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Susceptibility of Salmonella typhimurium and Salmonella typhi to oxygen metabolites
FEMS Microbiology Immunology 47 (1989) 279-284 Published by Elsevier
279
FIM 00052
Susceptibility of Salmonella typhimurium and Salmonella typhi to oxygen metabolites Yoshio Ishibashi a n d T o s h i h i k o Arai Department of Microbiology, Meiji College of Pharmacy, Nozawa, Tokyo, Japan Received 19 December 1988 Accepted 11 January 1989
Key words: Salmonella typhimurium; Salmonella typhi; Oxygen metabolites
1. SUMMARY The susceptibility of Salmonella typhimurium LT2 and of S. typhi 1079 to oxygen metabolites were compared. S. typhimurium LT2 and S. typhi 1079 were killed to an equal extent (about 40%) by the xanthine-xanthine oxidase (200 m U / m l ) system. Among the various scavengers of oxygen metabolites, catalase alone inhibited the killing of S. typhimurium LT2 and S. typhi 1079 by the xanthine-xanthine oxidase system, indicating that hydrogen peroxide contributed to the killing of Salmonellae. The respiratory burst of murine macrophages was efficiently triggered by the ingestion of S. typhimurium LT2, S. typhimurium SLll02, and S. typhi 1079 and all to the same extent. However, in the range of the concentration of hydrogen peroxide produced by murine macrophages, neither S. typhimurium LT2 nor S. typhi 1079 were killed. Only S. typhimurium SLll02, a rough mutant of S. typhimurium LT2, was markedly susceptible under these conditions. The findings suggest that both S. typhimurium LT2
Correspondence to: Yoshio Ishibashi, Department of Microbiology, Meiji College of Pharmacy, Nozawa, Setagaya-ku, Tokyo 154, Japan.
and S. typhi 1079 are resistant to oxygen-dependent killing mechanisms.
2. I N T R O D U C T I O N
Salmonella typhimurium is a facultative intracellular pathogen that causes a systemic infection in mice, whereas S. typhi, the agent of human typhoid, fails to cause disease in mice [1,2]. The mechanisms responsible for the species-specific pathogenicity of Salmonella remain unknown. Reactive oxygen metabolites such as superoxide anion (O£), hydrogen peroxide (H202) , hydroxy radical (-OH), and singlet oxygen (102) derived from the phagocytic cell respiratory burst are well known to play an important part in host resistance to microorganisms [3]. Recent studies have suggested that some intracellular pathogens may prevent microbicidal activity either by being resistant to oxygen metabolites or by inhibiting the generation of oxygen metabolites from phagocytes [4,8]. These findings give rise to the possibility that the species-specific pathogenicity of Salmonellae could be caused by differential susceptibility to oxygen metabolites or by a differential capacity to trigger the respiratory burst of macrophages. In this study, therefore, we attempted to
0920-8534/89/$03.50 © 1989 Federation of European Microbiological Societies
280 investigate the susceptibilities of S. typhimurium and S. typhi to oxygen metabolites, and the abilities of these strains to trigger the respiratory burst of macrophages.
3. M A T E R I A L S A N D M E T H O D S
3.1. Bacteria Salmonella typhimurium LT2 wild type and its LPS mutant strains SLl102, which possess no somatic repeating units and outer core [9], were used. S. typhi 1079 was a clinical isolate from a patient in China. Escherichia coli N I H J was used as a non-virulent strain. Bacteria were grown in L-broth at 3 7 ° C and harvested during exponential growth. Bacterial concentrations were determined by measuring the turbidity at 660 nm [10] and adjusted to the desired concentrations in Dulbecco's phosphate buffered saline (PBS).
3.2. Reagents Xanthine (Grade III), xanthine oxidase (Grade III), catalase, superoxide dismutase (SOD), and mannitol were purchased from Sigma Chemical Co., St. Louis, MO. Benzoate and hydrogen peroxide was obtained from Kanto Chemical Co., Ltd., Tokyo, Japan. 2,2,2-Diazabicyclooctane (DABCO) was purchased from Hani Chemical Co., Ltd., Tokyo, Japan.
3. 3. Cell free microbicidal assays The susceptibility of bacteria to oxygen metabolites generated by the xanthine-xanthine oxidase system was assayed as described previously [7]. Various concentrations of xanthine oxidase were added to bacterial suspensions (2 x 103 C F U / m l ) in a total volume of 0.5 ml of pBS containing 600/~m xanthine. After incubation for 1 h at 37 ° C, 0.1-ml aliquots were plated on L-agar plates in duplicate and the resulting colonies were counted. Susceptibility of bacteria to hydrogen peroxide was assessed in a similar way. A series of concentrations of hydrogen peroxide were added to the bacterial suspension (2 x 103 C F U / m l ) in a total volume of 0.5 ml of PBS and incubated for 1 h at 37 ° C. Bacterial viability was determined by the colony count.
