A complement-sensitive mutant of Pseudomonas aeruginosa

A complement-sensitive mutant of Pseudomonas aeruginosa

Ann. Microbiol. (inst. Pasteur) 1983, 134 A, 281-294 A COMPLEMENT-SENSITIVE OF PSEUDOMONAS MUTANT AERUGINOSA by C. Offredo-Hemmer, P. Berche and ...

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Ann. Microbiol. (inst. Pasteur) 1983, 134 A, 281-294

A COMPLEMENT-SENSITIVE OF

PSEUDOMONAS

MUTANT

AERUGINOSA

by C. Offredo-Hemmer, P. Berche and M. V6ron Laboratoire de Microbiologie, Facult~ de Mddecine Necker-En/anls Malades, 75730 Paris Cedex 15

SUMMARY The role of complement in the bactericidal activity of human serum against a m u t a n t strain of Pseudomonas aeruginosa used as a model was demonstrated. The involvement of complement in the bacterial destruction of P. aeruginosa, and the contribution of the alternative and classical pathways of the complement system were directly evidenced by using sera from complement-deficient patients. KEY-WOaDS: Pseudomonas aeruginosa, Serum bactericidal activity, Complement; Mutant, A,lternative pathway, Classical pathway.

INTRODUCTION Fresh serum from a number of animals and humans is a highly effective agent for non-specifically destroying many species of bacteria [29, 33]. There are strong indications that this is due mainly to the complement system, which represents about 10 % of human plasma proteins 119, 30]. Evidence suggests that the complement system participates in bacterial defenee in vivo as a mechanism against invasion of blood and dissemination of infection by Gram-nega~ive bacilli [13, 28]. Indeed, it is known that patients suffering from hereditary complement deficiencies or from chronic underlying diseases associated with impairment of complement activity are generally highly susceptible to-such infections as meningitis and septicaemia [3]. Pseudomonas aeruginosa is a Gram-negative microorganism which is wide-spread in the environment. Its importance as a human pathogen has increased over the past ten years [41] due to multiple factors, including M a n u s c r i t re~u le 24 d6cembre 1982, a c c e p t s le 17 f6vrier 1983.

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C. OFFREDO-HEMMER, P. BERCHE AND M. Vt~RON

the bacterial virulence of some strains, the impairment of the host immune system and the high resistance of this species to most antiseptics and antibiotics [22]. The susceptibility to fresh h u m a n serum of P. aeruginosa strains isolated from patients is v e r y heterogenous. It has been reported t h a t the majority of septicaemia- or endocarditis-producing strains are resistant to the bactericidal effect of normal h u m a n serum [24, 45]. On the other hand, some mucoid and non-mucoid strains of P. aeruginosa isolated from the s p u t u m of cystic fibrosis patients are much more sensitive to the complement bactericidal activity than the usual strains isolated from noncystic fibrosis patients [15, 18, 20, 38]. It is known that cystic fibrosis patients suffer from chronic pulmonary infection, mainly due to P. aeruginosa, and are rarely systemically infected [42]. Moreover, it has been found t h a t a serum-sensitive strain of P. aeruginosa does not produce endocarditis in an experimental model in the rabbit [4]. These findings suggest t h a t the complement system m a y play an i m p o r t a n t role as a host defence mechanism to prevent spreading of some P. aeruginosa infections. However, the involvement of the complement system in bacterial killing is still poorly understood. In the present work, the role of complement in the bactericidal activ i t y of h u m a n serum was investigated using a highly susceptible m u t a n t strain from P. aeruginosa.

