Vol. 135, No. 1
JOURNAL OF BACTERIOLOGY, July 1978, p. 153-160 0021-9193/78/0135-0153$02.00/0 Copyright X) 1978 American Society for Microbiology
Printed in U.S.A.
Cellular Lysis of Streptococcus faecalis Induced with Triton X-100 JAMES B. CORNETT* AND GERALD D. SHOCKMAN Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140 Received for publication 14 February 1978
Lysis of exponential-phase cultures of Streptococcus faecalis ATCC 9790 was induced by exposure to both anionic (sodium dodecyl sulfate) and nonionic (Triton X-100) surfactants. Lysis in response to sodium dodecyl sulfate was effective only over a limited range of concentrations, whereas Triton X-100induced lysis occurred over a broad range of surfactant concentrations. The data presented indicate that the bacteriolytic response of growing cells to Triton X100: (i) was related to the ratio of surfactant to cells and not the surfactant concentration per se; (ii) required the expression of the cellular autolytic enzyme system; and (iii) was most likely due to an effect of the surfactant on components of the autolytic system that are associated with the cytoplasmic membrane. The possibility that Triton X-100 may induce cellular lysis by releasing a lipid inhibitor of the cellular autolytic enzyme is discussed. Surface-active agents (surfactants) have been known for many years to possess bactericidal activity (46) and in some cases bacteriolytic activity as well (20). The bactericidal effects of ionic surfactants were attributed to their ability to electrostatically bind to and denature cellular enzymes (46) or to disrupt the cellular membrane, causing the leakage of vital cellular components to the surrounding medium (20), as observed during hemolysis of erythrocytes after treatment with detergents (26). The lytic effects of surfactants on bacteria, however, required a more complex interpretation due to the presence of a rigid peptidoglycan layer surrounding bacterial cells (47), which is not present in erythrocytes. The ability of surfactants to solubilze biological membranes (19) cannot adckiunt for the dissolution of bacterial cells whos'e cell wall peptidoglycan layer cannot be solubilized by a variety of detergents (32), including boiling sodium dodecyl sulfate (SDS) (47). Even gram-negative bacterial species, whose cellular envelope is largely composed of inner and outer membranes (13), are not susceptible to detergent-induced lysis unless the outer membrane and peptidoglycan layers are first disrupted with a lysozymeethylenediaminetetraacetic acid treatment (2, 17) or unless the cells are metabolically unbalanced by pretreatment with KCN (48) or UV irradiation (40). Among the gram-positive bacteria, whose peptidoglycan layer is considerably thicker than that of gram-negative organisms, some species
are very susceptible to surfactant-induced lysis. Beginning with the report by Lord and Nye (25) and subsequent studies by Avery and McCullen (1) and Dubos (14) on the deoxycholate-induced lysis of Streptococcus pneumoniae, it was realized that this surfactant did not actually lyse the cells, but rather initiated cellular lysis, an event which ultimately relied upon the action of cellular peptidoglycan hydrolytic (autolytic) enzymes. Dubos (14) clearly demonstrated that treatments which destroyed the autolytic enzyme activity of pneumococci (pH, iodination, heat, etc.) also prevented bacteriolysis upon subsequent exposure to deoxycholate and concluded that lysis resulted only when "an agent or a procedure not only kills the cel without destroying the autolytic enzymes, but also maintains conditions favorable for this activity." The requirement for functional autolytic enzyme activity during deoxycholate-induced lysis of S. pneumoniae has been considerably strengthened by the studies of an autolytic-defective strain of this bacterium (24, 42, 43). Bacteriolysis induced by ionic surfactants has been previously reported for such gram-positive cocci as S. pneumoniae (14) and Staphylococcus aureus (12), both of which are known to harbor potent autolytic enzyme systems (21, 39). Surfactants which can initiate cellular lysis by virtue of the cellular content of autolytic activity would provide a useful means by which to select for variant cell types with defective autolytic enzyme(s). In particular, such surfactants would be effective indicators of cellular autolytic activ153
