The synergistic action of lysozyme and trypsin in bacteriolysis

The Synergistic

Action of Lysozyme and Trypsin in Bacteriolysisl

M. E. Becker* and S. E. Hartsell From the Laboratories

of Bacteriology, Cniversity,

Department of Uioloyicnl Lajayette, Indiana

Sciences,

Purtlu(

Received September 20, 1954 INTRODUCTION !&al-enzyme syst’ems cont’aining lysozyme w&h certain proteolytic enzymes are capable of lysing heated cells of several species of bacteria resistant, t’o either enzyme alone (I). The fartors affecting total lysis and synergistic action of lysozyme and trypsin on 23 test organisms ha\-c> been delineat’ed further. Extensive lysis was also obtained following pretreatment with agents ot,her than heat. By proper selection of the physical and/or chemical agent, any of the test species was rendered lysable by t’he enzyme combi-

nation. Dilute alkali and certain lipide solvents were especially effective. MATERIALS

AND METHODS

Preparation of Cell Suspensions Cells were grown in Roux bottles for l&24 hr. ou yeast,-water, veal-infusion media (14). After washing twice with double-distilled water, suspensions were treated with various chemical and/or physical agents, rewashed, and incubated with test enzyme (8).

Pretreatment with Physical Agents 1. Heat. Distilled-water 15 min. (1).

suspensions were pasteurized in tubes at 70-80°C. for

r These data constitute, in part, a thesis submitted to the Graduate School of Purdue University in partial fulfillment of the requirements for the Doctor of Philosophy degree. Grateful appreciation is expressed to the Purdue R.esearch Foundation for the research grant while completing these studies. * Present address: Maryland State Department of Health, Baltimore, Mar,~land. 257

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E.

BECKER

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S. E.

HARTSELL

2. Alternate Freezing and Thawing. Cells were frozen at -25°C. in phosphate buffer, pH 7.0. After storage at -9°C. for 1 day, they were thawed in a 25°C. water bath. In some experiments, suspensions were quick frozen in tubes placed in a methanol-Dry Ice bath for 5 min. and thawed at 25°C. The process of freezing and thawing was repeated five times. 3. Drying. It was thought that the effects of dehydration on the slime layer and cell protein might also enhance tryptic and/or lysozymic activity. Thick cell suspensions were dried at 45°C. for 24 hr. To study the effects of temperature, per se, as distinct from those due to drying, aliquots were incubated in tightly stoppered tubes under the same conditions. 4. Ultraviolet Light. Quartz tubes containing 20 ml. of distilled-water suspensions were clamped to a Burrell wrist-action shaker at a mean distance of 10 cm. from a 15-w. General Electric germicidal lamp. The total incident irradiation at 2537 A. was calculated to be 447 X 10-e w./sq. cm. By shaking the tubes during the ultraviolet treatment, complete reproductive death of Escherichia coli and Micrococcus lysodeikticus resulted within 2 hr., provided the cell concentration was not greater than lO*/ml.

Pretreatment with Chemical Agents Cells were suspended in test reagents for periods up to 1 hr. at 253O”C., or 15 min. at 45”C., then washed several times with distilled water or buffer. Buffer was always used for resuspending alkali-treated cells. To evaluate any inhibitory effects on subsequent enzymatic activity, O.Ol-0.1.ml. portions of the test reagent were added to sensitive suspensions containing trypsin and/or lysozyme. If such cells lysed to the same extent as control suspensions containing no reagent, it was assumed that any chemical retained after washing was of no consequence. Pretreatment with water-insoluble agents, e.g., chloroform, involved the removal of the supernatant and centrifugation of distilled water suspensions, emulsification of the moist, packed cells with the test reagent. After the incubation period, water was added; the two liquid phases were separated by centrifugation, and the water layer, containing most of the cells, was removed with a pipet.