MATERIALS AND METHODS

Bacleria. Two strains of P. aeruginosa, called 72V0 (Collection de l'Institut Pasteur, Paris, CIP 1,005) and 72VA, were used in this work. The field strain 72V0 was isolated from a patient who died of septicaemia within 48 h of the onset [6]. The mutant strain 72VA was isolated from the original strain 72V0 after daily subcultures on trypticase soy broth at 37 ~ C over a one-year period. These strains were lyophilized and stored at room temperature. They were identified by the classical biochemical characters [7]. Their versatility towards 114 substrates as the only source of carbon was studied on synthetic medium ,, M70 )) as previously described [40]. Serotyping was carried out by seroagglutination [39], using antisera manufactured by the Institut Pasteur Production (iPP), according to Habs' typing system [14]. The minimal inhibitory concentrations (MIC) of various antibiotics (penicillin G, ampicillin, carbenicillin, gentamicin, tobramycin amikacin, colistin, tetracycline, sulfonamide, nalidixic acid) were determined by dilution in Mueller-Hinton broth (IPP).

Sera. All human and mouse sera used in the present work were aseptically collected at 4~ C. After clotting, centrifuged sera were rapidly dispensed into vials in 0.5 to 1.0 ml lots and immediately stored at -- 70 ~ C until required. When tested, they were quickly thawed once and rapidly used in experiments. Fresh human serum was prepared by pooling sera from 7 healthy donors MIC = minimal inhibitory concentration.

COMPLEMENT-SENSITIVE P. AERUGINOSA

283

(Centre de Transfusion, H6pital Necker-Enfants Malades, Paris). The haemolytic complement activity was titrated by determination of complement haemolysis 50 % (CH~o) according to a standardized method described elsewhere [181. The CH~o was estimated by Dr J. Leibowitch (H6pital Necker-Enfants Malades, Paris) at about 1,200 units/ml (100 % activity). The antibody titre against a heated suspension of P. aeruyinosa (strain 72V0) was 1/80 by agglutination the method in tubes [39]. Six human sera from patients with hereditary complement deficiencies, kindly supplied by Dr J. Leibowitch and Dr L. tialbwachs (H6pital Necker-Enfants Malades, Paris), were also used in this work: 4 sera from C2 deficiencies (~cPi ~, ~ Me ,,, ~ Be ,,, ,~ Bo ~); 1 serum from C3 deficiency (,c Ur ~); and 1 serum from C7 deficiency (,~ Ed ,~) described elsewhere [17]. The haemolytic complement activity (CHs0) was undetectable for C7 and C3 deficient sera and very low for C2 deficient sera (CHso < 10 %). Purified C2 fraction was a gift from Dr L. Halbwachs and Dr J. Leibowitch (HOpital Necker-Enfants Malades, Paris). Purified C7 fraction was obtained from Cordis Laboratories (Miami, Florida). A hypogammaglobulinaemic serum (,c Ch ~,) was collected from an untreated child suffering from Bruton-type agammaglobulinaemia. It was a gift from Dr C. Griscelli (H6pital Necker-Enfants Malades, Paris). Immunoglobulin levels in this serum were as follows: IgG 140 mg % (normal: 680-1,180 mg ~/o); IgM 13 mg % (normal: 50-115 mg %); IgA undetectable (normal: 66-134 mg %). CH~0 was estimated at 100 %. This serum did not contain any detectable agglutinin against the 72V0 strain. Fresh human serum was chelated by a solution of ethylene-glycol-tetraacetic acid (EGTA, Sigma Chemical Co., St. Louis, Missouri) plus magnesium ions (10 mM final) by the method of Fine el al. [11]. Fresh mouse serum was prepared as follows: thirty pathogen-free Swiss mice were bled from the retroorbital venous plexus; after clotting for about 1 h in ice, the serum was collected by centrifugation at 4 ~ C; the pooled mouse serum was not haemolysed and was stored at -- 70 ~ C in 0.2 ml volume aliquots.