3.4. Preparation of macrophage monolayers Resident peritoneal macrophages were obtained by washing the peritoneal cavity of B A L B / c mice with R P M I 1640 containing 5 /~g/ml of gentamicin and 5 U / m l of heparin as described previously [1]. Macrophages were washed three times and suspended at a concentration of 1 x 106/ml in R P M I 1640-GM supplemented with 10% fetal bovine serum (FBS). The macrophage suspensions were plated on 24-well tissue culture plates (Falcon Co., Ltd.) and incubated for 1 h at 37 °C. After incubation, macrophage monolayers (5 x 10 s cells/well) were rinsed with R P M I 1640G M to remove the nonadherent cells, and cultured with R P M I 1640-GM supplemented with 10% FBS for 2 days.
3.5. Assay for hydrogen peroxide generation by macrophages Generation of hydrogen peroxide by macrophages was assessed as follows. Bacteria were opsonized with 10% fresh serum for 30 min at 37 o C, washed with PBS and then suspended at a concentration of 5 × 108 C F U / m l in PBS. To the macrophage monolayers (5 x 10 s cells/well), were added 10/zl of the opsonized bacteria (5 × 108/ml) in a total volume of 0.5 ml of PBS, and the cells were then incubated at 3 7 ° C for 1 h. The concentration of hydrogen peroxide in the culture supernatant was measured using a peroxidase-absorbed carbon anode, a silver cathode, and o-dianisidine as the substrate of peroxidase as described previously [11].
3.6. Statistical analysis Statistical analysis was performed using Student's two-tailed t-test for independent means.
4. R E S U L T S
4.1. Susceptibility of Salmonellae to xanthine-xanthine oxidase Susceptibilities of S. typhimurium LT2, SLll02, and S. typhi 1079 to oxygen metabolites generated by the xanthine-xanthine oxidase system were examined. As shown in Fig. 1, S. typhimurium LT2 and S. typhi 1079 were killed by oxygen
281 100
more susceptible to oxygen metabolites than was S. typhimurium LT2 ( P < 0 . 0 5 ) . S. typhimurium SLl102, a rough mutant of S. typhimurium LT2, was considerably more susceptible to oxygen metabolites than S. typhimurium LT2, and approximately 60% of S. typhimurium SLl102 were killed by 200 m U / m l of xanthine oxidase ( P < 0.05). Xanthine oxidase or xanthine alone did not influence the viabilities of these bacteria (data not shown).
v
>, 50
I
I
i
I
2OO
0
Xanthine oxidase
I
400 ( mU/m| )
Fig. 1. Susceptibility of Salmonellae to xanthine-xanthine oxidase. Various concentrations of xanthine oxidase were added to the suspension containing 2 × 1 0 3 C F U / m l of S. typhimurium LT2 (@), SLl102 (©), or S. typhi 1079 (zx) in a total volume of 0.5 ml of PBS with 600 /~M xanthine, and incubated for 1 h at 3 7 ° C . Values represent the mean_+ standard error of five experiments.
metabolites in a dose-dependent manner. No difference was seen in the susceptibilities of these strains to up to 200 m U / m l of xanthine oxidase ( P > 0.05). The viabilities of S. typhimurium LT2 and S. typhi 1079 at 200 m U / m l of xanthine oxidase were 60.9 + 4.7% and 54.7 + 10.8%, respectively. At a high dose (400 m U / m l ) of xanthine oxidase, S. typhi 1079 was found to be
4.2. Effects of scavengers of oxygen metabolites on bacterial killing by xanthine-xanthine oxidase To determine which oxygen metabolite contribute to the killing of Salmonellae, we examined the effects of oxygen metabolite scavengers on killing by the xanthine-xanthine oxidase system. As shown in Table 1, only catalase, which reduces hydrogen peroxide to H 2 0 and 02, effectively inhibited killing. Heated catalase had no inhibiting effect. On the other hand, superoxide dismutase (SOD) (which promotes the conversion of O f to hydrogen peroxide), mannitol and benzoate (scavengers of OH), and DABCO (a quencher of 102) all failed to inhibit the bacterial activity of the xanthine-xanthine oxidase system. Mannitol, benzoate, and DABCO themselves did not influence the viability of the bacteria (data not shown). These results indicated that hydrogen peroxide contributed to the killing of Salmonellae sp.
