Pseudomonas aeruginosa lipopolysaccharide: factors influencing toxicity for isolated mitochondria and endotoxin properties1

G. GORDON GREER A N D F. H. MILAZZO Deprirtment of Microbiology and Immirnology, Qireen's University, Kingston, Ontario, Canada. K7L 3N6

Accepted March 2, 1976

GREER. G. G., and F. H. MILAZZO. 1976. Pserrdomonos aerlrainosrr l i~o~olvsacchar ide : factors . . . influencing toxicity for isolated mitochondria and endotoxin properties. Can. J. Microbiol. 22: 800-807.

In the present study Pse~rdotnotirrs nerrrgitlosn lipopolysaccharide (LPS) exhibited the follow- ingendotoxin properties: ( I ) toxicity for mice; (2) gelation of the Limirliis lysate; ( 3 ) induction ofa localized Shwartzman reaction in the skin of rabbits, and (4) anticomplementary activity.

Differences in LPS toxicity as measured with the rat liver mitochondrial assay system were found to be related to the nature of the bacterial growth media, the functional integrity of mitochondria, and the time and temperature of mitochondrial assay. The significance of these findings to P . nerirginosn infections is discussed, and it is concluded that LPS is a factor of importance.

GREER, G. G., e t F. H. MILAZZO. 1976. Pserrdomonns crerirginosa lipopolysaccharide: factors influencing toxicity for isolated mitochondria and endotoxin properties. Can. J. Microbiol. 22: 800-807.

Le lipopolysaccharide (LPS) de P . nerirginosa posstde les proprietts endotoxiques suivantes: ( I ) toxicite pour la souris; (2) gelification d'un lysat d e Limitlus; ( 3 ) induction d 'une reaction cutanee localisie d e Shwartzman chez le lapin, e t (4) activiti anticomplementaire.

D'apres les mesures faites dans un systeme utilisant des mitochondries de foie d e rat, les differences observees dans la toxicit6 du LPS son1 fonction de la nature du milieu d e culture des bacteries. de l'integrite fonctionnelle des mitochondries, ainsi que du temps et de la temperature de I'essai mitochondrial. Ces risultats sont discutes par rapport aux infections AP. aerirginosa et i l est conclu que le LPS est un facteur important.

[Traduit par le journal]

Introduction Although the lipopolysaccharide (LPS) of

many gram-negative organisms contributes to the pathogenesis of bacterial infections some in- vestigators have dismissed the importance of this material in P, aeruginosa related disease. This is due, in part, to the findings that killed cells and isolated LPS were not lethal for mice (20, 21); killed cells were incapable of inducing necrotic lesions in the skin of rabbits (31), and anti- exotoxin serum containing no agglutinating anti- bodies against whole cells protected against lethal infections in mice (22).

In contrast to these findings killed cells, cell walls, and P. aeruginosa LPS were all found to be lethal for mice (25) and isolated LPS elicited a Shwartzman reaction in the skin of rabbits (12). In respect to the protective capacity of anti- exotoxin sera (22), it is known that antibodies directed against P. aer~cginosa LPS are capable

'Received October 31, 1975.

of conferring passive protection against lethal infection in mice (10, 14).

Furthermore, Dyke and Berk (1) reported LPS extracted by the phenol-water procedure to be more lethal for mice than LPS extracted by four other extraction procedures. They suggested the inability of many investigators to isolate P. aeruginosa LPS in a toxic form might be related to the extraction procedure and (or) bacterial strain.

Recently using the rat liver mitochondria (RLM) system P. aer~~ginosa LPS was found to be toxic for RLM and to uncouple oxidative phosphorylation (8). However, it is very signi- ficant that methodology used to study LPS-RLM interaction is highly variable (6, 7, 1 I, 16, 17, 23) and since little data has been published to sup- port the validity of these methods it is difficult, if not impossible, to compare the relative effects of different LPS preparations. Also the evidence for a precise role of P. aer~rginosa LPS in pathogenesis is limited and contradictory.

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GREER AND MlLAZZO 80 1

The Dresent studv was undertaken to examine the influence of a variety of experimental con- ditions on toxicity measurements with the P. aeruginosa LPS-RLM system and to provide more definitive characterization of P. aeruginosa LPS using classical assays of endotoxicity.