Lytic Studies One-milliliter portions of cell suspensions were mixed with 7 ml: of 0.067 M phosphate buffer at plI 7.0. Then, 1 ml. of trypsin and/or lysozyme solution(s) was added, and the t.ot.al volume of all tubes was adjusted to 10 ml. with distilled water. The final conccnt,ration of each enzyme was 0.01 mg./ml. Lysis was followed spectrophotometrically (Coleman model 14) by noting lytic change after 1 hr. of incubation, or by lytic rate determinations. With the the initial turbidity of control exception of rsperiments on cell concentration, and test suspensions was adjusted to read between 20 and 35% transmission. The ctffcct of cnzymc action on subsequent solubilization of cell substance was also determined sp:ct.rophotometrically, after adjusting the pH to 9.5-10.5 with 1 X KaOII and further incubation at 45°C. for 15 min. The conditions in sequential enzyme experiments were the same as those involving concomit.ant act,ion, except that the incubation time with each enzyme was

SYNERGISTIC

ACTION

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half that employed in dual-enzyme experiments. incubation with the second enzyme.

259

Suspensions were washed before

RESULTS

E$ect of Pretreatment with Physical Agents The extent of lysis and synergism resulting from the concomitant action of lysozyme and trypsin on heated cells is influenced by the following factors: (a) pH, (b) cell and enzyme concentration, (c) age of cells, (d) test species, (e) severity of heat treatment, and (f) temperature of incubation. The last two factors were considered in a previous report (1). pH. With phosphate buffer, the greatest synergistic response occurred generally between pH 6.5 and 7.0, whereas either lysozyme or trypsin alone lysed optimally at pH 7.0 and 8.0, respectively. When the pH was increased above 8 by the use of Verona1 buffer, the lytic response of individual and of combined enzyme action decreased considerably. Buffers, other than phosphate, however, were generally less effective even at the same pH. It is of special interest that at each pH, maximum synergism often became evident at the lower incubation temperatures, as contrasted with the high optima of the enzymes individually (Fig. 1). The alkaline solubilization of cell substance after incubation with

0.7

LYW.CONC-IO-5g/ml TRYPSIN-

1’24ghl

t BUFFER- M/l5 PO4

k

f

‘I[

0 g ?

04

0 A A 0’

LYSOZYME TRYPSIN LYSO t TRYPSIN CALC. T t L

OM

FIG.

q \ h\/ da\i *

1

1

1. Effect of temperature

PH70 30 40 50 TEMPERATURE-C

60

0-0 I--. / PH 8.0 l-l304aww 20

and pH on lysis of heated E. coli.

260

M. E. BECKER AND 8. E. HABTSELL

PM 70 0

LYSjS Am3 (45 AT45C.)

q

FURTHER

INCW

WITH ENZYMES

LYSIS AFTER AIJWJ

*DnVON

LiT

FIG. 2. Effect of trypsin and lysozyme on alkaline solubility liaca .

of Sarcina UWUTZ-

enzyme(s) was useful in assessing otherwise inapparent changes. Often, trypsin and/or lysozyme produced no clearing until the cells were incubated further under alkaline conditions (Fig. 2). Using lysozyme alone on heated cells, the results were similar to those obtained by others with unheated cells (5,7, 11) ; i.e., lysozyme had a greater effect on subsequent alkaline solubility when cells had been preincubated at the more acid pH. However, the alkaline solubility was often reduced as much as 5075% by the heat treatment. Trypsin alone had much more effect on subsequent solubility when the pH was above 7. Using E. cdi as test resulting after simultaneous enzyme species, the alkaline solubility action was complete, regardless of the pH at which the enzymes had been incubated (Fig. 3). Cell Concentration. As determined both by lytic rate and total lysis, the greatest synergistic response occurred when cell concentrations of from 20 to 40% T were employed. Increasing the cell concentration to read less than 20 % T greatly decreased the lytic rate as determined by change in per cent T and/or density. Enzyme Concentration. Over the pH range 6-8, the optimum enzyme