Table 1 Effect of oxygen intermediate scavengers on killing by xanthine-xanthine oxidase Scavenger
None SOD (1000U/ml) Catalase (100 U / m l ) Inactivated catalase Mannitol (50 mM) Benzoate (10mM) DABCO (1 mM)
Viability (%)
S. typhimurium
S. typhimurium
S. typhi
LT2
SLl102
1079
45.9+ 9.3 41.6_+ 5.7 104.7 _+11.7 * * 29.7_+ 8.2 36.6 _+ 6.0 45.0_+ 8.6 38.4_+ 5.9
30.2_+ 2.2 35.4_+ 4.0 127.0 _+13.3 * * 25.2_+ 5.5 39.5 _+ 6.4 37.1_+ 4.5 32.6+ 1.8
30.6+10.8 16.2_+ 7.4 121.5 _+16.4 33.8_+ 15.7 21.9 _+ 7.9 17.8_+ 5.6 22.8_+ 6.5
The reaction mixtures containing bacteria (2 × 103 C F U / m l ) , xanthine oxidase (400 m U / m l ) , 600/xM xanthine, and scavengers at indicated concentrations in a total volume of 0.5 ml of PBS were incubated for 1 h at 37 o C. Values represent the mean + standard errors of four experiments. ** P < 0.01.
282
manner, and S. typhi 1079 was more susceptible to H 2 0 2 than S. typhimurium LT2 ( P < 0 . 0 1 ) . In contrast, approximately 60% of S. typhimurium SLl102 were killed by a low dose of H 2 0 2 (2.5 X 10 - 6 M ) . These findings were consistent with the results obtained by the xanthine-xanthine oxidase system.
4.4. Hydrogen peroxide generation by macrophages associated with the phagocytosis of Salmonellae
2.5xlff 6
2.Sxlff 5
H202
2.5x10 ~
( M )
Fig. 2. Susceptibility of Salmonellae to hydrogen peroxide. Various concentrations of hydrogen peroxide were added to the suspension containing 2 x 103 C F U / m l of S. typhimurium LT2 (O), SLll02 (o), or S. typhi 1079 (zx) in a total volume of 0.5 ml of PBS, and incubated for 1 h at 3 7 ° C . Values represent the mean + standard errors of five experiments.
4.3. Susceptibility of Salmonellae to hydrogen peroxide To confirm the effect of hydrogen peroxide on the killing of Salmonellae, the susceptibility of S. typhimurium LT2, SLl102, and S. typhi 1079 to hydrogen peroxide was examined. As shown in Fig. 2, both S. typhimurium LT2 and S. typhi 1079 were resistant to up to 1.25 x 10 -s M H 2 0 2. At higher concentration of H 2 0 2 , ( > 2.5 X 10 s M) these strains were killed in a dose-dependent
Table 2 Phagocytosis-induced hydrogen peroxide generation by macrophages nmol of hydrogen peroxide/ 5 × 105 cells/60 min
E. S. S. S.
coli NIHJ typhimurium LT2 typhimurium SLl102 typhi 1079
2.54+_0.38 2.15+_0.90 1.74_ 0.33 1.43 _+0.77
The macrophage monolayers (5 X 105 cells/well) were infected with opsonized bacteria (5 x 106 CFU) in a total volume of 500 ffl of PBS containing Ca 2+ and Mg 2+, and incubated for 1 h at 37 o C. Values represent the mean + standard errors of four experiments.
It has been demonstrated that some pathogens fail to trigger superoxide generation by macrophages [4,5,8]. We therefore examined the ability of S. typhimurium LT2, SLl102, and S. typhi 1079 to trigger hydrogen peroxide generation by murine macrophages in comparison with that of E. coli N I H J . As shown in Table 2, both S. typhimurium LT2 and S. typhi 1079 triggered the respiratory burst as efficiently as did E. coil N I H J . Although the hydrogen peroxide generation associated with phagocytosis of S. typhimurium SLl102 by macrophages was slightly lower than that of S. typhimurium LT2, the difference was not significant.