Assay of injection. Swiss outbred female 6-8-week old mice, pathogen-free, were used in all experiments. They were supplied by IFFA-CRED0 (Domaine des Oncins, 69210 L'Arbresle, France). This mouse strain did not display any complement deficiency [211. Mice were inoculated via a lateral tail vein with progressive doses of living bacteria of each strain. These bacteria were grown in trypticase soy broth (IPP), incubated for 5-6 h at 37 ~ C in a shaking water bath, and harvested while still in log phase. Survival of bacteria was followed in spleen and liver. Groups of 5 animals were killed by cervical dislocation 1/2 or 24 h after inoculation. The organs were removed aseptically and homogenized separately in sterile saline 0.15 M; 0.1 ml volume of 10-fold serial dilutions in saline were surface-plated on trypticase soy agar. Colonies were counted after a 24-h incubation at 37 ~ C. The lethal dose 50 % (LDso) was determined on groups of 5 mice after intravenous challenge by the log-probit method.

Baclericidal aelivily. Bacteria were grown in trypticase soy broth incubated in a shaking water bath for 18 h at 370 C. The bacterial suspension was then washed in NaCI 0.15 M by two centrifugations at 40 C (MSE 18 centrifuge), and adjusted at 108 bacteria/ml m phosphate-buffered saline, pH 7.2 (PBS, BD M6rieux, Marcy l'I~toile, France). The in vitro bactericidal activity of fresh sera was titrated in triplicate in plastic tubes containing 0.1 ml of the bacterial suspension in PBS (107/ml final) and 0 to 0.4 ml of fresh serum, in a final volume of 1 ml (PBS). The bactericidal

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C. OFFREDO-HEMMER, P. BEBCHE AND M. Vt~RON

activity of complement-deficient sera supplemented with purified complement fractions was studied by incubating, under the same conditions, a constant volume of 0.2 ml of deficient serum, to which was or was not added 0.2 ml of its corresponding (and lacking) concentrated, purified fraction (in excess) in a final volume of 1 ml (PBS). These tubes were incubated at 370 C in a low-speed roller apparatus (Roto-Torque, Cole Parmer Instrument Company, Chicago, Illinois). Fiftymicroliter samples were collected at progressive times with an ,, Eppendorf , micropipette. Bacterial counts were achieved by plating various dilutions of the bacterial suspension on trypticase soy agar (IPP). Bactericidal activity was assessed at given times by comparing the viable counts in serum assay to those obtained in serum-free medium. RESULTS

Bacterial character of wild and mutant strains of P. aeruginosa. The wild strain 72V0 displayed the typical aspect of P. aeruginosa cultures. After 24 h incubation at 37 ~ C on trypticase soy agar, colonies were irregular, rough, fried-egg and large (diameter 2-3 ram), as illustrated in figure 1 A. In contrast, colonies of the m u t a n t strain 72VA incubated under the same conditions appeared smaller (diameter 1-2 ram), regular, smooth and slightly mucoid (fig. 1 B). These two strains were strictly identical in their production of the pigments pyocyanin and pyoverdin, and in their classical biochemical characters. Moreover, among 114 substrates tested as the only source of carbon on synthetic medium, both strains were capable of growing on the same 78 substrates, including 12 carbohydrates and alcohols, 29 carboxylic acids, 5 aromatic compounds, 31 amino acids and amines and 1 hydrocarbon. They exhibited the same susceptibility to antibiofics: both were sensitive to carbenicillin, aminoglycosides and colistin, and resistant to the others antibiotics tested, as evidenced b y the observation of identical MIC. The strains were stable with respect to their colonial aspect and biochemical characteristics. The virulence of strains 72V0 and 72VA was also tested in mice and results are reported in table I. It was shown t h a t the LDso of 72VA was much higher than t h a t of 72V0. Moreover, 72VA was rapidly destroyed in spleen and liver 24 h after intravenous challenge of 106.~ bacteria, whereas bacterial survival or growth in organs was observed with 72VO under the same conditions.

