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J Med Microbiol 55 (2006), 505-510; DOI: 10.1099/jmm.0.46333-0
© 2006 Society for General Microbiology
ISSN 1473-5644

Dependence of the lethal effect of pore-forming haemolysins of Gram-positive bacteria on cytolytic activity

Isao Watanabe1, Takamasa Nomura1, Takanari Tominaga1, Kazuhiro Yamamoto1, Chikara Kohda2, Ikuo Kawamura1 and Masao Mitsuyama1

1 Department of Microbiology, Kyoto University Graduate School of Medicine, Yoshidakonoe-cho, Sakyo-ku, Kyoto 606-8501, Japan

2 Department of Bacteriology, Showa University School of Medicine, Tokyo 142-8555, Japan

Correspondence
Masao Mitsuyama
mituyama{at}mb.med.kyoto-u.ac.jp

Received 16 September 2005
Accepted 12 January 2006

Among bacterial haemolysins, cholesterol-dependent cytolysins (CDCs) produced by various Gram-positive bacteria are known to exhibit a lethal activity in mice. In this study, recombinant CDCs of streptolysin O, pneumolysin, ivanolysin O, listeriolysin O and several listeriolysin O mutants were constructed and the relationship between cytolytic activity and the lethal activity of each recombinant protein in mice was examined. Specific activity for cytolysis was determined by a quantitative haemolytic assay. Each protein was injected intravenously into mice and the lethal activity was evaluated by measuring the time until death of the mice. The four full-length CDC proteins exhibited lethal activity and their activities were highly proportional to their cytolytic activities. Inhibition of haemolytic activity resulted in the loss of lethal activity and non-haemolytic mutants of listeriolysin O did not exhibit any lethal activity. These data clearly indicate that the lethal effect of CDC proteins is dependent on the cytolytic activity.

Abbreviations: CDC, cholesterol-dependent cytolysin; HU, haemolytic unit; ILO, ivanolysin O; LLO, listeriolysin O; PLY, pneumolysin; SLO, streptolysin O.


   INTRODUCTION
TOP
 INTRODUCTION
METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Cholesterol-dependent cytolysins (CDCs) are a family of structurally related cytolytic protein toxins produced by different species of Gram-positive bacteria belonging to the genera Streptococcus, Listeria, Clostridium and others (Alouf, 1999; Billington et al., 2000). These toxins, traditionally known as haemolysins, are characterized by a highly conserved undecapeptide sequence (ECTGLAWEWWR) near the C terminus, which is essential for their cytolytic activity (Michel et al., 1990; Owen et al., 1994; Sekino-Suzuki et al., 1996). They are capable of binding directly to membrane cholesterol and forming transmembrane pores, resulting in cell lysis.

CDC proteins have also been known to exhibit lethal activity in experimental animals. Over 50 years ago, Howard & Wallace (1953b) reported the lethal action of streptolysin O (SLO). Since then, a similar lethal effect has also been observed in other CDC toxins – listeriolysin O (LLO), pneumolysin (PLY) and perfringolysin O (Bernheimer, 1976). The molecular mechanism of lethal activity has not been well elucidated, but the primary target of the lethal action appears to be the heart. In mice given an intravenous injection of a crude preparation of LLO, Kingdon & Sword (1970) observed a lethal arrhythmia as demonstrated by an electrocardiogram. Halbert et al. (1961) and Alouf & Palmer (1999) also showed that rabbits and mice given a lethal dose of SLO suffered from conduction defects and ventricular disturbances.

Although a number of previous reports suggest that cytolytic activity observed in vitro is related to the expression of lethal activity in vivo, there has been very limited information to show any direct evidence for a contribution of cytolytic activity to the lethal effect. In order to make this point clear, we constructed a variety of recombinant CDC proteins, including LLO, ivanolysin O (ILO), SLO, PLY, a non-cytolytic truncated LLO deficient for the C terminus and LLO mutants with replacement of 1 aa in the conserved undecapeptide region, which exhibited variable cytolytic activities. Each recombinant protein was examined for specific haemolytic activity in vitro and lethal activity in mice.


   METHODS
TOP
 INTRODUCTION
 METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Mice. Female C3H/HeN and C3H/HeJ mice were used for the experiments at 7 weeks of age. All experimental procedures on mice were approved by the Animal Ethics and Research Committee of Kyoto University Graduate School of Medicine.

