Antibacterial peptides and mitochonrial presequences affect mitochonrial coupling, respiration and protein import

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Eur. J. Biochem. 223, 1027-1033 (1994) 0 FEBS 1994

Antibacterial peptides and mitochondrial presequences affect mitochondrial coupling, respiration and protein import Marie HUGOSSON', David ANDREU3, Hans G. BOMAN' and Elzbieta GLASER'

'

Department of Biochemistry, Arrhenius Laboratories for Natural Sciences, Stockholm University, Sweden

* Department of Microbiology, Arrhenius Laboratories for Natural Sciences, Stockholm University, Sweden Department of Organic Chemistry, University of Barcelona, Barcelona, Spain (Received February 2WApril27, 1994) - EJB 94 0280/1

Cecropins A and P1, antibacterial peptides from insects and from pig and some related peptides released respiratory control, inhibited protein import and at higher concentrations also inhibited respiration. However, PR-39, an antibacterial peptide from pig intestine, was found to be almost inert towards mitochondria. The concentrations at which the three mitochondrial functions were effected varied for different peptides. Melittin, magainin and Cecropin-A-(1,13) -Melittin(l,l3)NH2, a hybrid between cecropin A and melittin, were most potent, while the two cecropins acted at higher concentrations. The biosynthesis of cecropin A is known and the intermediates are synthesized. We have used four peptides from this pathway to investigate their effects on coupling, respiration and protein import into mitochondria. Mature cecropin A followed by the preproprotein were most aggressive whereas the intermediates were less active or inert. The efficiency of different derivatives of cecropin A as uncouplers correlates well with their capacity to release membrane potential measured as fluorescence quenching of Rhodamine 123. Inhibition of respiration was found to be dependent on membrane potential and was most pronounced with mature cecropin A, less so with its three precursors. We also found that three peptides derived from mitochondrial presequences showed antibacterial activity. It is concluded that, there are similarities in the functions of antibacterial peptides and mitochondrial presequences, uncoupling activity in mitochondria cannot be correlated with the antibacterial activity (contrary to a previous suggestion), the processing of preprocecropin A may have evolved in such a way that there is a minimum of membrane damage from the intermediates in the pathway, and peptides produced for delivery outside of an animal have evolved to be more aggressive against mitochondria than peptides for delivery inside of the animal.

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During the last 13 years several classes of broadspectrum antibacterial peptides have been discovered in the animal kingdom, among them cecropins, defensins and magainins (reviewed by Boman, 1991; Zasloff, 1992). These three groups of peptides are well characterized, they have been cloned, their three-dimensional structures are known and for cecropins and defensins the gene structures have been determined. All three groups of peptides also form channels in artificial membranes but it is not yet known if channel formation is responsible for the lethal activity on bacteria. There are clear structural similarities between certain anti-

Correspondence to E. Glaser, Department of Biochemistry, Arrhenius Laboratories for Natural Sciences, Stockholm University, S-10691 Stockholm, Sweden Abbreviations. CA( 1,37)-NH,, amidated, natural cecropin A ; CA(1,37), cecropin A with a free C-terminal carboxyl group; CA(1,38), cecropin A extended by a C-terminal Gly; CA(-4,38), procecropin A; CA(-26,38), preprocecropin A ; CA(1,13)-Melittin( 1,13)-NH,, a cecropin-melittin hybrid peptide with C-terminal amide while CA(l,l3)-M(l,13) has a free C-terminal carboxyl group; pCOX IV(1,22) and pCOX IV(1,23), two peptides derived from the presequences of subunit IV of cytochrome-c oxidase; pF1P(1,25), a peptide derived from the prosequence of subunit F,P of the ATP synthase; ATIII, a helical peptide derived from the heparin-binding domain of antithrombin 111; CCCP, carbonylcyanide m-chlorohydrazone.

