t y o f biological activities. F o r t h i s reason, t h e a u t h o r considered t h a t p r o t e o l y t i c a c t i v i t y o f these m y c o p l a s m a s m u s t be verified using m u c h m o r e simplified or purified p r o t e i n s as s u b s t r a t e s . F u r t h e r m o r c , r e e x a m i n a t i o n of M. orale I m u s t be c o n d u c t ed, because a wide d i s t r i b u t i o n o f t h e a c t i v i t y in h u m a n m y c o p l a s m a s was suggested b y t h e p r e v i o u s r e p o r t ( W a t a n a b e et al., 1973). I n t h e p r e s e n t s t u d y , as the a u t h o r is i n t e r e s t e d in biological a c t i v i t y o f oral m y c o p l a s m a s , M. salivarium a n d M. orale I were e x a m i n e d for p r o t e o l y t i e a c t i v i t y in c u l t u r a l s u p e r n a t a n t s , whole cells, cell m e m b r a n e a n d soluble fractions. Materials and Methods Mycoplasma strains: M. salivarium 1T1, 1T4, 1T6, SP8, sPg, SP12, 2P2, 2P5 and PG20, and M. orale 1 BG1, BG2, BG10, BP2, BP5, BP6, BP7 and CH19299 strains were tested. PG20 and CH19299 strains were obtained through the courtesy of Dr. D. Taylor-Robinson (Clinical Research Centre, hIiddlesex, England). And the other strains were isolated from human oral cavities in our laboratory. Media and cultural conditions: Liquid medium utilized for propagation of the organisms consisted of Difco PPLO broth (without crystal violet) supplemented with Difco yeast extract (1~ w/v), Difco PPLO serum fraction (1~ v/v), arginine-HC1 (I~ w/v), phenol 9 Med. Microbiol.Immunol., Vol. 161

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red (0.0020/0, w/v), penicillin G (1000 u/ml) a n d thallium acetate (0.050/0, w/v). And the reaction was adjusted to p H 7.0. A 1000-ml liquid medium was inoculated with 10 nil of stock cultures containing approximately 100 CFU of organisms. All cultures were contained in screw-capped bottles a n d incubated a t 37 ~ for 3 days. Whole cells: The cells were harvested b y centrifuging 1000 ml of b r o t h cultures a t 8000 g for 30 rain, washed 3 times in PBS (pH 7.0 phosphate buffered saline), a n d resuspended in 10 mI of the same buffer. The cell suspensions obtained were stored a t -- 70 ~ until used. Cell m e m b r a n e a n d soluble fractions: The cells were disrupted by osmotic shock a n d fractionated following the methods described b y Hollingdale a n d Lemcke (1969), and Cirillo a n d Razin (1973). Crude enzymes isolated from cultural s u p e r n a t a n t s : Three-day-old b r o t h cultures were centrifuged a t 100000 g for 30 rain, a n d 561 g of a m m o n i u m sulfate was added for 1 litre of supernatants. The precipitated protein was used as crude enzymes, after collected b y centrifugation at 15 000 g for 30 min, dissolved in the minimum volume of PBS, a n d dialyzed against a large volume of PBS. Protein measurement: Protein content was determined b y the m e t h o d described b y Lowry a n d his colleagues (Lowry et al., 1951). Proteolytic activity assay: The following three methods were applied for proteolytic activity assays. As substrates, casein (nach H a m m a r s t e n ; Mark & Co., Inc., Germany), bovine albumin (Cohn Fraction V; Daiichi Pure Chemicals Co., Ltd., Tokyo, Japan), and horse serum (Shiihashi Shoran Co., Ltd., Tokyo, J a p a n ) were used. Horse serum was heated a t 56 ~C for 30 rain before used.