Construction and purification of various recombinant proteins. We generated various CDC proteins as 6xHis-tagged recombinant proteins as listed in Table 1Go. Briefly, the gene encoding the mature CDC protein was amplified by PCR using the primers shown in Table 2Go and ligated into the pQE31 vector (Qiagen). For the construction of LLO mutant with replacement of 1 aa in the undecapeptide region, the region containing the undecapeptide to the C terminus was amplified by PCR with a mutagenic primer and the 3' LLO primer (Table 2Go). A secondary PCR was performed with the 5' LLO primer and the PCR product in which the mutation was introduced as the 3' primer. The resultant second PCR product was ligated into the pQE31 vector. The recombinant plasmid was electroporated into Escherichia coli SG13009. The recombinant E. coli clone was cultured in tryptic soy broth at 25 °C for 4–6 h in the presence of 1 mM IPTG. Bacterial lysate was prepared and recombinant protein was purified with a Ni–nitrilotriacetic acid agarose column (Qiagen). The buffer was replaced with PBS by passing through PD10 desalting columns (Amersham Pharmacia Biotech AB). The level of LPS was determined by a Limulus colour KY test (Wako Pure Chemical Industries). Purity was determined by Coomassie brilliant blue staining after SDS-PAGE. Each recombinant protein prepared in this study showed a single band of the predicted molecular mass following SDS-PAGE analysis (data not shown).


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  		Table 1. Recombinant cytolysins used in this study

 

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  		Table 2. Oligonucleotide primers used in this study

 
Assay for haemolytic activity of recombinant cytolysins. Recombinant protein was diluted twofold with PBS and incubated with an equal volume of 1 % sheep erythrocytes for 30 min at 37 °C. Supernatant was collected and the A415 of released haemoglobin was measured. One haemolytic unit (HU) was defined as the amount of recombinant protein required for 50 % haemolysis.

Determination of lethal toxicity of CDCs. Five C3H/HeN mice per group were injected intravenously with LLO, ILO, SLO, PLY or LLO mutants and survival time (t) was measured in seconds. Lethal activity of CDCs was defined as 1000/t. In some experiments, LLO was treated with 10 µg cholesterol ml–1 overnight at 4 °C or by heating at 94 °C for 15 min to inhibit haemolytic activity. To exclude the possible contribution of contaminating LPS to the lethal effect of CDCs, we employed LPS-non-responsive C3H/HeJ mice for measurement of lethal activities.

Statistical analysis. Student's t-test was used to determine the statistical significance of the values obtained and a value of P<0·05 was considered statistically significant.


   RESULTS AND DISCUSSION
TOP
 INTRODUCTION
 METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Dose-dependent expression of the lethal activity of LLO

In order to determine whether the lethal effect of LLO was exhibited in a dose-dependent manner, mice were injected intravenously with graded doses of LLO (Fig. 1Go). The lethal effect was not observed in mice injected with less than 12·5 pmol LLO. After injection of 100 pmol LLO (equivalent to 5·6 µg), mice quickly succumbed with a mean survival time of 55 s. The survival time decreased gradually with an increase in the dose of LLO. Mice given 800 pmol died as early as 26 s after the injection.


Figure 1
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  		Fig. 1. Dose-dependent lethal activity of LLO. Graded doses of LLO were injected intravenously into C3H/HeN mice and survival time (t) was monitored in seconds (n=5 for each group). Lethal activity was expressed as 1000/t. Data represent the mean±SD and are representative of three independent experiments.

 
It is known that LPS induces a lethal shock to mice by inducing high titres of endogenous cytokines (Pfeffer et al., 1993; Rothe et al., 1993). As the LLO prepared in this study contained a small amount of LPS [0·188 ng (µg LLO protein)–1], the contaminating LPS might have been involved in the lethal activity of LLO. To rule out this possibility, we injected 100 pmol LLO into LPS-non-sensitive C3H/HeJ mice and monitored survival time. There was no significant difference between the mean survival time of C3H/HeN (55±6·7 s) and C3H/HeJ (51±6·7 s) mice, indicating that contaminating LPS was not involved in the lethal effect of LLO. In addition, mice did not die without showing any symptoms when given 5 ng E. coli LPS, roughly five times higher than the dose of LPS contaminating the standard injecting dose of LLO in this study.