5. D I S C U S S I O N In this study we have examined the susceptibility of S. typhimurium and S. typhi to oxygen metabolites. The susceptibility of S. typhi 1079 to the xanthine-xanthine oxidase system was similar to that of S. typhimurium LT2, although S. typhi 1079 was more susceptible to oxygen metabolites than was S. typhimurium LT2 at a high concentration of xanthine oxidase (400 m U / m l ) . We found that among the various oxygen metabolites, hydrogen peroxide contributed to the killing of Salmonellae. However, both S. typhimurium LT2 and S. typhi 1079 were resistant to up to 1.0 x 10 s M of hydrogen peroxide. Although it is difficult to estimate the concentration of hydrogen peroxide within the phagocytic vacuole, hydrogen peroxide produced by phagocytes is known to exist in an equilibrium between vacuole, cytosolic, and extracellular milieus, because hydrogen peroxide can permeate the cytoplasmic membrane and diffuse extracellularly [12]. Therefore, the extracellular concentration of hydrogen peroxide may reflect
283 the intraphagosomal concentration of h y d r o g e n peroxide. Tsunawaki et al. [13] have reported that PMA-stimulated mouse peritoneal macrophages are capable of generating 2 n m o l of HzOz/90 r a i n / 1 0 6 cells. We have an almost similar result in that S. typhimurium LT2 and S. typhi 1079-infected macrophages p r o d u c e d 1.4-2.2 n m o l of hydrogen p e r o x i d e / 5 × 10 5 cells/60 min, which is equivalent to 2.8-4.4 × 10 6 M. In the range of this concentration of hydrogen peroxide, neither S. typhimurium LT2 nor S. typhi 1079 were killed. W e c a n n o t rule out the possibility that the concentration of h y d r o g e n peroxide in phagosomes m a y be transiently higher than that in extracellular medium. However, Weiss et al. [14] have reported that the neutrophils from a patient with chronic granulomatous disease (unable to generate a respiratory burst) kill ingested S. typhimurium almost as effectively as the neutrophils f r o m a normal individual, and that depletion of O 2 b y N 2 flushing has no effect on the killing of S. typhimurium by neutrophils. These observations support our finding that b o t h S. typhimurium and S. typhi were resistant to oxygen-dependent killing mechanisms. It therefore seems likely that the species-specific pathogenicity of Salmonellae is not due to differential susceptibility to o x y g e n metabolites. Cell wall m u t a n t s of S. typhimurium have been reported to be highly sensitive to complement c o m p o n e n t s [15] and bactericidal permeability-increasing protein [16]. In this study, we have demonstrated that S. typhimurium SLl102, a rough m u t a n t of LT2, was sensitive to h y d r o g e n peroxide. It has been demonstrated that Mycobacterium leprae, Toxoplasma gondii, and Histoplasma capsulatum fail to trigger superoxide generation f r o m macrophages [4,6,8]. We found that S. typhimurium LT2, S. typhimurium SLl102, and S. typhi 1079 efficiently triggered h y d r o g e n peroxide generation by murine macrophages. This result suggested that the capacity of Salmonellae to trigger the respiratory burst is not correlated with pathogenicity. C o m p l e m e n t c o m p o n e n t s in fresh serum play an important role in non-specific defense mechanisms to bacterial infections [17]. However, we found that both S. typhimurium LT2 and S. typhi
1079 were resistant to 20% fresh mouse serum, whereas S L l 1 0 2 was markedly susceptible to fresh mouse serum (data not shown). Thus, the speciesspecific pathogenicity of Salmonellae might not be due to differential susceptibility to fresh serum components. In conclusion, our present data demonstrated that species-specific pathogenicity of Salmonellae m a y be due neither to differential susceptibility to oxygen metabolites nor to the differential capacity to trigger the respiratory burst of macrophages. Different degrees of susceptibility to the oxygen-independent killing mechanisms m a y therefore be responsible for the specific pathogenicity of Salmonellae, and we are currently exploring this possibility.
REFERENCES [1] Lissner, C.R., Weinstein, D.L. and O'Brien, A.D. (1985) Mouse chromosome 1 Ity locus regulates microbicidal activity of isolated peritoneal macrophages against a diverse group of intracellular and extracellular bacteria. J. Immunol. 135, 544-547. [2] O'Brien, A.D. (1982) Innate resistance of mice to Salmonella typhi infection. Infect. Immun. 38, 948-952. [3] Beaman, L. and Beaman, B.L. (1984) The role of oxygen and its derivatives in microbial pathogenesis and host defense. Ann. Rev. Microbiol. 38, 27-48. [4] Eissenberg, L.G. and Goldman, W.E. (1987) Histoplasma capsulatum fails to trigger release of superoxide from macrophages. Infect. Immun. 55, 29-34. [5] Holzer, T.J., Nelson, K.E., Schauf, V., Grispen, R.G. and Andersen, B.R. (1986) Mycobacterium leprae fails to stimulate phagocytic cell superoxide anion generation. Infect. Immun. 51,514-520. [6] Murray, H.W. and Cohn, Z.A. (1979) Macrophage oxygen-dependent antimicrobial activity. I. Susceptibifity of Toxoplasma gondii to oxygen intermediates. J. Exp. Med. 150, 938-949. [7] Murray, H.W. (1981) Susceptibility of Leishmania to oxygen intermediates and killing by normal macrophages. J. Exp. Med. 153, 1302-1315. [8] Wilson, C.B., Tsai, V. and Remington, J.S. (1980) Failure to trigger the oxidative metabolic burst by normal macrophages. Possible mechanism for survival of intracellular pathogens. J. Exp. Med. 151, 328-346. [9] Nakano, M. and Saito, K. (1969) Chemical components in the cell wall of Salmonella typhimurium affecting its virulence and immunogenicity in mice. Nature 222, 1085-1086. [10] Stendahl, O., Tagesson, C., Magnusson, K.E. and Edebo, L. (1977) Physico-chemical consequences of opsonization of Salmonella typhimurium with hyperimmune IgG and complement. Immunol. 32, 11-18.