154
J. BACTERIOL.
CORNETT AND SHOCKMAN
exponentially growing cultures of S. faecalis (about 0.1 mg of cellular dry weight per ml) resulted in a decrease in turbidity, followed by resumption of growth (Fig. 1A). As the concentration of SDS was increased, rates of cellular lysis decreased (Fig. 1A), and growth did not recover. The rather narrow range of SDS concentration observed to induce lysis of growing cultures appeared to be related to the ratio of SDS to cells (weight/weight) rather than to SDS concentrations per se (Fig. 1B). Effects of TX on exponentially growing cultures. In contrast to SDS, the lytic response induced by TX continued to increase with increasing detergent concentration (Fig. 2A). Similar to SDS, lysis in response to TX treatment was related to the ratio of TX to cells calculated (37). For some experiments (as indicated), cultures on a weight-to-weight basis (Table 1). For exwere grown in medium (S broth) consisting of 1% ponentially growing cultures (0.2 mg of cells per tryptone (Difco Laboratories), 1% yeast extract ml), ratios of TX to cells (weight/weight) of (Difco), 2% glucose, and 0.3 M sodium phosphate (pH approximately 0.2 or more stopped further 7). The turbidity of experimental cultures was moni- growth and resulted in cellular lysis. Cellular tored at 675 nm with a Coleman spectrophotometer. growth could eventually resume with lower levFor determining cell mass, optical density (OD) values els of TX (as seen with SDS), whereas even were adjusted to agree with Beer's Law, yielding ad- lower levels of TX merely reduced cellular justed OD (AOD) units (41) where an AOD value of growth. 1.0 is equivalent to 0.39 mg of cellular dry weight per Effect of TX on activity of the S. faecalis ml. Cellular lysis. Cells from exponentially growing autolytic peptidoglycan hydrolase. Unlike cultures were filtered (MiUipore Corp., 0.45-,um pore SDS, which rapidly inactivates wall-bound audiameter), washed on the filters with ice-cold, double- tolytic enzyme (38), relatively high concentra-
ity in nongrowing cells, a condition in which penicillin and other cell wall antibiotics are not effective (5, 9, 11, 16, 30). As noted by Dubos (14), surfactants of this type must be able to initiate cellular autolysis without reducing the activity of the endogenous autolytic enzyme(s). This report describes aspects of the susceptibility and the resistance of cultures of S. faecalis ATCC 9790 to lysis induced by the nonionic detergent Triton X-100 (TX). The results reported here clearly indicate that the ability of TX to induce cellular lysis requires the participation, and expression, of an active autolytic enzyme system. MATERIALS AND METHODS Growth of bacteria. S. faecalis ATCC 9790 was routinely grown at 37°C in chemically defined medium
distilled water, resuspended into the appropriate lysis buffer at room temperature, and then incubated at 37°C. Lysis due to the action of surfactants or penicillin G was induced by the addition of these agents directly to the growing cultures in growth medium. All detergent concentrations are given in percentage values (wt/vol). Except where noted otherwise, cellular autolysis was measured by the decrease in turbidity at 450 nm (Bausch and Lomb Spectronic 20). Rates of cellular autolysis were calculated from the linear portion of the curve describing the exponential decrease in turbidity with time (27) and were expressed in units (k, in h-1) describing the time required for the culture turbidity to decrease by one-half. For cultures which increased in turbidity (i.e., growth), k was determined on a similar basis and given as a negative value. Other procedures. Crude soluble autolysin was extracted in the latent (proteinase-activable) form from aqueous suspensions of isolated S. faecalis walls (20 mg/ml) at 0°C with 0.01 N NaOH (10) or obtained as supernatant fluids of trypsin-speeded autolysates of isolated cell walls (38). Protoplasts of S. faecalis were prepared exactly as described by Roth et al. (31). SDSinactivated cell wall substrate for the assay of autolytic enzyme activity was prepared as described previously (38). TX was purchased from Rohm and Haas (Philadelphia, Pa.) or Sigma Chemical Co. (St. Louis, Mo.). SDS was purchased from Fisher Scientific Co. (Fair Lawn, N.J.).
RESULTS Effects of SDS on exponentially growing cultures. The addition of SDS (10 ,ug/ml) to
A
B 0
0~~~~~~~1
00.1
50.