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6.0

ACTION

65

pH DURING

FIG. 3. Effect

of trypsin

IN

and lysozyme

261

BACTERIOLYSIS

I

1.0

l.6

ENZYME

INCUBATION

on the alkaline

solubility

of heated

E. co&.

concentration in dual-enzyme systems was 1O-2mg./ml. for each enzyme. Increasing the concentration of either one, while holding the other constant at 10e2 mg./ml., failed to increase the over-all lytic response. In fact, lysozyme concentrations greater than 0.1 mg./ml. were less effective either alone or in combination with trypsin because of the formation of a fine precipitate. Chemical analysis indicated that a lysozymenucleic acid complex was responsible for the turbidity increase. This precipitation of nucleic acids by lysozyme has been reported by other authors (4, 9, 10). A definite stoichiometric amount of lysozyme appeared to be necessary to produce the effect. No measurable turbidity increase occurred with supernatants from heated control cells or those from unheated suspensions treated with lysozyme, and therefore it seemed probable that heat was required to labilize the bonds joining the lysozyme substrate with cell constituents, thereby removing interfering layers of nucleic acid (3). Age of Cells. If cultures were incubated on agar for extended periods, their sensitivity to test enzymes progressively decreased. Heated 8-day cells of E. coli were absolutely insensitive to lysozyme alone over the pH range 6-8, although 18-hr. cells elicited the usual 10-15 % change in transmission. A similar relationship obtained with heated M. EysodeiktiCUS,although it was generally lysed completely by the enzyme combination, regardless of age. The aging effect was noted only if cells were incu-

262

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S. E. HARTSELL

bated for several days on agar; for washed, 18-hr. suspensions, stored at 6-8°C. in distilled water or buffer for similar periods, were as sensitive as fresh suspensions of Whr. cells. In fact, refrigeration for periods longer than 5 days considerably enhanced lysability. Since storage at higher temperatures for an even shorter period of time was equally effective, it was assumed that the increase in sensitivity was conditioned by autolytic changes. Test Species. Of the 23 strains of bacteria tested, 17 manifested some degree of synergism (Fig. 4) when preheated 15 mm. at 70-80°C. Gramnegative species, especially of the genera Aerobacter, Escherichia, Salmonella, and Neisseria, elicited the greatest synergistic response as well as the most complete total lysis. Among heated gram-positive organisms, M. lysodeikticus and certain bacilli were extremely sensitive. Suspensions of bacilli consisted predominantly of vegetative cells, for spores were refractory altogether. Micrococcus pyogenes var. albus was also nonlysable either before or after heat treatment, although its resistance disappeared completely following treatment with dilute alkali. Note the variability among strains, especially those of Escherichiu and Micrococcus (Fig. 5). INCUB. I HR. AT 25- 27C * INCUB. I HR AT45 C HEAT TREATMENT 15’AT 70 OR 60 C PH70 I SYNERGISM 60.

Jo-

; g 40: 2 u 30-

20 -

IO -

o-

FIG. 4. Total on heated cells.

lytic

change and synergism

produced

by lysozyme

+

trypsin

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IN BACTERIOLYSIS

ALKALINE TREATMENT1 60 - PREVIOUS l/2 HR AT 25C. ALKALI THEN NELITRALIZED AND CELLS WASHED,

I.*1

50-

:

40-

5 I u I-

30-

INCUBATION WITH ENZYMES: I HR. AT 45C,pH 7.0 (PO4). THEN pH ADJUSTED TO 9.5 AND CELLS INCUBATED IS’LONGER.