Materials and Methods Cultural Conditions and L P S Extraction

The conditions used for the growth of P. aeruginosa KcIIR and the methods used for LPS extraction and purification were identical with those previously described (8). Media used for growth were as follows: medium 1, tryptic soy broth (Difco); medium 2, glucose-glutamate inorganic salts (12); medium 3, citrate-ammonium salts (9); and medium 4, King's medium A (18). LPS derived from cells grown in these media were designated LPS-I, LPS-2, LPS-3, and LPS-4 respectively. Escherichia coli LPS prepared by the phenol-water procedure was a commercial preparation obtained from the Sigma Chemi- cal Co.

Mitochondria1 Assay Rat liver mitochondria (RLM) were isolated and

assayed polarographically for oxygen consumption a s previously described (8). LPS toxicity was determined following LPS-RLM interaction for 1.5 min a t 30°C before the addition of substrate (final concentration 3.3 m M glutamate plus 33 m M malate). When succinate was used 0.2 pg rotenone (Sigma Chemical Co.) was included in the reaction medium. After 2 min of substrate oxidation 500 n M of adenine 5'-diphosphate (ADP) was added. LPS toxicity was defined as the pg LPS/mg RLM protein required to reduce the respiratory control ratio (RCR) to 1 (6). To study the influence of RLM assay conditions on LPS toxicity the incubation time before substrate addition, the assay temperature, or the substrate concentration was altered while all other conditions were identical with those described above. LPS (125 pg/mg RLM protein) was used since this amount produced about a 50% reduction in a RCR of 10 with 3.3 m M glutamate plus 33 m M malate using a 1.5-min incubation time a t 30 "C.

Mouse Lethality Tests T o determine the lethality of viable P. aeruginosa the

organism was cultured in medium 2 as previously de- scribed. Before injection cells were washed three times in sterile 0.85% NaCI. Serial 10-fold dilutions were prepared and 0.5-ml injections were made intraperitoneally into 20 g, male, Swiss white mice. Ten mice were injected at each concentration and 10 saline injected controls were included. Deaths were recorded at-48 h and the LD,, determined by the method of Reed and Muench (27). In determining the toxicity of LPS for mice identical procedures were used with the exception that serial two- fold dilutions of LPS were prepared.

Limulus Assay Gelation of the amebocyte lysate from Limulus poly-

phemus was determined according to the procedures of Jorgensen and Smith (15). All glassware used was

pyrogen-free. Serial 10-fold dilutions of LPS were pre- pared in sterile, pyrogen-free, distilled water (Travenol Laboratories Inc.). A volume of 0.1 ml of LPS was added to 0.1 ml of Limulus lysate (Sigma Chemical Co.) and incubation was carried out for 60min a t 37°C. The formation of a firm gel constituted a positive reaction.

Anticomplementary Activity Serial twofold dilutions of LPS were prepared in guinea

pig complement and incubation carried out for 60 min a t 37 "C. Complement in the absence of LPS was included as control. After incubation residual complement activity was titrated by mixing 0.2 ml of the contents of each tube with 0.2 ml of a 1% solution of sensitized sheep red blood cells and incubating a t 37 "C for 60 min. Percentage of lysis was determined spectrophotometrically at 541 nm and the percentage of inhibition of lysis was computed by subtraction. Under these conditions of assay the com- plement control (absence of LPS) gave about 55% lysis.

Localized S/ztvarizman Reaction An initial dose of 0.3 to 500 pg LPS in 0.1 ml sterile

saline was injected intradermally into the shaved flanks of 10-week-old New Zealand white rabbits. A challenge dose of 1 mg LPS in 0.1 rnl of sterile saline was injected into the marginal ear vein 24 h later. The appearance (within 24 h) of necrotic lesions at the intradermal injection sites was considered a positive result.

Results Effect of Bacterial Growth Medium on LPS

Toxicity Figure 1 shows the relative toxicities of LPS

derived from cells grown in different culture media. LPS-2 was found to be the most toxic, 78 pg/mg RLM protein, while LPS-1 and 3 showed similar toxicities, 167 and 169 pg/mg RLM protein, and although not shown LPS-4 was the least toxic. This preparation 156 pg LPS/mg RLM protein could only reduce the RCR from 6.5 to 5.2. In consideration of these findings, batch amounts of LPS-2 (medium 2) were prepared and used throughout the remainder of the investigation. Henceforth, this material will be referred to simply as LPS.