284 [11] Iwai, H. and Akihama, S. (1982) Determination of hydrogen peroxide with peroxidase adsorbed carbon electrode. Yakugaku Zasshi 102, 1120-1123. [12] Test, S.T. and Weiss, S.J. (1984) Quantitative and temporal characterization of the extracellular H202 pool generated by human neutrophils. J. Biol. Chem. 259, 399-405. [13] Tsunawaki, S. and Nathan, C.F. (1984) Enzymatic basis of macrophage activation. Kinetic analysis of superoxide production in lysates of resident and activated mouse peritoneal macrophages and granulocytes. J. Biol. Chem. 259, 4305-4312. [14] Weiss, J., Stendahl, O. and Elesbach, P. (1982) Killing of gram-negative bacteria by polymorphonuclear leukocytes. Role of an 02-independent bactericidal system. J. Clin. Invest. 69, 959-970.
[15] Joiner, K.A., Hammer, C.H., Brown, J.E., Cole, R.J. and Frand, M,M. (1982) Studies on the mechanism of bacterial resistance to complement-mediated killing. I. Terminal components are deposited and released from Salmonella minnesota $218 without causing bacterial death. J. Exp. Med. 155, 797-808. [16] Rest, R.F., Cooney, M.J. and Spitznagel, J.K. (1977) Susceptibility of lypopolysaccharide mutants to the bactericidal action of human neutrophil lysosomal fractions. Infect. Immun. 16, 145-151. [17] Joiner, K.A., Brown, E.J. and Frank, M.M. (1985) Complement and bacteria. Chemistry and biology in host defense. Ann. Rev. Immunol. 2, 461-491.
279
FIM 00052
Susceptibility of Salmonella typhimurium and Salmonella typhi to oxygen metabolites Yoshio Ishibashi a n d T o s h i h i k o Arai Department of Microbiology, Meiji College of Pharmacy, Nozawa, Tokyo, Japan Received 19 December 1988 Accepted 11 January 1989
Key words: Salmonella typhimurium; Salmonella typhi; Oxygen metabolites
1. SUMMARY The susceptibility of Salmonella typhimurium LT2 and of S. typhi 1079 to oxygen metabolites were compared. S. typhimurium LT2 and S. typhi 1079 were killed to an equal extent (about 40%) by the xanthine-xanthine oxidase (200 m U / m l ) system. Among the various scavengers of oxygen metabolites, catalase alone inhibited the killing of S. typhimurium LT2 and S. typhi 1079 by the xanthine-xanthine oxidase system, indicating that hydrogen peroxide contributed to the killing of Salmonellae. The respiratory burst of murine macrophages was efficiently triggered by the ingestion of S. typhimurium LT2, S. typhimurium SLll02, and S. typhi 1079 and all to the same extent. However, in the range of the concentration of hydrogen peroxide produced by murine macrophages, neither S. typhimurium LT2 nor S. typhi 1079 were killed. Only S. typhimurium SLll02, a rough mutant of S. typhimurium LT2, was markedly susceptible under these conditions. The findings suggest that both S. typhimurium LT2
Correspondence to: Yoshio Ishibashi, Department of Microbiology, Meiji College of Pharmacy, Nozawa, Setagaya-ku, Tokyo 154, Japan.
and S. typhi 1079 are resistant to oxygen-dependent killing mechanisms.
2. I N T R O D U C T I O N
Salmonella typhimurium is a facultative intracellular pathogen that causes a systemic infection in mice, whereas S. typhi, the agent of human typhoid, fails to cause disease in mice [1,2]. The mechanisms responsible for the species-specific pathogenicity of Salmonella remain unknown. Reactive oxygen metabolites such as superoxide anion (O£), hydrogen peroxide (H202) , hydroxy radical (-OH), and singlet oxygen (102) derived from the phagocytic cell respiratory burst are well known to play an important part in host resistance to microorganisms [3]. Recent studies have suggested that some intracellular pathogens may prevent microbicidal activity either by being resistant to oxygen metabolites or by inhibiting the generation of oxygen metabolites from phagocytes [4,8]. These findings give rise to the possibility that the species-specific pathogenicity of Salmonellae could be caused by differential susceptibility to oxygen metabolites or by a differential capacity to trigger the respiratory burst of macrophages. In this study, therefore, we attempted to
0920-8534/89/$03.50 © 1989 Federation of European Microbiological Societies
280 investigate the susceptibilities of S. typhimurium and S. typhi to oxygen metabolites, and the abilities of these strains to trigger the respiratory burst of macrophages.