* 20-

IO-

oE’IG.

t L T C,T. UNTREATED CELLS

.c CELLS PRETREATED WITH ALKALI

5. Effect of previous treatment with lysis of M. albus by trypsin and lysozyme.

4

ALKl

lysozyme

AFTER 2ND I ADDITION

in alkali

on subsequent

E$ect of Pretreatment with Physical Agents Other Than Heat Alternate Freezing and Thawing. In general, freezing and thawing effected only moderate changes in enzyme susceptibility. Lysozyme action was enhanced by only 5-15 %, while trypsin susceptibility usually remained unaltered, unless cells were frozen and thawed at least five times. Certain species, normally very resistant to the trypsin-lysozyme combination, e.g., M. pyogenes var. albus, were made to lyse 25-50% by this system after one freezing. The lysability of Sarcina aurantiaea remained unchanged after five freezings. E. coli became somewhat more sensitive after one freezing, but lysability was not appreciably enhanced by additional freezings. Drying. The drying of thick suspensions of E. coli at 45°C. in Petri plates had no significant effect on subsequent enzyme sensitivity. The usual effects of autolysis upon enzymic lysis were evident, i.e., an enhancement of lo-20% transmission in tryptic or lysozymic lysis and an additive response by the dual-enzyme system. Ultraviolet Light. Irradiation of E. coli and M. lysodeikticus suspensions for a period of 2 hr. failed to alter their enzyme sensitivity, even though the treatment had effected complete reproductive death. Thus, contrary to popular belief, death in this instance did not necessarily render protoplasm more susceptible to enzymatic digestion.

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Pretreatment with Chemical Agents Alkali. On gram-negative species, alkali usually increased subsequent trypsin response more than that of lysozyme. Lysis by the dual-enzyme system never quite equaled that on heated cells, however, and synergism was rarely evident. It was inadvisable to treat gram-negative bacteria with alkali more concentrated than 0.1 N, for above this concentration the reagent itself caused considerable clearing (cf. also the studies of Kaplan and Kaplan (8) on alkali and gram-negative cells). Moderate response was obtained with alkali as dilute as 0.001 N, although the optimum was generally 0.1 N under the preincubation conditions (0.5-l hr. at 25-27°C. or 15 min. at 45°C.). Gram-positive species, although generally much more resistant to the alkali alone than gram-negative types, exhibited t.he more striking response when subsequently incubated wit.h enzymes. M. pyogenes var. albus, normally an extremely resistant species, was completely lysed by the dual-enzyme system, and even lysozyme alone effected very considerable clearing (Fig. 5). Synergism was especially in evidence. It will be noted also that lysozyme action alone had conditioned alkaline solubility.

METHANOL PRmEATYENTINCUB. I HR Al 45 C.

l/2

HR Al 3OoC

60 I

!wNEReISY

50

40

f; 30

20

lo

Fra. 6.

Effectof methanolon subsequentlysis

of S. autantiaca.

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Trypsin was inert altogether on this organism. S. aurantiuea was lysed only if the alkali required at concentration was increased to 0.5 N. Again, subsequent tryptic lysis remained unaltered. The trypsin apparently became active on this organism only when present with lysozyme. In contrast M. pyogenes var. aureus became very sensitive to trypsin following alkali treatment. Alcohol. Pretreatment of gram-negative species with ethanol gave results similar to those obtained with alkali. The degree of synergism approached that obtained with heated cells. The time that the alcohol was allowed to react was found, as in the case of alkali, to make little difference, since the lytic response obtained after 15 min. was about 90 % as great as that after 1 hr. Of the gram-positive species tested, Bacillus megatherium and S. aurantiaca, both elicited moderate response to lysozyme plus trypsin. M. pyogenes var. albus was unaffected by alcohol pretreatment, even if the cells had previously been heated and/or disrupted. Generally, with gram-positive species, trypsin action was influenced much more than that of lysozyme. This is in contrast with results on alkali-treated cells of M. pyogenes var. albus and S. aurantiaca. Methanol. With E. coli and S. aurantiaca as test species, methanol effected extensive lysability by the dual-enzyme system (Fig. 6). About

FIG. 7. Effect

of CHCla

on subsequent

lysis

of S. aurantiaca.