1. Plate Test Agar (Oxoid agar No. 1) was suspended in P B S a t each of 1.8~ a n d 1.0~ concentrations, autoclaved a t 121 ~ for 15 min, and cooled down to 56~ C-agar containing casein (1 ~ w/v) was prepared b y combining 50 ml of 1.8~ agar a n d 50 ml of 2~ casein solution (in PBS), a n d BA-agar containing bovine albumin (! ~ w/v) by combining 90 ml of 1 ~ agar and 10 m l of 1 0 % bovine albumin solution (in PBS). Into 9 cm diameter Petri dishes was poured 10 ml of each of C- a n d BA-agar. Drops (0.05 ml) of whole cell suspensions were placed on these agar plates, which were incubated at 37 ~ for 18 hrs. After incubation, C-agar plates were overlayed with a s a t u r a t e d a m m o n i u m sulfate solution, and BA-agar plates with 1~ sulfosalicylic acid. Since these reagents precipitated intact proteins unaffected b y proteinases, the area in which proteins had been decomposed appeared as a clear zone.

2. Di/]usion Test A 0.9o/0 agar solution was prepared as above-mentioned, and, in an a m o u n t of 3 ml, poured into 9 cm diameter P a t t i dishes to produce a t h i n agar-layer. Penicillin cups were placed on t h e t h i n agar-layer, and 7 ml amounts of C-, BA- and HS-agar, which was prepared by combining 80 ml of 1.1 ~ agar a n d 20 ml of horse serum, were poured. After agar was solidified, cups were t a k e n away to form wells (8 y, 2 ram). The wells were filled with 0.1 ml of crude enzymes prepared from cultural supernatants, undiluted and diluted in PBS in serial 2-fold steps. After incubated a t 37 ~ for 18 hrs, the agar plates were treated as above-mentioned. HS-agar plates were treated just as C-agar plates. Proteolytie activity was expressed as the w i d t h of clear zones produced around t h e wells.

3. Tube Test Caseinolytic activity was assessed b y measuring the a m o u n t of amino groups liberated in a given time following the m e t h o d described b y K u n i t z (1947) basically except for some modifications. Six test tubes were n u m b e r e d from 1 to 6. Each of t h e m was prepared to contain reaction mixtures as follows. Tube No. 1 (Blank), 1.2 ml of PBS a n d 1.2 ml of 10~ trichloroacetic acid (TCA). Tube No. 2 (Enzyme control), 1.0 ml of PBS, 1.2 ml of 10~ TCA a n d 0.2 ml of a sample. Tube No. 3 (Substrate control), 1.0 ml of 0.50/0 casein solution (in PBS), 1.2 ml of 10~ TCA a n d 0.2 ml of a sample. Tube No. 4 (0 hr incubation), 1.0 nd of 0.50/0 casein, 1.2 mI of 1 0 % TCA a n d 0.2 ml of a sample. Components of the content in tube No. 2 to 4 were p i p e t t e d a n d mixed in the order described. Tube No. 5 (2 hrs incubation) a n d 6

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(4 hrs incubation) were prepared to contain 1.0 ml of 0.50/0 casein and 0.2 ml of a sample. Tube :No. 5 received 1.2 ml of 10~ TCA after 2 hrs incubation and tube No. 6 after 4 hrs incubation a t 37~ After TCA was added, reaction mixtures were shaken well and allowed to s t a n d a t room t e m p e r a t u r e for 30 rain. Then, t h e mixtures were centrifuged a t 15000 g for 30 rain to remove precipitates. The s u p e r n a t a n t s were colored by the phenol reagents and read in a spectrophotometer a t 750 m#. After corrected by subtracting readings of enzyme a n d substrate control~, readings a t O, 2, a n d 4 hrs incubation were plotted in a diagram. Samples tested b y this method were whole cells, cell m e m b r a n e a n d soluble fractions.

Results I n t h e first place, PG20 (M. salivarium) a n d CH19299 (~I. orale 1) were examined for proteolytic activity. In consequence, the following results were obtained. On agar plates containing each of casein and bovine albumin, the whole (.ells of these two strains split b o t h of the substrates, which was manifested as production of zones of clearing around a film of t h e cells (Fig.l), On t h e other hand, by tube test. also, caseinolytic activity was ~bund in whole cells b y measuring amino groups liberated by breakdown of casein, and the level of t h e activity appeared higher in P(~20 t h a n CH19299 (Fig. 2). The above findings gave risc to a question whether the activity existed in both of membranes a n d intracellular materials released by disruption of cells or either of them. In order to elucidate this question, cell membrane and soluble fractions were prepared and examiued for the activity. In consequence, the activity was found in both of the fractions (Fig.3).