Effect of RCR on L P S Toxicity It should be stressed that although identical

~rocedures and conditions were used for mito- chondrial isolation these organelles varied in respect to their RCR. This was reflected in variations in toxicity of the same LPS prepara- tion (Fig. 2). Further, LPS was more toxic for RLM with lower RCR than for preparations with a higher RCR. In view of these findings the results in Tables 1 t o 4 were recorded as per- centage of reduction in RCR.

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/ Lq L P S / m g R L M P R O T E I N

FIG. 1 . The effect of P. cieruginosn LPS derived from cells grown in different culture media on the RCR of RLM. LPS 1 (O), LPS 2 (O), or LPS 3 (A) was added to the RLM and allowed to react 1.5 min at 30 "C before the addition of 3.3 m M glutamate plus 33 m M malate. Con- trol RLM had a mean RCR of 6.5.

I17J~re17ce of It1c~ibatiot7 Titne on LPS Toxicity The data in Table 1 (RCR) suggest that control

mitochondria were tightly coupled for the first 2 min of incubation before substrate addition. This was followed by a reduction in RCR with increased incubation time, indicating some loss in mitochondrial integrity. In the presence of 125 pg LPS/mg RLM protein RCR were reduced to the same extent at time intervals from 0 to 1.5 min. Increased incubation times resulted in enhanced toxicity and after 15 min of incubation respiratory control was eliminated.

It is also important to note that when LPS, RLM, and substrate were added to the reaction vessel simultaneously (0 time) there was a 49x reduction in the RCR. This would suggest that LPS can readily alter some mitochondrial functions.

Effect of Assay Tetnperature on LPS Toxicity In Table 2 it can be seen that control RLM

had relatively high RCR at 25 to 30 "C and that decreased RCR were found at temperatures

1OL. VOL. 22. 1976

R C R O F C O N T R O L R L M

FIG. 2. The relation of the RCR of control RLM to the toxicity of P. aer~igirzosa LPS. Assay conditions were identical with those described for Fig. 1.

TABLE 1. The effect of incubation time on the RCR of control and LPS-treated RLMa

- - - - -

RCR Time, - %reduction minb Control RLM RLM + LPSc in RCR

"Assay temperature was 30°C and substrate concentralion was 3.3 m M glutamate + 33 m M malate.

bTime of incubation before subslrate addition. cLPS added to give a final concentration of 125 pg/rng RLM

protein.

outside this range. Although 125 pg LPS/rng RLM protein did not completely remove re- spiratory control between 20 and 30 "C it eliminated R C R a t temperatures above 35 "C.

Effect of Substrate Concentration on LPS Toxicity The R C R of control R L M was dependent

upon substrate concentration a t lower concen- trations of glutamate plus rnalate (Table 3). Under these conditions LPS toxicity was not

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TABLE 2. The effect of assay temperature on the RCR of control and LPS-treated RLM"

RCR Temp., 4, reduction

"C Control RLM RLM + LPSb in RCR

.Time of incubation before substrate addition was 1.5 min and substrate concentration was 3.3 m M glutamate + 33 m M malate.

bLPS added to give a final concentralion of 125 pglmg RLM proteln.

affected by substrate concentration. When sub- strate concentration was increased the RCR of control RLM essentially became constant. Al- though LPS toxicity decreased at higher levels of substrate, this effect was diminished as LPS concentration was also increased.

The interpretation of these data may have

been complicated by the presence of the two substrates glutamate plus malate. To overcome this problem experiments were carried out using succinate as substrate with rotenone to inhibit any reduced nicotinamide adenine dinucleotide phosphate (NADH) linked oxidations. The data presented in Table 4 with succinate plus rotenone confirmed the results obtained using glutamate plus malate.

Endotoxin Properties of LPS Viable P. aeruginosa grown in medium 2 had

an LD,, for mice of 7.4 x lo6 organisms and LPS derived from these cells had an LD,, of 2.5 mg. Furthermore, LPS-injected mice showed typical manifestations of endotoxin shock: en- crusted eyes and loss of mobility.

Table 5 shows the comparative Lit71i1Ii1~ activity of E. coli and P. aeruginosa LPS. Escherichia coli LPS (0.01 ng/ml) demonstrated greater activity than P. aerirginosa LPS (0.1 ng/ml).