Sensitivity of the strains to serum bactericidal activitg. The bactericidal activity of fresh h u m a n serum collected from healthy donors (pooled sera) was evaluated on strains 72VO and 72VA. Bacterial

FIG. 1. - - Colonies of the wild ( A ) and mutant ( B ) strains of P . a e r u g i n o s a . C o l o n i e s s h o w n a r e t h o s e of t h e w i l d s t r a i n 7 2 V O (A) a n d t h e m u t a n t o n t r y p t i e a s e s o y a g a r w e r e i n c u b a t e d 24 h a t 37 ~ C.

s t r a i n 7 2 V A (13). C u l t u r e s

FIG. 1

COMPLEMENT-SENSITIVE

TABLE I.

- -

P. AERUGINOSA

Virulence of mutant 72VA.

Loglo Bacteria per organ (a) Spleen Strains

LD~o (1)

1/2 h

Original 72VO M u t a n t 72VA

106.a 10 e.2

4.8• 4.7 5:0.2

287

Percentage of bacterial survival (a)

Liver 24 h

4.5• <12.0

1/2 h

24 h

Spleen

Liver

6.2:t:0.1 6.5d:0.2

7.7=t=1.1 2.9•

70 % ~1/1,000

>100 % 1/4,000

(1) T h e lethal dose 50 % was d e t e r m i n e d on groups of 5 Swiss mice after i. v. challenge. (2) Bacterial c o u n t s in spleen a n d liver were assessed 1/2 h and 24 h after i. v. challenge of 106.5 bacteria per m o u s e ( m e a n • s t a n d a r d deviation, f r o m 5 organs). (a) Ratios: ioglo bacteria per organ within 1/2 h/loglo bacteria per organ within 24 h, e s t i m a t e d after i. v. challenge (106.5 bacteria per mouse).

'7

............................................................

// ..............

o

6

5.

,=,, a,.

,<

~4_ uJ I--

~ 3 _

2'0

40

s'o

a'o

16o Oo TIME~M,,~u;~s)

///

2~,o

FIG. 2. - - Bactericidal activity o/ /resh h u m a n serum on the wild slrain ( 7 2 V 0 ) and the mutant strain ( 7 2 V A ) of P. aeruginosa. Bacterial s u s p e n s i o n s of 72VO (o) a n d 72VA ( e ) were i n c u b a t e d in 20 % h u m a n s e r u m - s u p p l e mented medium ( ) or in serum-free m e d i u m ( . . . . ).

288

C. OFFBEDO-HEMMEB, P. BERCHE AND M. VEBON

suspensions (107/ml final) were incubated in vitro at 370 C with fresh h u m a n serum (20 % final). The bacterial survival was followed at progressive times (0, 1, 2 and 4 h), compared Lo serum-free controls. Results are illustrated in figure 2. It is shown t h a t bacteria of the m u t a n t 72VA were almost completely destroyed by h u m a n serum in 1 h, as opposed to bacteria of the original 72VO which were to:ally resistant to serum bactericidal activity. After 4 h, most bacteria from 72VA were killed, with a bacterial survival rate of less t h a n 10 -5 at thaL time. Different concentrations of fresh h u m a n serum ranging from 1 to 40 % were also tested (fig. 3). After a 4 h-incubation, the bactericidal effect 50 % was obtained with a serum concentration of 4 % final. The maximal effect was reached at a concentration of 20 % serum or mord~ This concentration was used in all the experiments which followed. 100

r t~

50.

/

/

/

f 1'0 CONCENTRATION

2'0 OF

3'0 HUMAN

40 SERUM ~

F l a . 3. - - Bactericidal activity of progressive concentrations of h u m a n serum on the mutant 7 2 V A of I3. a e r u g i n o s a . B a c t e r i a l s u s p e n s i o n s w e r e i n c u b a t e d f o r 4 h a t 37 ~ C w i t h p r o g r e s s i v e c o n c e n t r a t i o n s of f r e s h h u m a n s e r u m . T h e p e r c e n t a g e of b a c t e r i c i d a l a c t i v i t y w a s c a l c u l a t e d b y c o m p a r i n g t h e n u m b e r o f s u r v i v i n g b a c t e r i a in h u m a n s e r n m - s u p p ] e m e n t e d m e d i u m w i t h t h e n u m b e r of b a c t e r i a in s e r u m - f r e e m e d i u m .

