Metabolically controlled killing of Pasteurella septica by antibody and complement

598 Biochimica et Biophysica Acta, 362 (1974) 598--602

© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands BBA Report BBA 21397 METABOLICALLY CONTROLLED KILLING OF P A S T E U R E L L A S E P T I C A BY ANTIBODY AND COMPLEMENT

ELWYN GRIFFITHS National Institute for Medical Research, Mill Hill, London, NW7 1AA (U.K.)

(Received June 24th, 1974)

Summary Recent work on the killing of Pasteurella septica by antibody and complement has shown that the process has features not previously associated with serum bactericidal reactions. In the present work it is shown that uncouplers and inhibitors of oxidative phosphorylation protect the bacteria against the action of antibody and complement. The results suggest that the bactericidal reaction is dependent upon bacterial oxidative energy metabolism.

The mechanism of action of antibody and complement on bacteria is a highly complicated process, involving not only the binding of the antibody to the bacterial cell but the subsequent attachment and activation of complement. Although much is known about the individual steps in the complement reaction sequence, it is still not known how the combined action of antibody and complement damages and kills a bacterial cell [1--4]. A widely held assumption is that the mechanism is similar to that involved in lysis of the red blood cell [ 5,6]. It is also assumed that the mechanism responsible for killing avirulent bacteria is the same as that responsible for killing highly virulent organisms [6,7,8]. Little attention has been paid to the possibility that they may be different. Recent work on the mechanism of action of protective horse antibody against the highly virulent organism, Pasteurella septica, has shown that the process, which appears to involve complement, operates by interfering with the biochemistry of the bacterial cell [9,10,11]. This leads to a rapid intracellular degradation of ribosomal RNA, loss of ribosomes, and cell death. Both killing and the degradation of RNA can be prevented by the addition of haematin [ 10,11 ], or by inactivating the complement by heating the serum at 56°C for 30 min. The addition of acetyltyrosine ethyl ester, which inhibits

599 the complement sequence at C3 [12,13], and destruction of C3 and C4 by ammonia treatment [14] also abolishes the antibacterial effects (Griffiths, E., unpublished results). A feature of the action of antibody and complement on P. septica is the delay which elapses between the time of addition of antiserum to the bacteria growing rapidly in fresh serum, and the onset of RNA degradation and cell death [11]. The length of this time interval, about 40 min, suggests that some metabolic event has to occur before RNA breakdown and killing can be initiated. Since neither chloramphenicol nor rifampicin affected the time course of RNA breakdown it was concluded that the synthesis of a new protein, such as a nuclease, was not involved [11]. In the present work, the effects of uncouplers and inhibitors of oxidative phosphorylation have been studied. The results suggest that the bactericidal action of antibody and complement on P. septica depends upon bacterial oxidative energy metabolism. Fig. 1 shows the effect of 2,4 dinitrophenol on the killing of P. septica by specific horse antibody and complement. It can be seen that addition of dinitrophenol 5 min after the addition of antiserum abolished the bactericidal effect (Fig. la) and also prevented the degradation of RNA (Fig. lb). Addition of dinitrophenol 35 min after the antiserum, but before the onset of RNA breakdown and cell death, had little or no effect (Fig. 1), either on the bactericidal event or on RNA degradation. Separate experiments showed that dinitrophenol did not interfere with the binding of the antibody to P. septica. Bacteria exposed to antiserum for 5 min at 37°C both in the presence or 7.C

b r

~oo-~-~-~-

o

~

o

o

o - 0

\o

80

a

~

E 0

o6.C

.

7,

~'e

~~ 6o

~

o

o-

0

;

~40

C 5.(

20

.m > \

O0L

I

20

I

40

I

~o

I

8o

Time (rain)

I

loo

I

20

2o

6'o

8'0

1~,o

T i m e (rain)

Fig.l. Effect of dinitrophenol on the action of P. septica antiserum. Specific antiserum [i0] was added at zero time to bacteria, prelabeiled with [ 14C]t~racil [11], growing in normal horse serum under controlled conditions [i 0,11 ]. Viability and loss of radioactive label from the ceils were determined as before [11]. (a) Viability. ~, control, n o dinitrophenol; o dinitrophenol added 5 rain after the antiserum; e, dinitrophenol added 35 rain after the antiserum. (b) B r e a k d o w n of R N A . ~, control, no added dinitrophenoL 1 0 0 % = 4 0 0 0 c p m ; 0 dinitrophenol added 5 rain after antiserum, 1 0 0 % = 3 9 0 0 cpm; e, dinitrophenol added 35 rain after antiserum, 1 0 0 % = 3 9 5 0 cpm. Final dinitrophenol concentration in serum

was 4.10 -3

M in each

case.