3. M A T E R I A L S A N D M E T H O D S
3.1. Bacteria Salmonella typhimurium LT2 wild type and its LPS mutant strains SLl102, which possess no somatic repeating units and outer core [9], were used. S. typhi 1079 was a clinical isolate from a patient in China. Escherichia coli N I H J was used as a non-virulent strain. Bacteria were grown in L-broth at 3 7 ° C and harvested during exponential growth. Bacterial concentrations were determined by measuring the turbidity at 660 nm [10] and adjusted to the desired concentrations in Dulbecco's phosphate buffered saline (PBS).
3.2. Reagents Xanthine (Grade III), xanthine oxidase (Grade III), catalase, superoxide dismutase (SOD), and mannitol were purchased from Sigma Chemical Co., St. Louis, MO. Benzoate and hydrogen peroxide was obtained from Kanto Chemical Co., Ltd., Tokyo, Japan. 2,2,2-Diazabicyclooctane (DABCO) was purchased from Hani Chemical Co., Ltd., Tokyo, Japan.
3. 3. Cell free microbicidal assays The susceptibility of bacteria to oxygen metabolites generated by the xanthine-xanthine oxidase system was assayed as described previously [7]. Various concentrations of xanthine oxidase were added to bacterial suspensions (2 x 103 C F U / m l ) in a total volume of 0.5 ml of pBS containing 600/~m xanthine. After incubation for 1 h at 37 ° C, 0.1-ml aliquots were plated on L-agar plates in duplicate and the resulting colonies were counted. Susceptibility of bacteria to hydrogen peroxide was assessed in a similar way. A series of concentrations of hydrogen peroxide were added to the bacterial suspension (2 x 103 C F U / m l ) in a total volume of 0.5 ml of PBS and incubated for 1 h at 37 ° C. Bacterial viability was determined by the colony count.
3.4. Preparation of macrophage monolayers Resident peritoneal macrophages were obtained by washing the peritoneal cavity of B A L B / c mice with R P M I 1640 containing 5 /~g/ml of gentamicin and 5 U / m l of heparin as described previously [1]. Macrophages were washed three times and suspended at a concentration of 1 x 106/ml in R P M I 1640-GM supplemented with 10% fetal bovine serum (FBS). The macrophage suspensions were plated on 24-well tissue culture plates (Falcon Co., Ltd.) and incubated for 1 h at 37 °C. After incubation, macrophage monolayers (5 x 10 s cells/well) were rinsed with R P M I 1640G M to remove the nonadherent cells, and cultured with R P M I 1640-GM supplemented with 10% FBS for 2 days.
3.5. Assay for hydrogen peroxide generation by macrophages Generation of hydrogen peroxide by macrophages was assessed as follows. Bacteria were opsonized with 10% fresh serum for 30 min at 37 o C, washed with PBS and then suspended at a concentration of 5 × 108 C F U / m l in PBS. To the macrophage monolayers (5 x 10 s cells/well), were added 10/zl of the opsonized bacteria (5 × 108/ml) in a total volume of 0.5 ml of PBS, and the cells were then incubated at 3 7 ° C for 1 h. The concentration of hydrogen peroxide in the culture supernatant was measured using a peroxidase-absorbed carbon anode, a silver cathode, and o-dianisidine as the substrate of peroxidase as described previously [11].
3.6. Statistical analysis Statistical analysis was performed using Student's two-tailed t-test for independent means.
4. R E S U L T S
4.1. Susceptibility of Salmonellae to xanthine-xanthine oxidase Susceptibilities of S. typhimurium LT2, SLll02, and S. typhi 1079 to oxygen metabolites generated by the xanthine-xanthine oxidase system were examined. As shown in Fig. 1, S. typhimurium LT2 and S. typhi 1079 were killed by oxygen
281 100
more susceptible to oxygen metabolites than was S. typhimurium LT2 ( P < 0 . 0 5 ) . S. typhimurium SLl102, a rough mutant of S. typhimurium LT2, was considerably more susceptible to oxygen metabolites than S. typhimurium LT2, and approximately 60% of S. typhimurium SLl102 were killed by 200 m U / m l of xanthine oxidase ( P < 0.05). Xanthine oxidase or xanthine alone did not influence the viabilities of these bacteria (data not shown).
v
>, 50
I
I
i
I
2OO
0
Xanthine oxidase
I
400 ( mU/m| )
Fig. 1. Susceptibility of Salmonellae to xanthine-xanthine oxidase. Various concentrations of xanthine oxidase were added to the suspension containing 2 × 1 0 3 C F U / m l of S. typhimurium LT2 (@), SLl102 (©), or S. typhi 1079 (zx) in a total volume of 0.5 ml of PBS with 600 /~M xanthine, and incubated for 1 h at 3 7 ° C . Values represent the mean_+ standard error of five experiments.