The bactericidal effect of fresh mouse serum was also tested under the same conditions. It was found t h a t fresh mouse serum totally lacked bactericidal a c t i v i t y upon both 72VO and 72VA (data not shown).

COMPLEMENT-SENSITIVE

P. AERUGINOSA

289

Role of complement in serum bactericidal activity. The role of complement in serum bactericidal activity was studied on the m u t a n t 72VA. Bactericidal assays were carried out by incubating bacterial suspensions with various sera for 4 h at 37 ~ C. Bacterial viability was estimated at t h a t time and results are reported in table II. No bacterial destruction was observed with heated normal serum (30 min, 560 C), indicating t h a t bactericidal activity was heat-sensitive. Direct evidence t h a t complement was involved in bacterial killing was provided by the results of bactericidal assays on complement-deficient sera: C3 deficient serum ~ Ur ~ as well as C7 deficient serum ~ Ed ~, were totally inefficient in destroying bacteria. The addition of purified C7 component completely restored bactericidal activity in the case of C7-deficient serum. These results strongly suggest t h a t bactericidal activity is due to the complement system. TABLE II. - -

Sensitivity of the mutant 72VA to fresh h u m a n serum.

Loglo bacteria/ml S o u r c e of s e r a

Treatment of s e r a

0 h

4 h

Bacterial s u r v i v a l (*)

No serum (control)

None

7.17

7.25

> 100 %

Normal

Untreated 30 m i n , 5 6 o C Mg-EGTA

7.21 7.16 7.52

0.50 7.44 3.30

10 --6.7 > 100 % 10 -~.~

Untreated

7.00

6.90

_ 100 %

Untreated A- p u r i f i e d C7 (**)

7.45 7.14

7.47 2.39

_ 100 % 10 -4.7

Untreated Untreated Untreated Untreated + p u r i f i e d C2 (**)

6.85 7.44 7.23 6.84 6.65

3.82 4.54 4.85 3.38 0.50

10 -~.9 10-z. 4 10 -s.* 10 -6.6

Untreated 30 m i n , 5 6 o C

6.81 6.74

0.40 6.67

10 -6.4 _ 100 %

serum

C3-deficient serum ,~ U r ,, C7-deficient serum , E d ,, C2-deflcient sera ~cB o ,, ,~ P i ,, ~, M e d ~, ~ B e ,, Hypoagammaglobulinaemic s e r u m ,, C h ,,

A l l s e r a w e r e u s e d a t 20 % f i n a l ( v / v ) . E a c h e x p e r i m e n t w a s p e r f o r m e d a t l e a s t in t r i p l i c a t e ; s t a n d a r d d e v i a t i o n (*) A f t e r a 4 - h i n c u b a t i o n a t 37 o C. (**) T h e p u r i f i e d c o m p l e m e n t f r a c t i o n s w e r e u s e d a t 2 0 % f i n a l ( v / v ) .

1 0 -~.6

~ 0.20.

The role of the classical and alternative pathways was then investigated (table II). Normal serum was chelated with a solution of magnesium ions (10 mM) plus E G T A (10 mM final). This solution alone had no effect on bacterial viability (data not shown). Bactericidal activity of Mg-EGTAAnn. Microbiol. (Inst. Pasteur), 134 A, n ~ 3, 1983.