Fig.1. Caseinolytic activity in whole cells of M. orale 1 CH19299 (1) and M. ,vdicaruim PG20 (2). Zones of clearing (A) can be seen around a fihn of cells (B)

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3

4

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Fig.2. Caseinolytic activity in whole cells of M. orale 1 CH19299 and M. salivarium PG20. a protein content ([zg) of samples in reaction mixtures Fig.3. Caseinolytic a c t i v i t y in cell m e m b r a n e (A) a n d soluble fractions (B) of M. orale 1 CH19299 a n d M. salivarium PG20. a protein content ([zg) of samples in reaction mixtures 9*

130

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(mm)

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6-

b

o(pH)

~3 r

,

,

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0Dilution. 1 2(321r Fig.4

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Incubation time (hour) Fig.5

Fig.4. Protcolytic activity in cultural supernatants of M. orale I CH19299 (..... ) and M. sali. varium PG20 ( ). 9 horse serum proteins,, casein, 9 bovine albumin Fig.5. Relationship of cascinolytic activity in cultural supernatants with incubation time and growth stage expressed as a pH value of cultures

?"---SP12 550 //~:::'" SP9 550 "1 T4 550 "1T1 550 :"-1T6 550 P5 500 "~ 0.1 , ~ . , : " 2 "'SP8 450 o

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b

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Fig.7

Fig.6. Caseinolytic activity in whole cells of M. sal%arium (strains isolated from oral cavities) a strains tested, b protein content ([zg) of samples in reaction mixtures Fig.7. Caseinolytic activity in whole cells of M. orale 1 (strains isolated from oral cavities) a strains tested, b protein content (~g) of a samp[e in reaction mixtures The cultural supernatant was, also, examined for the activity, and evidenced to contain active principles capable of decomposing horse serum proteins, casein and bovine albumin. However, there was some difference between these strains in the affinity for substrates. That is, PG20 digcsted horse serum proteins more actively than casein, while CH19299 the latter than the former, although the lowest affinity for bovine albumin common to these two strains (Fig. 4). Digestion of horse serum proteins by CH19299 was so feeble that the clear zones produced around the wells were very indistinct and might be overlooked unless scrutinized intensively. I n the meantime, cultural supernatants are considered to contain two types of active principles. That is, one of them is produced early in the growth of cultures and therefore presumably extraceIlular. And the other is found in older cultures, supposed to be liberated by disruption of cells, and therefore presumably intracellular. In PG20 and CH19299, the activity was detected in supernatants of cultures regarded to be at an early stage of growth judging from a p H value of cultures (Fig.5). This finding suggests that the active principles exist extracellularly. Besides, the presence of intraccllular active principles is, also, suggested by the fact that the activity in cultural supernatants increases in parallel with ageing of the cultures. In the last place, as representative strains of .M. salivarium and M. orale I werc demonstrated to possess proteolytic activity, examination was extended to isolates from oral cavities identified with these mycoplasmas. As a result, the activity was detected in whole cells (Figs. 6 and 7) and cultural supernatants of all the strains tested (Table 1).

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Table 1. Caseinolytic activity in cultural supernatants Mycoplasma strains M. salivarium 1T1 1T4 1T6 SP8 SP9 SP12 2P2 2P5