Critical requirement of haemolytic activity for the lethal effect of LLO

A previous report has shown that the lethal effect of LLO is prevented by neutralization of the haemolytic activity (Geoffroy et al., 1987). When 100 pmol LLO was subjected to treatment with cholesterol or was heated at 94 °C for 15 min, the haemolytic activity was reduced from 538 to 15·6 and 1·0 HU, respectively. These treatments completely abolished the lethal activity of LLO in mice (data not shown). Furthermore, we investigated the lethal effect of LLO 415, a truncated LLO mutant that was deficient for the C-terminal domain 4 and did not bind cholesterol (Kohda et al., 2002). The truncated LLO 415 showed neither haemolytic activity nor lethal activity, indicating that the cytolytic activity of LLO is exclusively required for expression of the lethal effect in vivo. Geoffroy et al. (1987) reported that purified native LLO showed a lethal toxicity to mice and that this toxicity was inhibited completely by treatment with cholesterol or antiserum or by heating, which is consistent with our present findings.

Lethal effect and cytolytic activity in other CDC proteins

It is known that various protein toxins of the CDC family cause haemolysis by a common mechanism (Alouf, 1999; Billington et al., 2000). If the cytolytic process of LLO is necessary for the lethal effect in vivo, it is possible that other CDC toxins also exhibit a lethal effect in proportion to their haemolytic activities. To address this point, we generated recombinant ILO, SLO and PLY. Based on the lethal activity of LLO, we injected 100 pmol each toxin and monitored the survival time (Fig. 2Go). As expected, all CDCs exerted a lethal effect; however, there were differences in activity among the recombinant CDC proteins examined. LLO exerted the strongest lethal effect, whilst the activity of ILO was somewhat weaker than LLO. SLO and PLY showed a significantly weaker activity than LLO. Interestingly, these lethal activities paralleled their haemolytic activities, as LLO induced a strong haemolysis, with the activity of ILO being somewhat weaker than that of LLO, and SLO and PLY showing markedly weaker haemolytic activities compared with LLO. These data indicated that there is a strong relationship between haemolytic activity and the lethal effect of CDCs.


Figure 2
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  		Fig. 2. Lethal effects of several CDCs. LLO, ILO, SLO or PLY (100 pmol) was injected intravenously into C3H/HeN mice and survival time (t) was monitored in seconds. Dots indicate the lethal activities of recombinant CDC proteins. Bars indicate the mean of lethal activities. The haemolytic activity of each 100 pmol haemolysin is also indicated (HU). Data are representative of three independent experiments.

 
To confirm this further, we generated several mutant LLOs exhibiting different cytolytic activities. An undecapeptide located in domain 4 in the C-terminal region is believed to be critical for binding to cholesterol on the cell membrane and for cytolysis (Michel et al., 1990; Owen et al., 1994; Sekino-Suzuki et al., 1996; Baba et al., 2001). The requirement of domain 4 was clearly shown by the loss of cytolytic and lethal activities in a preparation of LLO containing only domains 1–3 (LLO 415) (Fig. 3Go). It has been reported that replacement of a unique cysteine residue located within the undecapeptide of LLO by site-directed mutagenesis resulted in just a minor loss of haemolytic activity. In contrast, a remarkable decrease in the cytolytic activity was demonstrated after replacement of one of the three hydrophobic tryptophan residues in the undecapeptide (Michel et al., 1990). Thus, we generated four LLO mutants: LLO C484S, which showed a minor decrease in the haemolytic activity, and LLO W489A, LLO W491A and LLO W492A, in which the cytolytic activity was mostly greatly diminished. Although LLO C484S retained its lethal activity, its mean survival time was extended twofold (Fig. 3Go). The two non-haemolytic mutant proteins, LLO W489A and LLO W491A, did not exhibit any lethal activity. In the group of mice given LLO W492A, in which the haemolytic activity was severely affected, six out of 10 mice survived. Based on these results, it seems that the lethal effect of CDCs is highly dependent on the cytolytic process of CDCs. In order to show more clearly the correlation between the lethal effect and haemolytic activity, we plotted the lethal activity (1000/t) data against the cytolytic activity (HU) of CDCs and LLO mutants and found that the correlation coefficient (r=0·911) was significant. Furthermore, Howard & Wallace (1953a, b) showed that rabbits were more sensitive to the lethal action of SLO than mice and that rabbit erythrocytes were also sensitive to cytolysis by SLO compared with mouse erythrocytes. Halbert et al. (1963) reported a similar potency of SLO. These reports support the correlation between cytolytic and lethal activities.