20

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C. OFFREDO-HEMMER, P. BERCHE AND M. V~RON

chelated serum was significantly impaired. The bacterial survival rates dropped from 10 -6.7 (untreated normal serum) to 10 _4.2 (chelated normal serum). Since the Mg-EGTA t r e a t m e n t is known to block the classical p a t h w a y [13], these data suggested t h a t the alternative p a t h w a y was involved. However, the residual bactericidal activity of chelated serum was still very effective. This observation favors the view t h a t the classical p a t h w a y is also involved in bactericidal activity. This fits in with the finding t h a t the bactericidal activity of C2-deficient sera was significantly diminished but still effective. The bacterial survival rates obtained after incubation with these C2-deficient sera ranged from 10 -2.4 to 10 -3.4, whereas t h a t of normal serum was 10 -6., . The bactericidal capacity of C2-defieient sera was totally lost by heating these sera for 30 rain at 560 C (data not shown). In only one case (serum ~ Be ~), was it possible to add purified C2. The serum bactericidal activity was then totally restored (surviving bacteria: 10-6."). This indicates t h a t the C2 component, which is involved in the classical pathway, is required for bacterial killing. The role of antibodies was also investigated by testing h y p o g a m m a globulinaemic serum (table II). This untreated serum was as effective as normal serum in destroying bacteria (bacterial survival rate: 10-6.4). Again the bactericidal activity was totally abrogated b y prior heating (30 min, 56 ~ C). DISCUSSION A non-induced m u t a n t isolated b y subcultures from a virulent, serumresistant strain of P. aeruginosa was used in this work as a model for studying bacterial destruction of P. aeruginosa by fresh serum. This m u t a n t was highly sensitive to the bactericidal activity of fresh h u m a n serum and displayed a low virulence in mice, although fresh mouse serum did not destroy bacteria in vitro. This discrepancy between in vivo rapid bacterial killing in mice and in vitro resistance to fresh mouse serum m a y be explained by several hypotheses. It has long been believed t h a t fresh mouse serum, contrary to human, rabbit or guinea-pig sera, lacks complem e n t activity. Indeed, this absence of complement activity in some inbred strains is due to a C5 deficiency [21]. Even in mouse strains with no detectable C deficiency, complement activity is still difficult to measure by standard haemolytic assay; however, more sensitive assays have been found to be successful in demonstrating mouse complement activity in this case [8]. Therefore, it was plausible t h a t the same difficulty in measuring complement activity would be encountered in our bactericidal assay. It can also be argued t h a t mouse complement acts in vivo, b y killing bacteria through an indirect mechanism such as opsonization [10 I. The acquired susceptibility of the m u t a n t 72VA to fresh h u m a n serum was mediated by the complement system. This conclusion is supported b y the finding t h a t normal heat-treated serum and C-deficient sera either totally or partially failed to destroy bacteria. Direct evidence t h a t comple-

COMPLEMENT-SENSITIVE P. AERUGINOSA

291

ment was involved in bacterial lysis was brought out by the observation that the bactericidal activity of complement-defcient sera was specifically restored by addition of the corresponding purified components C2 and C7. The respective roles of the alternative and classical pathways of the complement system were first investigated by chelating normal human serum with Mg-EGTA. It was found that chelation of normal human serum significantly reduced bactericidal activity, but thal this reduction was not complete. This chelation is known to block the antibody-dependent classical pathway through binding of Ca 2+ ions, but allows the Mg2+-requiring alternative pathway. Consequently, these data indicate that the classical pathway is required for bacterial killing. However, the alternative pathway also plays an important role, since the bactericidal activity of serum after Mg-EGTA chelation was still partially effective. Moreover, the bactericidal activity of C2-deficient sera was impaired and then completely restored by the C2-purified component. Since the C2 component is implicated only in the classical pathway, the role of this pathway is confirmed. On the other hand, it was found that the bactericidal activity of hypogammaglobulinaemic serum with no detectable amount of auti-P, aeruginosa agglutinating antibodies was as effective as that of normal serum. Although it cannot be ruled out that even in hypogammaglobulinaemic serum only minute quantities of specific antibodies participate in bacterial killing through the classical pathway, this result favors the view that total bacterial destruction can be completed through the alternative pathway alone. Our data also suggest that the killing capacity of human serum toward the m u t a n t 72VA is considerable. Indeed, bacterial destruction proceeded quite rapidly and efficiently even when the classical complement pathway was blocked or deficient. However, the specific contribution of each of the two pathways to serum killing activity could not he evaluated under our experimental conditions. It is inferred that both pathways participate synergistically in bacterial lysis. It has been reported that most mucoid and non-mucoid serum-sensitive strains of P. aeruginosa isolated from cystic fibrosis patients were destroyed only via the classical pathway [38]. Moreover, some strains isolated from various P. aeruginosa infections have been reported to be susceptible to both alternative and classical pathways [12, 18]. However, the role of complement and the contribution of each pathway in the 'bactericidal activity of human serum against P. aeruginosa has been evaluated only through indirect evidence, based mainly on the chelation procedure. This work provides the first direct evidence of complement involvement and the possible role of the two pathways in bacterial killing of P. aeruginosa by human serum. The importance of the antibody-independent alternative pathway as a killing mechanism of non-specific defence [32] is therefore confirmed. Biochemical changes in the outer membrane components of Gramnegative bacteria have been correlated with bacterial serum resistance. This has been demonstrated primarily in various strains of Escherichia coli and Salmonella sp., including changes in lipopolysaccharide composition [2, 27, 36, 37], protein profile [9, 35] and lipid composition [1]. Such