Width (mm) of clear zones

lYIycoplasma strains

Width (mm) of clear zones

4.5 4.0 4.8 5.5 5.3 5.0 5.5 6.0

M. orale 1 BG1 BG2 BG5 BGI0 BP2 BP5 BP6 BP7

6.0 4.5 5.5 4.0 4.0 5.5 5.5 4.5

Discussion

Both of PG20 (M. salivarium) and CH19299 (M. orale 1) were substantiated to posses proteolytie activity in cultural supernatants, whole cells, cell membrane and soluble fractions. This suggests t h a t these strains have proteolytic enzymes associated with and free from cell membranes. I n addition, easeinolytic activity was demonstrated to exist in cultural supernatants and whole cells of all strains tested of M. salivarium and M. orale 1, which were isolated from human oral cavities. Therefore, proteolytic activity is considered to be a property which is not confined to exclusively limited strains, but characterize most strains of these mycoplasmas. Based on these facts, it is speculated t h a t part of nutritional requirements of these mycoplasmas is met by decomposition of horse serum proteins or some other native proteins supplemented essentially in media, although proteins in media have been believed to act as detoxifiers (Rodwell, 1960; Rodwell and Abott, 1961) and regulate sterol-uptake (Smith and Boughton, 1961). There were some differences between M. salivarum and M. orale I in substrate specificity. That is, M. salivarium decomposed horse serum protiens more readily than casein, and, on the contrary, M. orale 1 casein than horse serum proteins. Presumably, this is one of the reasons why the author and his colleagues (Watanabe et al., 1973) could not detect the activity in M. orale I in the previous study. Besides, it is also likely that the method applied in the study was not sensitive enough to detect the feeble lysis of horse serum proteins by M. ora/e 1. At the beginning of the present study, to examine whole cells for proteolytic activity by tube test, the content in the test tubes was read in a spectrophotometer at 280 m~ without colorization by the phenol reagents. But this method was found not to be applicable, because the content showed the maximum ultraviolet absorbanee not at 280 m~, but a t 265 mlz. This was supposed to be due to the intraeellular materials liberated by disruption of ceils in reaction mixtures. Similarly, the activity of crude enzymes prepared from cultural supernatants was assayed b y tube test, which was not successful either. After this failure, it was found that the crude enzymes contained a great amount of TCA-

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soluble substances, which could not be excluded b y persistent dialyzation to a large volume of buffer, was colored b y the phenol reagents, and showed the maximum ultraviolet absorbance at 280 m~. References Aluotto, B.B., Wittier, R. G., Williams, C. 0., Faber, J.E.: Standardized bacteriological techniques for the characterization of mycoplasma species. Int. J. Syst. Bact. 20, 35--58 (1970) Czekalowski, W., Hall, D. A., Woolclock, P.R.: Studies of proteoly~ic activity of myco. plasmas: Gelatinolytic property. J. gem Microbiol. 75, 125--133 (1973) Freundt, E. A.: The 5iycoplasmataceae (The pleuropneumonia group of organisms, morphology, biology and taxonomy). Copenhagen: Munksgaard 1958 Lowry, O. H., Rosenbrough, I~. J., Farr, A. L., Randal, R. J.: Protein measurement with the folin phenol reagent. J. biol. Chem. 193, 265--273 (1951) Razin, S., Oliver, Ofra.: Morphogenesis of mycoplasma and bacterial L-form colonies. J. gen. ~Iicrobiol. 24, 225--237 (1961) Rodwell, A. W.: Nutrition and metabolism of Mycoplasma mycoides vat. mycoides. Ann. N. Y. Acad. Sci. 79, 105--116 (1960) Rodwell, A. W., Abbot, A.: The function of glycerol, choresterol and long-chain fatty acids in the nutrition of Mycoplasma mycoides. J. gen. Microbiol. 25, 201--214 (1961) Smith, P. F., Boughton, J. E.: Role of protein and phospholipid in the growth of pleuropneumonia-like organisms. J. Bact. 80, 851--860 (1961) Watanabe, T., Mishima, K., Horikawa, T.: Proteolytie activity of human mycoplasmas. Jap. J. Microbiol. 17, 151--153 (1973) Watanabe, T . : Proteolytic activity of human oral mycoplasmas. Jap. J. Bact. 29, 269 (1974) Dr. Tsuguo Watanabe Department of Bacteriology School of Dental Medicine Tsurumi University 2-1-3, Tsurumi, Tsurumi-ku Yokohama Kanagawa Japan 230