266

M. E. BECKER AND S. E. HARTSELL

80% of the response in the latter case was synergistic. IMethanol was slightly more effective than ethanol; its optimum concentration was about 100 %, as contrasted with 70 % for ethanol. Chloroform. Although M. pyogenes var. albus was unaffected by pretreatment with this reagent, all other test species (8. aurantiaca, Sarcina Mea, and E. coli) were rapidly lysed when the two enzymes were allowed to act concomitant.ly. Tryptic action was enhanced more than that of lysozyme. In the case of S. aurantiuca, however, virtually the entire dual-enzyme response was synergistic (Fig. 7). DISCUSSION

Experimentation with lysozyme and trypsin on both chemically and physically pretreated cells has shown that this particular enzyme combination is capable of completely lysing many species of bacteria. The lytic rate, as well as the total lysis effected by the combination, was often greater than that which could be accounted for on the basis of additive action of individual enzymes and was, therefore, considered as being a synergistic response. The conditioning of enzymatic lysis by the various pretreating agents probably involves complex physical and/or chemical changes in cell components. Any explanation of the synergistic response would, therefore, require a consideration first of the possible changes resulting from pretreatment, and second, of how these changes might have influenced the action of individual enzymes. Autolysis could not have contributed toward the over-all lytic change, since Warburg studies and specific enzyme assays indicated that these cells were enzymatically inactive (1). Loss of permeability is commonly thought to be responsible for the enhanced tryptic digestibility of heated cells. Experimentation on disintegrated cells (1) does not support this hypothesis, however. Moreover, although tryptic activity is enhanced by pasteurization, lysozymic lysis, especially of very sensitive strains, is greatly decreased by this treatment. It would be difficult to explain the latter change on the basis of permeability. If we accept the theory that denaturation of protein in some way renders the lysozyme substrate unavailable (6), we still must formulate a new hypothesis to account for the increased lysozyme sensitivity of E. coli after heating. Perhaps, in this species, the heat, by removing nucleic acids, exposes underlying layers of mucopolysaccharide to enzymic attack (2, 3). It is noteworthy that, with the exception of Pseudomonas pulrejaciens

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267

and two species of Bacillus, trypsin was inactive altogether on unheated cells. In view of the fact that trypsin is most active on denatured protein, it is possible that denaturation is responsible for the increased tryptic lysis of heated cells. Reagents, such as alcohol and dilute alkali may also have similarly affected cell proteins, at least of gram-negative types, for both agents enhanced tryptic lysis, especially on gram-negative cells. These reagents did not affect tryptic lysis of M. pyogenes var. albus or S. aurantiaca, however, although these organisms were rendered moderately sensitive to lysozyme alone and were completely lysed by t’he dual-enzyme system. In the case of certain gram-positive types, then, it would appear that the degree of protein denaturation was not a critical factor, since tryptic digestion was not enhanced by the above treatment. The fact that the synergistic action of lysozyme and trypsin completely lysed these cells, would suggest that the lysozyme substrate was, itself, interfering with tryptic action, perhaps as a bound form with protein. That such interference of trypic activity does occur is further supported by experiments on the sequential rather than concomitant action of the t’wo enzymes. When lysozyme was added first to alkali-t#reated cells of M. pyogenes var. albus or S. aurantiaca, the over-all lytic change was twice as great as that which resulted when trypsin was added first, followed by lysozyme. The sequential enzyme action on certain heated gram-negative bacteria has given similar results (1). The sensitivity of bacteria to lysozyme is sometimes attributed to the amount and availability of specific acetylaminopolysaccharide substrate within the cell. M. pyogenes var. albus, for example, is normally completely refractory to lysozyme, and attempts to extract specific substrate have failed (6) ; yet, as has been noted, this species was quite extensively lysed by lysozyme alone, provided the cells were first treated with dilute alkali. This sensitivity, in itself, is strong evidence that, lysozyme substrate is present in substantial amounts. In fact, by the proper selection of a pretreating agent, it was possible to demonstrate lysozyme sensitivity with every species tested. It is quite apparent from t#heseresults, and t’hose of Peterson (II), that the lysozyme substrate is much more widely di&ributed in the cell and among species than was formerly believed. Salton (12) has postulated that melting of cell wall lipides by heat could enhance tryptic activity by allowing for better enzyme-substrate contact. Perhaps this theory might be extended to include lysozyme sensitivity as well, since the lipide solvents often enhanced the activit,y of