In Fig. 3 the inactivation of complement by

TABLE 3. The effect of glutamate + malate concentration on the RCR of control and LPS-treated RLM"

Glutamate +

malate,' mM

0.2 0.4 0.8 1.6 3.3

- - --- -

78 0 (Control)

%reduction RCR RCR in RCR

4.1 1.4 66 6.8 3.3 60 8.6 4.4 61 9.1 3.1 66 9.8 9.0 6

125

4, reduction RCR in RCR

%reduction RCR in RCR

T i m e o f incubation before substrate addition was 1.5 rnin and temperature of assay was 30 "C. OFinal concentration of LPS in pg/mg RLM protein. (Final concentration of glutamate + 10 x final concentration o f malate.

TABLE 4. The effect of succinate concentration on the RCR of control and LPS-treated RLM"

LPSb

156 202 0 (Control) -------------- -----------

Succinate, ---- % reduction %reduction m M RCR RCR in RCR RCR in RCR

'Time o f incubation before substrate addition was 1.5 min and temperature of assay was 30 "C. Rotenone (0.2 pg) was included in the reaction medium.

*Final concentralion o f LPS in pglmg R L M protein.

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804 CAN. J. MICROBIOL. VOL. 22, 1976

TABLE 5. Limulus lysate gelation by LPS

LPS

ng LPS/ml E. coli P. aeruginosa

'Formation o f a firm gel after 60 rnin o f incu- bation at 37 "C.

and E. coli LPS is illustrated in Fig. 4. Although both preparations produced necrosis in the skin of rabbits only 2.5 pg of E. coli LPS was required to induce a necrotic lesion of equal severity to that observed with 500 pg P. aeruginosa LPS. Further, the minimal intradermal doses to pro- duce a positive reaction were 2.5 and 125 pg for E. coli and P. aeruginosa LPS respectively. Rabbits injected with E. coli LPS showed signs of severe shock within 3 h of the intravenous injection and death resulted in 24 h. In contrast rabbits injected with P. aeruginosa LPS showed

l O O r no signs of endotoxin shock. I

FIG. 3. Anticomplementary activities of E. coli (0) and P. aeruginosa (0) LPS. Residual hemolytic complemenr activity was titrated following LPS-complement inter- action for 60 min at 37 "C. Lysis of sensitized sheep red blood cells was determined spectrophotometrically at 541 nM.

P. aeruginosa and E. coli LPS is compared. Although only 35 pg of E. coli LPS was sufficient to produce a 50% reduction in the hemolytic complement activity, 500 pg of P. aeruginosa LPS was required to produce the same effect.

The Shwartzman reactivity of P. aeruginosa

FIG. 4. Localized were 2.5 (I), 5 (2), Saline control (5). Bar = 2 cm.

Discussion

The results reported in this study have shown that the medium used for bacterial growth as well as the mitochondrial assay conditions signi- ficantly influenced measurements ofP. aeruginosa KcIIR LPS toxicity. In addition it was firmly established that this LPS exhibited the following biological properties: (1) toxicity for Swiss, white mice; (2) gelation of the Limulus lysate; (3) induction of a localized Shwartzman reaction in the skin of rabbits, and (4) anticomplementary activity.

Initially it was found that the composition of the bacterial growth medium appeared to in- fluence the toxicity of P. aeruginosa LPS for RLM. LPS derived from cells grown in one of the chemically defined media (glucose-glutamate inorganic salts) was more toxic for RLM than were LPS isolated from cells grown in three other media. This data adds support to the findings of Fukushi et al. (3) who, using a variety of assays measuring endotoxicity, found that the bacterial strain, cultural conditions, and method of ex- traction influenced the chemical composition and biological activities of several enterobacterial LPS.

In relation to the effects of time and tempera- ture of mitochondrial assay, the results of the present study as well as those reported by Mela et a[ . (23) were obtained by direct addition of LPS to the reaction vessel containing RLM. Furthermore, the present work showed that even when LPS, RLM, and substrate were added to

Shwartzman reaction induced by LPS in the skin of rabbits. Intradermal injections 30 (3), 60 (4) pg E. coli LPS, and 125 (6), 250 (7), 500 (8) pg P. aeruginosa LPS. Reactions were recorded within 24 h of an intravenous injection of 1 mg LPS.