Figure 3
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  		Fig. 3. Lethal effects of LLO and LLO mutants. LLO or an LLO mutant (100 pmol) was injected intravenously into C3H/HeN mice and survival time (t) was monitored in seconds. Dots indicate the lethal activities of LLO and the LLO mutants. Bars indicate the mean of lethal activities. Haemolytic activities of LLO and the LLO mutants injected into mice are indicated (HU). Data are representative of three independent experiments.

 
As mentioned above, cholesterol-treated LLO displayed no detectable lethal effect, despite retaining a haemolytic activity of 15·6 HU. In contrast, the injection of similar cytolytic activity (18 HU) of the LLO C484S mutant protein resulted in a clear, albeit reduced, lethal effect in this experiment. In this regard, it was also conflicting that LLO W492A exhibiting only 1·6 HU displayed some lethal activity. Judging from the dose-dependency of the lethal activity of LLO shown in Fig. 1Go, there appears to be a threshold level of ~100 pmol of active molecule required for lethal activity. Therefore, it is possible that a small number of active cytolytic molecules remaining intact after cholesterol treatment were not able to cause the final cardiotoxicity, while all of the molecules of mutant proteins with a very low specific activity were engaged in affecting cardiac function. A second possible explanation for the discrepancy in the activity of cholesterol-treated LLO may be an altered in vivo distribution after injection. As cholesterol treatment results in the formation of protein–cholesterol complexes in vitro, it is conceivable that, due to hydrophobic interactions, such complexes form a microaggregation with which the remaining active LLO molecules associate and then are cleared from circulation more quickly than other soluble mutants. A third possibility is that a small proportion of active LLO remaining after cholesterol treatment is more susceptible to the blocking effect of plasma cholesterol in the circulation compared with the susceptibility of mutants with far lower specific activity. At present, we have no direct evidence to explain the abolished lethality following cholesterol treatment whilst retaining some haemolytic activity. Due to the difficulty in completely abolishing haemolytic activity with in vitro cholesterol treatment, it seems that more than one of the above-mentioned mechanisms may be involved.

Many studies have suggested that CDCs affect cardiac function (Halbert, 1970; Kingdon & Sword, 1970; Bernheimer, 1976). It has been found that certain substances with anti-serotonin activity protect animals against the acute lethal effects of SLO (Halbert et al., 1963). The extremely rapid alteration in cardiac function elicited by SLO might result from the release of vasoactive substances rather than from the direct cytotoxic action of the protein itself (Alouf & Palmer, 1999). Kingdon & Sword (1970) reported that creatine phosphokinase levels in plasma increased after an injection of haemolysin, suggesting that haemolysin causes damage to the myocardium. In the present study, it was shown clearly that the cytolytic activity of any of the CDC proteins was indispensable for the expression of lethal activity in vivo. However, the molecular mechanism for the cardiotoxicity is yet to be determined at the molecular level. The various recombinant CDC proteins constructed in this study may be useful tools in this line of study.

We have shown here that CDCs are capable of exhibiting lethal activity in mice when injected intravenously in the form of purified recombinant protein. In the case of infection with bacteria producing CDC, however, it is open to argument whether CDC protein contributes to pathogenesis by exhibiting such a direct lethal effect. For instance, LLO, a major virulence factor of Listeria monocytogenes, enables the bacterium to escape from macrophage phagosomes. It has been reported that the cytolytic activity is exerted only in acidic conditions and not in neutral to alkaline conditions as observed in the cytoplasm (Geoffroy et al., 1987). Therefore, it is unlikely that LLO, even if released into the circulation, affects the cardiac function of the host infected with L. monocytogenes. On the other hand, it has been shown that PLY is a virulence determinant of Streptococcus pneumoniae and contributes to its pathogenesis by exerting a haemolytic activity (Jounblat et al., 2003). In addition, SLO is known to be required for the virulence of Streptococcus pyogenes; however, mutation in the gene encoding SLO resulted in little effect on lethality in infected mice and SLO may contribute to virulence by an unknown mechanism other than lethal toxicity (Limbago et al., 2000). Taken together, these results indicate that the direct engagement of cardiotoxicity of the CDC protein does not seem to be essential for the lethality in the infected host, although this activity has long been known as one of the biological activities of the CDC family of toxins.