600 absence of dinitrophenol (4- 10- 3 M) were killed and their RNA degraded when t h e y were resuspended in fresh serum without antibody and dinitrophenol. Cyanide (5.10-3 M) was also effective in abolishing the antibacterial action of antiserum. The addition of KCN 5 min after the addition of antiserum abolished the bactericidal effect and prevented the degradation of RNA in exactly the same way as dinitrophenol. Similarly, when KCN was added 35 min after the antiserum but again before the onset of RNA breakdown and cell death, it had little or no effect, either on the bactericidal action or on RNA breakdown. Abolition of the antibacterial action of antiserum was also obtained with carbonylcyanide m-chlorophenylhydrazone (2.10- 3 M). This is another uncoupler of oxidative phosphorylation. The same result was also obtained under anaerobic conditions. Anaerobic conditions were produced by replacing the usual gas mixture of (5% CO2 + 95% air) and (5% CO2 + 95% N2 ) [10,11] with 02 free (5% CO2 + 95% N2 ), and the last traces of 02 removed by adding ascorbic acid (0.1%, w/v). Since Fe 2÷ is known to abolish the action of antiserum on P. septica [15], it was important to eliminate the possibility that the ascorbic acid was reducing Fe 3÷ on the transferrin and producing free Fe 2÷ in the serum. A separate experiment, carried out with the help of P.A. Charlwood and P.H. Jarritt, showed that ascorbic acid (0.1%, w/v; buffered at pH 7.3) did not remove iron from s9 Fe-transferrin. These results show that uncouplers and inhibitors of oxidative phosphorylation protect P. septica against the action of antibody and complement. They also suggest that the bactericidal reaction is dependent upon the metabolic state of the bacterial cell. On the basis of these results with energy inhibitors, the following tentative scheme is proposed for the pathway of action of antibody and complement on P. septica:

Bacteria +

f antibody + complemen

.--~ Stage I

Energy

Stage II (promotes RNA degradation and cell death)

Stage I, in which antibody and perhaps some components of complement bind to the bacteria, does not require energy and does not cause any apparent physiological damage. Energy appears to be required to convert Stage I to Stage II, the stage in which the physiological damage occurs. Energy inhibitors have no effect when added during Stage II. The present results do not, of course, show whether complement acts specifically at Stage I or later at Stage II, nor whether all the components of the complement system are required. However, the observation that the action of antibody and complement on P. septica depends on cellular oxidative energy metabolism means that the bacteria are taking an active part in their own death.