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806 CAN. J. MICROBIOL. VOL. 22. 1976

the reaction vessel simultaneously a reduction of about 50% in the RCR was observed. In con- trast to these findings both Harris et 01. (1 1) using Bordetella LPS and Kato (16, 17) using Salmonella and E. coli LPS reported that LPS had no effect upon mitochondria unless it was preincubated with mitochondria before assay. Harris et al. (11) used 5-min preincubation periods at 37 "C while Kato (16, 17) used 15-min preincubation periods at 20°C. However, the present investigation showed that at temperatures outside the range of 25 to 30 "C or a t times of incubation exceeding 1.5 min mitochondrial functions were impaired (reduced RCR). In light of this the results reported by both Harris et al. (11) and Kato (16, 17) may have been influenced by their experimental conditions which altered the susceptibility of mitochondria to LPS.

Using the present criteria, the toxicities of S. typhit~i~iri~i1~1 (6), E. roli (2 3), and P. aer~igitiosa LPS (8) would be 23, 80, and 200 pg LPS/mg RLM protein respectively. Further, Fig. 1 showed that this same P. aeruginosa LPS (LPS 2) had a toxicity of 78 pg LPS/mg RLM protein. From these results it would appear that not only was S. typliiiiiriri~it~7 LPS more toxic but that the same preparation of P. aer~igit7osa LPS varied in toxicity. However, the present results clearly showed mitochondrial preparations with higher RCR were more resistant to LPS (Fig. 2). It should be noted that the RCR of control RLM in studies with S . typhinluriun7 (6) and E. coli (23) LPS were 4.6 and 6 to 7 respectively. Also control RLM had RCR of 6.5 (Fig. 1 ) and 10.0 (6) in studies with P. aer~iginosa LPS. When these differences in the RCR of control RLM are interprcted in view of the present findings all of the above LPS would have comparable toxicities.

In contrast to previous work in which substrate protccted RLM against LPS (6) the present study showed that LPS toxicity for RLM was not altered over a wide concentration range of glutamate plus malate or succinate. Only at higher levels of these substrates was LPS toxicity found to decrease but this effect was diminished as LPS concentration was also increased.

Collectively the results have stressed the im- portance of a standard mitochondrial assay system and have shown that without careful consideration of methodology erroneous con- clusions result regarding LPS toxicity for RLM.

In regard t o endotoxin properties it should be emphasized that P. aeruginosa LPS had charac-

teristics common to many LPS. The LD,, of viable P. aerugitiosa KcIIR (7.4 x lo6 cells) indicates that this strain was comparatively pathogenic (19, 26, 29). Extracted LPS had a n LD,, for mice of 2.5 mg indicating a lower toxicity than the value of 0.84 mg reported by Dyke and Berk (1). Although Liu (20) stated that 2 to 3 mg of purified P. aeruginosa LPS did not kill a mouse, comparisons with the present findings are difficult since experimental condi- tions were not given. Also it has been shown that LPS lethality was affected by the bacterial strain and extraction procedure (I , 3).

Subsequent studies of other endotoxin prop- erties, carried out in the present investigation, showed the Litnulus lysate gelation, anti-comple- mentary and localized Shwartzman activities of E. coli LPS were far greater than those observed with P. aeruginosa LPS. The interpretation of this data, however, may have been misleading since the E. coli LPS was a commercial prepara- tion and its method of purification was unknown. In respect to this it should be stressed that E. coli LPS was exceedingly more soluble in water o r saline than P. aerugitiosa LPS and that the biological activities of LPS are dependent upon its solubility and physicochemical state (4, 5,28).

It is noteworthy that complement-LPS inter- actions have been implicated as factors con- tributing to the pathophysiological changes ob- served during endotoxemia (24) including both the localized and generalized Shwartzman reac- tions (2). Furthermore, a reported manifestation of P. aer~rgitiosn infections is the appearance of hemorrhagic necrotic lesions (1 3, 30, 32). In the present work similar lcsions were induced in the skin of rabbits by P. aer~iginosa LPS. Since complement activation may be involved in the production of these lesions and since P. aeru- ginosa LPS consumes complement in vitro it is not unreasonable to assume that LPS contributed to the severity of necrotic lesions observed in vivo during infection.

After due consideration of this data it would appear that future studies of factors contributing to the lethality of P. aeruginosa infections should include LPS.

Acknowledgment The authors gratefully acknowledge the finan-

cial support of the National Research Council of Canada.

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