metabolites in a dose-dependent manner. No difference was seen in the susceptibilities of these strains to up to 200 m U / m l of xanthine oxidase ( P > 0.05). The viabilities of S. typhimurium LT2 and S. typhi 1079 at 200 m U / m l of xanthine oxidase were 60.9 + 4.7% and 54.7 + 10.8%, respectively. At a high dose (400 m U / m l ) of xanthine oxidase, S. typhi 1079 was found to be
4.2. Effects of scavengers of oxygen metabolites on bacterial killing by xanthine-xanthine oxidase To determine which oxygen metabolite contribute to the killing of Salmonellae, we examined the effects of oxygen metabolite scavengers on killing by the xanthine-xanthine oxidase system. As shown in Table 1, only catalase, which reduces hydrogen peroxide to H 2 0 and 02, effectively inhibited killing. Heated catalase had no inhibiting effect. On the other hand, superoxide dismutase (SOD) (which promotes the conversion of O f to hydrogen peroxide), mannitol and benzoate (scavengers of OH), and DABCO (a quencher of 102) all failed to inhibit the bacterial activity of the xanthine-xanthine oxidase system. Mannitol, benzoate, and DABCO themselves did not influence the viability of the bacteria (data not shown). These results indicated that hydrogen peroxide contributed to the killing of Salmonellae sp.
Table 1 Effect of oxygen intermediate scavengers on killing by xanthine-xanthine oxidase Scavenger
None SOD (1000U/ml) Catalase (100 U / m l ) Inactivated catalase Mannitol (50 mM) Benzoate (10mM) DABCO (1 mM)
Viability (%)
S. typhimurium
S. typhimurium
S. typhi
LT2
SLl102
1079
45.9+ 9.3 41.6_+ 5.7 104.7 _+11.7 * * 29.7_+ 8.2 36.6 _+ 6.0 45.0_+ 8.6 38.4_+ 5.9
30.2_+ 2.2 35.4_+ 4.0 127.0 _+13.3 * * 25.2_+ 5.5 39.5 _+ 6.4 37.1_+ 4.5 32.6+ 1.8
30.6+10.8 16.2_+ 7.4 121.5 _+16.4 33.8_+ 15.7 21.9 _+ 7.9 17.8_+ 5.6 22.8_+ 6.5
The reaction mixtures containing bacteria (2 × 103 C F U / m l ) , xanthine oxidase (400 m U / m l ) , 600/xM xanthine, and scavengers at indicated concentrations in a total volume of 0.5 ml of PBS were incubated for 1 h at 37 o C. Values represent the mean + standard errors of four experiments. ** P < 0.01.
282
manner, and S. typhi 1079 was more susceptible to H 2 0 2 than S. typhimurium LT2 ( P < 0 . 0 1 ) . In contrast, approximately 60% of S. typhimurium SLl102 were killed by a low dose of H 2 0 2 (2.5 X 10 - 6 M ) . These findings were consistent with the results obtained by the xanthine-xanthine oxidase system.
4.4. Hydrogen peroxide generation by macrophages associated with the phagocytosis of Salmonellae
2.5xlff 6
2.Sxlff 5
H202
2.5x10 ~
( M )
Fig. 2. Susceptibility of Salmonellae to hydrogen peroxide. Various concentrations of hydrogen peroxide were added to the suspension containing 2 x 103 C F U / m l of S. typhimurium LT2 (O), SLll02 (o), or S. typhi 1079 (zx) in a total volume of 0.5 ml of PBS, and incubated for 1 h at 3 7 ° C . Values represent the mean + standard errors of five experiments.
4.3. Susceptibility of Salmonellae to hydrogen peroxide To confirm the effect of hydrogen peroxide on the killing of Salmonellae, the susceptibility of S. typhimurium LT2, SLl102, and S. typhi 1079 to hydrogen peroxide was examined. As shown in Fig. 2, both S. typhimurium LT2 and S. typhi 1079 were resistant to up to 1.25 x 10 -s M H 2 0 2. At higher concentration of H 2 0 2 , ( > 2.5 X 10 s M) these strains were killed in a dose-dependent
Table 2 Phagocytosis-induced hydrogen peroxide generation by macrophages nmol of hydrogen peroxide/ 5 × 105 cells/60 min
E. S. S. S.
coli NIHJ typhimurium LT2 typhimurium SLl102 typhi 1079
2.54+_0.38 2.15+_0.90 1.74_ 0.33 1.43 _+0.77
The macrophage monolayers (5 X 105 cells/well) were infected with opsonized bacteria (5 x 106 CFU) in a total volume of 500 ffl of PBS containing Ca 2+ and Mg 2+, and incubated for 1 h at 37 o C. Values represent the mean + standard errors of four experiments.