bacterial peptides and mitochondrial presequences, in particular these two types of peptides are membrane active and can form positively charged amphipathic a helices (for reviews on protein import into mitochondria, see Hart1 et al., 1989; Baker and Schatz, 1991). Two antibacterial peptides from the frog Xenopus, magainin and PGLa, were found to release the respiratory control in rat liver mitochondria and it was suggested that this could be the mechanism by which bacteria were killed (Juretic et al., 1989; Westerhoff et al., 1989 a and b). We have decided to extend these studies for the following reasons. Westerhoff et al. only recorded the action on mitochondria for two closely related frog skin peptides. We wanted to compare clearly different peptides from different sources for their action on mitochondria and to compare natural antibacterial peptides with peptides derived from mitochondrial presequences. We also wanted to investigate whether the precursors of a membrane-active peptide have evolved in such a way that they do not interfere with the intracellular membranes present in the organelles of the cells that make the peptide. Intracellular membranes are present chiefly in mitochondria, in the endoplasmic reticulum, the Golgi apparatus and the nucleus. We consider mitochondrial membranes as model targets for the study of different membrane reactions caused by antibacterial peptides. Cecropins are known to lyse

1028 bacterial membranes within minutes, while they do not cause any detectible lysis of mitochondria. Since mitochondria are endosymbionts of microbial origin they may reveal biochemical reactions caused by cecropins that cannot be detected on live bacteria. In the present work we show that cecropins act as uncouplers, but at higher concentrations than magainin and the bee venom toxin melittin. Some of the antibacterial peptides also inhibited respiration albeit at quite different concentrations. The same was found for two synthetic peptides corresponding to the N-terminal part of mitochondrial precursor proteins. Three such peptides showed a moderate antibacterial activity against Bacillus meguterium. Comparison of the precursors of cecropin A show that they are less aggressive to mitochondria than the mature peptide. A preliminary report of some of our results has appeared in a symposium volume (Hugosson et a]., 1992).

Protein import into mitochondria The procedure of Whelan et al. 1990 was followed. Spinach (Spinacia oleracea L. cv. Medania) leaf mitochondria were purified as described in Hamasur et al. (1990). Labelled Neurosporu pre-FJ, the catalytic subunit of the mitochondrial ATP synthase, was prepared by in vitro transcription and translation of linearized pGem3 vector using SP6 RNA polymerase (Melton et al., 1984) and rabbitt-reticulocyte lysate (Pelham and Jackson, 1976) with ["Slmethionine. Peptides were incubated for 5 min with spinach mitochondria prior to import experiments. The samples were centrifuged at maximal speed in an Eppendorf centrifuge at 4"C, and the peptide-treated mitochondria were used for in vitro import experiments. After import and proteinase K treatment, mitochondria were washed once in import medium. All samples were then analyzed by SDSPAGE using a gradient of 1217% polyacrylamide (Laemmli, 1970) followed by autoradiography.

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MATERIALS AND METHODS

Mitochondria, respiratory measurements and membrane potential

Potato tuber mitochondria were purified according to (Glaser et al., 1989). Pig heart mitochondria were purified according to (Lee and Ernster, 1967). The protein content was measured according to (Peterson, 1977). Respiratory rate of mitochondria was measured at 23°C with a Hansatech 0, electrode in 0.5 ml respiration medium (0.3 M sucrose, 10 mM KC1, 5 mM MgCl,, 0.1% BSA, 10 mM potassium phosphate, pH 7.2) with 1.2 mM NADH (exogen substrate for plant mitochondria) as substrate for potato tuber mitochondria and 10 mM malate and 10 mM glutamate as substrate for pig heart mitochondria. Cecropins and other peptides were added directly to the 0,-electrode chamber. Uncoupling results are given as the relative increase in oxygen consumption, using as reference respiration with only NADH. Inhibition is given as a percentage of maximal respiration in the presence of carbonylcyanide m-chlorophenylhydrazone (CCCP). Mitochondria1 energizing was monitored by fluorescence quenching of Rhodamine 123 (Emaus et al., 1986).