601

In this and certain other respects, the bactericidal action of antiserum on P. septica shows interesting similarities to the action of colicins on bacteria and some instructive comparisons can be made. The colicins and antibody and complement can be considered as highly specific antibacterial proteins. After binding to surface receptors, the colicins induce various lethal biochemical events in the bacterial cell [16--22] and, in the case of P. septica, antibody and complement induce the breakdown of RNA and cell death. The action of colicins, like that of P. septica antiserum, is also apparently dependent upon bacterial oxidative energy metabolism. Thus, uncouplers and inhibitors of oxidative phosphorylation protect bacteria against colicin action [ 1 8 , 2 3 , 2 4 ] . Furthermore, the antibacterial action of colicins also proceeds through at least two successive stages [23,24,25 ]. Whilst these resemblances may be general and point simply to a common action on the cell membrane, it is clear that current concepts about the action of antibody and complement on bacteria will have to be modified, at least as far as they apply to P. septica. Whether or not other highly virulent bacteria are dealt with in a similar way remains to be seen. References 1 K o l b , W.P., H a x b y , J . A . , A r r o y a v e , C.M, and MiJller-Eberhard, H.J. ( 1 9 7 2 ) J. E x p . Med. 1 3 5 , 549--566 2 R o m m e l , F . A . a n d M a y e r , M.M. ( 1 9 7 3 ) J. I m m u n o l . 1 1 0 , 6 3 7 - - 6 4 7 3 M a y e r , M.M. ( 1 9 7 2 ) P r o c . Natl. A c a d . Sci. U.S. 69, 2 9 5 4 - - 2 9 5 8 4 MiJller-Eberhard, H.J. (1972) The Harvey Lectures 66, 75--104 5 C o o m b s , R . R . A , and L a c h m a n n , P.J. ( 1 9 6 8 ) Br. Med. Bull. 24, 1 1 3 - - 1 1 7 6 R o w l e y , D. ( 1 9 7 3 ) J . I n f e c t . Dis. 1 2 8 , $ 1 7 0 - - S 1 7 5 7 H u m p h r e y , J . H , and D o u r m a s h k i n , R . R . ( 1 9 6 9 ) Adv. I m m u n o l . 11, 7 5 - - 1 1 5 8 M u s c h e l , L . H . ( 1 9 6 5 ) Ciba F o u n d a t i o n S y m p o s i u m o n C o m p l e m e n t , Churchill, L o n d o n , 1 5 5 - - 1 6 9 9 G r i f f i t h s , E. ( 1 9 7 1 ) Nat. N e w Biol. 2 3 2 , 89---90 1 0 G r i f f i t h s , E. ( 1 9 7 1 ) Ettr. J. B i o c h e m . 23, 6 9 - - 7 6 11 G r i f f i t h s , E. ( 1 9 7 4 ) Bioehim. B i o p h y s . Acta 3 4 0 , 4 0 0 - - 4 1 2 1 2 Basch, R.S. ( 1 9 6 5 ) J. I m m u n o l . 9 4 , 6 2 9 - - 6 4 0 1 3 S h i n , H.S. a n d M a y e r , M.M. ( 1 9 6 8 ) B i o c h e m i s t r y 7, 3 0 0 3 - - 3 0 0 6 1 4 L a c h m a n n , P.J., H o b a r t , M.J. and A s t o n , W.P. ( 1 9 7 3 ) in H a n d b o o k of E x p e r i m e n t a l I m m u n o l o g y , (Weir, D.M., ed.), C h a p t . 5, B l a c k w e l l Scientific Publications, O x f o r d 1 5 Bullen, J . J . , R o g e r s , H . J . and Lewin, J . E . ( 1 9 7 1 ) I m m u n o l o g y 20, 3 9 1 - - 4 0 6 1 6 N o m u r a , M. ( 1 9 6 3 ) C o l d Spring Harbor S y r u p . Q u a n t . Biol. 28, 3 1 5 - - 3 2 4 17 N o m u ~ a , M. ( 1 9 6 7 ) A n n u . Rev. M i c r o b i o l . 21, 2 5 7 - - 2 8 4 1 8 Reeves, P. ( 1 9 7 2 ) The Bacteriocins, Vol. 1 1 , Mol. Biol. B i o c h e m . B i o p h y s . , Springer Verlag, Berlin 19 Fields, K . L . a n d L u r i a , S.E. ( 1 9 6 9 ) J. B a c t e r i o l . 9 7 , 5 7 - - 6 3 2 0 H o l l a n d , I.B. ( 1 9 6 8 ) J. Mol. Biol. 31, 2 6 7 - - 2 7 5 21 N o s e , K. a n d M i z u n o , D. ( 1 9 6 8 ) J. B i o c h e m . 64, 1 - - 6 2 2 K o n i s k y , J. a n d N o m u r a , M. ( 1 9 6 7 ) J. Mol. Biol. 2 6 , 1 8 1 - - 1 9 5 23 H o l l a n d , E.M. a n d H o l l a n d , I.B. ( 1 9 7 0 ) J. G e n . M i c r o b i o l . 6 4 , 2 2 3 - - 2 3 9 2 4 De G r a a f , F . K . ( 1 9 7 3 ) A n t o n i e van L e e u w e n h o e k 39, 1 0 9 - - 1 1 9 2 5 Plate, C.A. a n d L u r i a , S.E. ( 1 9 7 2 ) P r o c . N a t l . A c a d . Sci. U.S. 6 9 , 2 0 3 0 - - 2 0 3 4