It has been demonstrated that some pathogens fail to trigger superoxide generation by macrophages [4,5,8]. We therefore examined the ability of S. typhimurium LT2, SLl102, and S. typhi 1079 to trigger hydrogen peroxide generation by murine macrophages in comparison with that of E. coli N I H J . As shown in Table 2, both S. typhimurium LT2 and S. typhi 1079 triggered the respiratory burst as efficiently as did E. coil N I H J . Although the hydrogen peroxide generation associated with phagocytosis of S. typhimurium SLl102 by macrophages was slightly lower than that of S. typhimurium LT2, the difference was not significant.
5. D I S C U S S I O N In this study we have examined the susceptibility of S. typhimurium and S. typhi to oxygen metabolites. The susceptibility of S. typhi 1079 to the xanthine-xanthine oxidase system was similar to that of S. typhimurium LT2, although S. typhi 1079 was more susceptible to oxygen metabolites than was S. typhimurium LT2 at a high concentration of xanthine oxidase (400 m U / m l ) . We found that among the various oxygen metabolites, hydrogen peroxide contributed to the killing of Salmonellae. However, both S. typhimurium LT2 and S. typhi 1079 were resistant to up to 1.0 x 10 s M of hydrogen peroxide. Although it is difficult to estimate the concentration of hydrogen peroxide within the phagocytic vacuole, hydrogen peroxide produced by phagocytes is known to exist in an equilibrium between vacuole, cytosolic, and extracellular milieus, because hydrogen peroxide can permeate the cytoplasmic membrane and diffuse extracellularly [12]. Therefore, the extracellular concentration of hydrogen peroxide may reflect
283 the intraphagosomal concentration of h y d r o g e n peroxide. Tsunawaki et al. [13] have reported that PMA-stimulated mouse peritoneal macrophages are capable of generating 2 n m o l of HzOz/90 r a i n / 1 0 6 cells. We have an almost similar result in that S. typhimurium LT2 and S. typhi 1079-infected macrophages p r o d u c e d 1.4-2.2 n m o l of hydrogen p e r o x i d e / 5 × 10 5 cells/60 min, which is equivalent to 2.8-4.4 × 10 6 M. In the range of this concentration of hydrogen peroxide, neither S. typhimurium LT2 nor S. typhi 1079 were killed. W e c a n n o t rule out the possibility that the concentration of h y d r o g e n peroxide in phagosomes m a y be transiently higher than that in extracellular medium. However, Weiss et al. [14] have reported that the neutrophils from a patient with chronic granulomatous disease (unable to generate a respiratory burst) kill ingested S. typhimurium almost as effectively as the neutrophils f r o m a normal individual, and that depletion of O 2 b y N 2 flushing has no effect on the killing of S. typhimurium by neutrophils. These observations support our finding that b o t h S. typhimurium and S. typhi were resistant to oxygen-dependent killing mechanisms. It therefore seems likely that the species-specific pathogenicity of Salmonellae is not due to differential susceptibility to o x y g e n metabolites. Cell wall m u t a n t s of S. typhimurium have been reported to be highly sensitive to complement c o m p o n e n t s [15] and bactericidal permeability-increasing protein [16]. In this study, we have demonstrated that S. typhimurium SLl102, a rough m u t a n t of LT2, was sensitive to h y d r o g e n peroxide. It has been demonstrated that Mycobacterium leprae, Toxoplasma gondii, and Histoplasma capsulatum fail to trigger superoxide generation f r o m macrophages [4,6,8]. We found that S. typhimurium LT2, S. typhimurium SLl102, and S. typhi 1079 efficiently triggered h y d r o g e n peroxide generation by murine macrophages. This result suggested that the capacity of Salmonellae to trigger the respiratory burst is not correlated with pathogenicity. C o m p l e m e n t c o m p o n e n t s in fresh serum play an important role in non-specific defense mechanisms to bacterial infections [17]. However, we found that both S. typhimurium LT2 and S. typhi
1079 were resistant to 20% fresh mouse serum, whereas S L l 1 0 2 was markedly susceptible to fresh mouse serum (data not shown). Thus, the speciesspecific pathogenicity of Salmonellae might not be due to differential susceptibility to fresh serum components. In conclusion, our present data demonstrated that species-specific pathogenicity of Salmonellae m a y be due neither to differential susceptibility to oxygen metabolites nor to the differential capacity to trigger the respiratory burst of macrophages. Different degrees of susceptibility to the oxygen-independent killing mechanisms m a y therefore be responsible for the specific pathogenicity of Salmonellae, and we are currently exploring this possibility.
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