Jr-
JOURNAL OF BACTERIOLOGY, July 1978, p. 153-160 0021-9193/78/0135-0153$02.00/0 Copyright X) 1978 American Society for Microbiology
Printed in U.S.A.
Cellular Lysis of Streptococcus faecalis Induced with Triton X-100 JAMES B. CORNETT* AND GERALD D. SHOCKMAN Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140 Received for publication 14 February 1978
Lysis of exponential-phase cultures of Streptococcus faecalis ATCC 9790 was induced by exposure to both anionic (sodium dodecyl sulfate) and nonionic (Triton X-100) surfactants. Lysis in response to sodium dodecyl sulfate was effective only over a limited range of concentrations, whereas Triton X-100induced lysis occurred over a broad range of surfactant concentrations. The data presented indicate that the bacteriolytic response of growing cells to Triton X100: (i) was related to the ratio of surfactant to cells and not the surfactant concentration per se; (ii) required the expression of the cellular autolytic enzyme system; and (iii) was most likely due to an effect of the surfactant on components of the autolytic system that are associated with the cytoplasmic membrane. The possibility that Triton X-100 may induce cellular lysis by releasing a lipid inhibitor of the cellular autolytic enzyme is discussed. Surface-active agents (surfactants) have been known for many years to possess bactericidal activity (46) and in some cases bacteriolytic activity as well (20). The bactericidal effects of ionic surfactants were attributed to their ability to electrostatically bind to and denature cellular enzymes (46) or to disrupt the cellular membrane, causing the leakage of vital cellular components to the surrounding medium (20), as observed during hemolysis of erythrocytes after treatment with detergents (26). The lytic effects of surfactants on bacteria, however, required a more complex interpretation due to the presence of a rigid peptidoglycan layer surrounding bacterial cells (47), which is not present in erythrocytes. The ability of surfactants to solubilze biological membranes (19) cannot adckiunt for the dissolution of bacterial cells whos'e cell wall peptidoglycan layer cannot be solubilized by a variety of detergents (32), including boiling sodium dodecyl sulfate (SDS) (47). Even gram-negative bacterial species, whose cellular envelope is largely composed of inner and outer membranes (13), are not susceptible to detergent-induced lysis unless the outer membrane and peptidoglycan layers are first disrupted with a lysozymeethylenediaminetetraacetic acid treatment (2, 17) or unless the cells are metabolically unbalanced by pretreatment with KCN (48) or UV irradiation (40). Among the gram-positive bacteria, whose peptidoglycan layer is considerably thicker than that of gram-negative organisms, some species
are very susceptible to surfactant-induced lysis. Beginning with the report by Lord and Nye (25) and subsequent studies by Avery and McCullen (1) and Dubos (14) on the deoxycholate-induced lysis of Streptococcus pneumoniae, it was realized that this surfactant did not actually lyse the cells, but rather initiated cellular lysis, an event which ultimately relied upon the action of cellular peptidoglycan hydrolytic (autolytic) enzymes. Dubos (14) clearly demonstrated that treatments which destroyed the autolytic enzyme activity of pneumococci (pH, iodination, heat, etc.) also prevented bacteriolysis upon subsequent exposure to deoxycholate and concluded that lysis resulted only when "an agent or a procedure not only kills the cel without destroying the autolytic enzymes, but also maintains conditions favorable for this activity." The requirement for functional autolytic enzyme activity during deoxycholate-induced lysis of S. pneumoniae has been considerably strengthened by the studies of an autolytic-defective strain of this bacterium (24, 42, 43). Bacteriolysis induced by ionic surfactants has been previously reported for such gram-positive cocci as S. pneumoniae (14) and Staphylococcus aureus (12), both of which are known to harbor potent autolytic enzyme systems (21, 39). Surfactants which can initiate cellular lysis by virtue of the cellular content of autolytic activity would provide a useful means by which to select for variant cell types with defective autolytic enzyme(s). In particular, such surfactants would be effective indicators of cellular autolytic activ153