   ACKNOWLEDGEMENTS
 
This study was supported by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology and a Grant-in-Aid for Scientific Research (B, C) from the Japan Society for the Promotion of Sciences, Japan.


   REFERENCES
TOP
 INTRODUCTION
 METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Alouf, J. E. (1999). Introduction to the family of the structurally related cholesterol-binding cytolysins (‘sulfhydryl-activated’ toxins). In The Comprehensive Sourcebook of Bacterial Protein Toxins, pp. 443–456. Edited by J. E. Alouf & J. H. Freer. London: Academic Press.

Alouf, J. E. & Palmer, M. W. (1999). Streptolysin O. In The Comprehensive Sourcebook of Bacterial Protein Toxins, pp. 457–475. Edited by J. E. Alouf & J. H. Freer. London: Academic Press.

Baba, H., Kawamura, I., Kohda, C., Nomura, T., Ito, Y., Kimoto, T., Watanabe, I., Ichiyama, S. & Mitsuyama, M. (2001). Essential role of domain 4 of pneumolysin from Streptococcus pneumoniae in cytolytic activity as determined by truncated proteins. Biochem Biophys Res Commun 281, 37–44.[CrossRef][Medline]

Bernheimer, A. W. (1976). Sulfhydryl activated toxins. In Mechanisms in Bacterial Toxinology, pp. 85–97. Edited by A. W. Bernheimer. New York: Wiley.

Billington, S. J., Jost, B. H. & Songer, J. G. (2000). Thiol-activated cytolysins: structure, function and role in pathogenesis. FEMS Microbiol Lett 182, 197–205.[CrossRef][Medline]

Geoffroy, C., Gaillard, J.-L., Alouf, J. E. & Berche, P. (1987). Purification, characterization, and toxicity of the sulfhydryl-activated hemolysin listeriolysin O from Listeria monocytogenes. Infect Immun 55, 1641–1646.[Abstract/Free Full Text]

Halbert, S. P. (1970). Streptolysin O. In Microbial Toxins, vol. III, pp. 69–98. Edited by T. C. Montie, S. Kaids & S. J. Ajl. New York: Academic Press.

Halbert, S. P., Bircher, R. & Dahle, E. (1961). The analysis of streptococcal infections. V. Cardiotoxicity of streptolysin O for rabbits in vivo. J Exp Med 113, 759–784.[Abstract/Free Full Text]

Halbert, S. P., Bircher, R. & Dahle, E. (1963). Studies on the mechanism of the lethal toxic action of streptolysin "O" and the protection by certain antiserotonin drugs. J Lab Clin Med 61, 437–452.[Medline]

Howard, J. G. & Wallace, K. R. (1953a). The comparative resistances of the red blood cells of various species to haemolysis by streptolysin O and by saponin. Br J Exp Pathol 34, 181–184.[Medline]

Howard, J. G. & Wallace, K. R. (1953b). The comparative resistances of rabbits, guinea pigs and mice to the lethal actions of streptolysin O and saponin. Br J Exp Pathol 34, 185–190.[Medline]

Jounblat, R., Kadioglu, A., Mitchell, T. J. & Andrew, P. W. (2003). Pneumococcal behavior and host responses during bronchopneumonia are affected differently by the cytolytic and complement-activating activities of pneumolysin. Infect Immun 71, 1813–1819.[Abstract/Free Full Text]

Kingdon, G. C. & Sword, C. P. (1970). Cardiotoxic and lethal effects of Listeria monocytogenes hemolysin. Infect Immun 1, 373–379.[Abstract/Free Full Text]

Kohda, C., Kawamura, I., Baba, H., Nomura, T., Ito, Y., Kimoto, T., Watanabe, I. & Mitsuyama, M. (2002). Dissociated linkage of cytokine-inducing activity and cytotoxicity to different domains of listeriolysin O from Listeria monocytogenes. Infect Immun 70, 1334–1341.[Abstract/Free Full Text]

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Michel, E., Reich, K. A., Favier, R., Berche, P. & Cossart, P. (1990). Attenuated mutants of the intracellular bacterium Listeria monocytogenes obtained by single amino acid substitutions in listeriolysin O. Mol Microbiol 4, 2167–2178.[CrossRef][Medline]

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