292

C. OFFREDO-HEMMER, P. BERCHE AND M. VERON

biochemical differences between serum-sensitive and serum-resistant strains of P. aeruginosa have not yet been studied, but it is expected that the chemical membrane composition of 72VA strain may be altered as compared to the original 72V0. On the other hand, it is known t h a t various plasmids confer increased complement resistance upon their hosts [9, 22, 25, 26, 31]. Such a hypothesis for a genetic mechanism remains to be investigated in

P. aeruginosa. The mechanism whereby complement proteins kill Gram-negative bacteria is still obscure. The complement proteins m a y act on specific sites located in the outer membrane, but the nature of their primary target remains uncertain [5, 43, 44]. Such a complement-membrane interaction might ultimately result in the loss of wall material and an impairm e n t of the supportive function of this structure.

RI~SUMI~ MUTANT

DE

~r P S E U D O M O N A S

SENSIBLE

AERUGINOSA ~

AU COMPLI~MENT

Cette 6tude montre le r61e du compl6ment dans l'activite bact6ricide du serum humain sur une souche m u t a n t e de Pseudomonas aeruginosa utilis6e comme mod61e. La preuve directe de ce r61e, ainsi que celle de la participation des deux voies, classique et alterne, est apport6e grace l'utilisation de serums de malades atteints de deficits en certains facteurs du syst~me du complement. MOTS-CLES : Pseudomonas aeruginosa, Bact~ricidie s6rique, Compl6m e n t ; Mutant, Voie alterne, Voie classique.

REFERENCES [1] AKIYAMA,Y. & INOUE, K., Isolation and properties of complement-resistant strains of E. coll. In/eel. I m m u n . , 1977, 18, 446-453. [2] ALLEN, R. J. • SCOTT, G. K., Human serum complement requirements for bacterial killing and protoplast lysis of E. coli ML 308 225. J. gen. Microbiol., 1981, 123, 179-181. [3] ALPER, C. A. & ROSEN, F. S., Human complement deficiencies, in (( Mechanisms of immunopathology 7)(S. Cohen, P. H. Ward & R. T. McCluskey) (p. 289-305). John Wiley & Sons, Chichester, 1979. [4] ARCHER, G. & FEKETY, F. R., Experimental endocarditis due to Pseudomonas aeruginosa. - - I. Description of a model. J. in/ed. Dis., 1976, 134, 1-7. [5] BAYER, M. E., The fusion sites between outer membrane and cytoplasmic membrane of bacteria: their role in membrane assembly and virus infections, in ,( Bacterial outer membranes ,~ (M. Inouye) (p. 167-202). John Wiley & Sons, Chichester, 1979.

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