154
J. BACTERIOL.
CORNETT AND SHOCKMAN
exponentially growing cultures of S. faecalis (about 0.1 mg of cellular dry weight per ml) resulted in a decrease in turbidity, followed by resumption of growth (Fig. 1A). As the concentration of SDS was increased, rates of cellular lysis decreased (Fig. 1A), and growth did not recover. The rather narrow range of SDS concentration observed to induce lysis of growing cultures appeared to be related to the ratio of SDS to cells (weight/weight) rather than to SDS concentrations per se (Fig. 1B). Effects of TX on exponentially growing cultures. In contrast to SDS, the lytic response induced by TX continued to increase with increasing detergent concentration (Fig. 2A). Similar to SDS, lysis in response to TX treatment was related to the ratio of TX to cells calculated (37). For some experiments (as indicated), cultures on a weight-to-weight basis (Table 1). For exwere grown in medium (S broth) consisting of 1% ponentially growing cultures (0.2 mg of cells per tryptone (Difco Laboratories), 1% yeast extract ml), ratios of TX to cells (weight/weight) of (Difco), 2% glucose, and 0.3 M sodium phosphate (pH approximately 0.2 or more stopped further 7). The turbidity of experimental cultures was moni- growth and resulted in cellular lysis. Cellular tored at 675 nm with a Coleman spectrophotometer. growth could eventually resume with lower levFor determining cell mass, optical density (OD) values els of TX (as seen with SDS), whereas even were adjusted to agree with Beer's Law, yielding ad- lower levels of TX merely reduced cellular justed OD (AOD) units (41) where an AOD value of growth. 1.0 is equivalent to 0.39 mg of cellular dry weight per Effect of TX on activity of the S. faecalis ml. Cellular lysis. Cells from exponentially growing autolytic peptidoglycan hydrolase. Unlike cultures were filtered (MiUipore Corp., 0.45-,um pore SDS, which rapidly inactivates wall-bound audiameter), washed on the filters with ice-cold, double- tolytic enzyme (38), relatively high concentra-
ity in nongrowing cells, a condition in which penicillin and other cell wall antibiotics are not effective (5, 9, 11, 16, 30). As noted by Dubos (14), surfactants of this type must be able to initiate cellular autolysis without reducing the activity of the endogenous autolytic enzyme(s). This report describes aspects of the susceptibility and the resistance of cultures of S. faecalis ATCC 9790 to lysis induced by the nonionic detergent Triton X-100 (TX). The results reported here clearly indicate that the ability of TX to induce cellular lysis requires the participation, and expression, of an active autolytic enzyme system. MATERIALS AND METHODS Growth of bacteria. S. faecalis ATCC 9790 was routinely grown at 37°C in chemically defined medium
distilled water, resuspended into the appropriate lysis buffer at room temperature, and then incubated at 37°C. Lysis due to the action of surfactants or penicillin G was induced by the addition of these agents directly to the growing cultures in growth medium. All detergent concentrations are given in percentage values (wt/vol). Except where noted otherwise, cellular autolysis was measured by the decrease in turbidity at 450 nm (Bausch and Lomb Spectronic 20). Rates of cellular autolysis were calculated from the linear portion of the curve describing the exponential decrease in turbidity with time (27) and were expressed in units (k, in h-1) describing the time required for the culture turbidity to decrease by one-half. For cultures which increased in turbidity (i.e., growth), k was determined on a similar basis and given as a negative value. Other procedures. Crude soluble autolysin was extracted in the latent (proteinase-activable) form from aqueous suspensions of isolated S. faecalis walls (20 mg/ml) at 0°C with 0.01 N NaOH (10) or obtained as supernatant fluids of trypsin-speeded autolysates of isolated cell walls (38). Protoplasts of S. faecalis were prepared exactly as described by Roth et al. (31). SDSinactivated cell wall substrate for the assay of autolytic enzyme activity was prepared as described previously (38). TX was purchased from Rohm and Haas (Philadelphia, Pa.) or Sigma Chemical Co. (St. Louis, Mo.). SDS was purchased from Fisher Scientific Co. (Fair Lawn, N.J.).
RESULTS Effects of SDS on exponentially growing cultures. The addition of SDS (10 ,ug/ml) to
A
B 0
0~~~~~~~1
00.1
50.
Jr-