268

M.

E.

BECKER AND 5. E. HABTSELL

both enzymes. Chloroform and methanol, for example, were especially active in conditioning the enzymatic lysis of E. wli, an organism whose cell wall consists essentially of lipide materials (13). It, is probable that most of the pretreating agents used in these experiments, including heat, alkali, or lipide solvents, could have effectively removed such lipide materials. Undoubtedly, all factors included in the above discussion contribute to the synergism observed in these dual-enzyme investigations. It is believed that each organism is unique with regard to each one of these factors, and would become lysable by similar dual-enzyme systems if subjected t.o the proper pretreating agent. ACKNOWLEDGMENTS The authors aish for his assistance in New York, and Dr. Engineering, Purdue

to express their sincere appreciation to Dr. Warren Menke the calibration of the ultraviolet lamp and to Carl Demrick, Burton Wilner, Department of Chemical and Metallurgical University, for the Italian translations. SUMMARY

The degree of synergism resulting from the concomitant action of lysozyme and trypsin on heated cells was influenced by cell and enzyme concentration, age, and pH of incubation. The enzyme system completely lysed species of the following genera: Aerobacter, Eschmichia, Neisseria, Salmonella, Sarcina, Micrwmwus, and Bacillus. Moderate response was elicited by species of Proteus, Serratia, Pseudomonas, and Streptomyces. A similar synergistic response has been observed on cells pretreated with agents other than heat. Certain chemicals, e.g., dilute alkali and lipoidal solvents, conditioned the enzymatic lysis of M. pyogenes var. aureus, M. pyogenesvar. albus and S. aurantiaca, species which hitherto could not be lysed by the lysozyme-trypsin combination. The implications of these studies as regards the presence and disposition of cellular substrate have been discussed. REFERENCES 1.

M., AND HARTSELL, S. E., drch.Biochem. and Biophys. 6!l, 402-10 (1954). L., Boll. ist. sieroterap. milan. N, 364-72 (1950). L., Bull. World Health Org. 8. 3-17 (1952). P., AND CARTA, R., Riu. biol. (Perugia) 43.547-64 (1951). 5. CONGRAM,L., Master’s Thesis, Purdue Univ., Lafayette, Indiana, 1951. 6. EPSTEIN, L., AND CHAIN, E., Brit. J. Ezpll. Pathol. !U. 339-55 (1940).

BECKER, 2. CALIFANO, 3. CALIFANO, 4. CASELLI,

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7. GRULA, E., ANDHARTSELL,S. E., J. Bacterial. 88,302-6 (1954). 8. KAPLAN,M., ANDKAPLAN,L., J. Bactetiol. 26, 309-21 (1933). 9. KLOTZ, I., ANDWALKER,F., Arch. Biochem. 18, 319-25 (1948). 10. NIHOUL, E., MASSART,L., ANDVAN HEEL, G., Arch. intern. pharmacodynamie 88, 123-5 (1951). 11. PETERSON, R., Master’s Thesis, Purdue University, Lafayette, Indiana, 1954. 12. SALTON,M., J. Gem Microbial. 9, 512-23 (1953). 13. SALTON,M., Biochim. et Biophys. Acta 10, 512-23 (1953). 14. SMOLELIS,A., ANDHARTSELL,S. E., J. Bactetiol. 68, 731-6 (1949).