Antibacterial peptides and mitochondrial presequences All peptides were made by the solid-phase method (Merrifield, 1986) except cecropin A with a free C-terminal carboxy group [CA(1,37)] which was made as a fusion protein from a synthetic gene (Callaway et al., 1993) Natural cecropin A, [CA(1,37)-NH,] which has a C-terminal amide group, and its precursors (Boman et al., 1989a) and melittin were synthesized at the Merrifields laboratory (New York). Magainin 2 amide was obtained from M. Zasloff (now at Magainin Pharmaceutical Inc.). The hybrid CA(1,13)-Melittin(l,13)-NH2 (Boman et al., 1989b) and its C-terminal carboxy homolog~ewere synthesized by Magainin Pharmaceutical Inc. and A. Engstrom (University of Uppsala), respectively. The peptide corresponding to residues 1-25 of subunit F,P of the ATP synthase, pF,,8(1,25), was synthesized by one of us. The peptides derived from the presequences of subunit IV of cytochrome-c oxidase, pCOX IV(1,22) and pCOX IV(1,23) and antithrombin ATIII were obtained from M. Cumsky, University of California, Irvine. PR-39 was purified from pig small intestine (Agerberth et al., 1991).

Antibacterial activity assay

An inhibition-zone assay was used with thin agarose plates seeded with about 10' viable cells (Hultmark et al., 1983). For each peptide, a dilution series was applied to small wells (3 pl) and after incubation overnight the diameters of the inhibition zones were used to calculate the lethal concentration (the LC value), the lowest concentration that will inhibit bacterial growth.

RESULTS The peptides used There are certain structural similarities between antibacterial peptides and mitochondrial presequences, in particular both are membran-active structures and they can form amphipathic a helices which are positively charged. The sequences of the peptides used are given in Fig. 1. Cecropins are key factors in cell-free immunity in insects and their biosynthesis was experimentally settled with the synthetic precursors given in Fig. 1. A cecropin has also been found in porcine small intestine (Lee et al., 1989). Insect cecropins are composed of two different domains linked by a hinge region (Holak et al., 1988), while the porcine cecropin is an extended helix with a flexible segment (Sipos et al., 1992). Melittin is a bee venom toxin that can form dimers. As a monomer it has a structure that resembles the cecropins but the polarity is reversed. The hybrid molecule CA( 1 - 13)melittin( 1- 13)-NH, with one domain from cecropin A [CA(l-13)] and another from melittin [melittin(l- 13)] is a potent antibacterial and antimalarial agent (Boman et al., 1989b). The solution stucture is also known for this peptide (Sipos et al., 1991). Magainin 2 is an antimicrobial peptide from the skin of Xenopus luevis (Zasloff, 1987) and PR-39 is a proline-arginine rich antibacterial peptide from pig small intestine (Agerberth et al., 1991). Also included in Fig. 1 are two peptides with sequences derived from the presequences of subunit IV of cytochrome-c oxidase, pCOX TV(1-22) and pCOX IV(1-23), and one derived from subunit FJof the ATP synthase, pF,/3(1-25). Finally, ATIII is an a-helical peptide derived from antithrombin 111by an inversion of residues 125-146.

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Sequences of antibacterial peptides used:

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Cecrapin A [ 1 , 3 7 ) :

KWKLFKKIEK VGQNIRDGII K A G P A V A W G QATQIAK-NH2

Cecropin A(1,38):

KWKLFKKIEK VGQNIRDGII K A G P A V A W G QATQIAKG-OH

Cecropin P1:

SWLSKTAKKL ENSAKKRISE GIAIAIQGGP R-OH

0

."7

GIGKFLHSAK KFGKAFVGEI MNS-NH2

Me1 ittin :

GIGAVLKVLT TGLPALISWI KRKRQQ-NH2

CA(l,l3)M(l,l3):

KWKLFKKIEK VGQGIGAVLK VLTTGL-NH2

PR-39:

RRRPRPPYLP RPRPPPFFPP RLPPRIPPGF PPRFPPRFP-NH2

0

CA(1,38)

d

E

'-

Magainin 2 amide:

Prepro-sequence of cecropin

4-

CA(-26,38)

3.5

-x- -x CA(-4,38)

A

pSignal---Pro-

-20

-26

-10

-4

-5

MNFSRI FFFVFACLTA LAMVNA

-

-1

AP

-

EP -

Peptldes derived from mitochondrial presequences :

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pF1P(1,25):

0

MASRRLLASL LRQSAQRGGG LISRS

pCOX IV(1.23): MLSLRQSIRF FKPATRTLCS SRY

Peptide derived from antithrombin 111: RNASVLKSSK NAKRYLRCNL KA

Fig. 1. Amino acid sequences of cecropin, magainin, melittin, the cecropin-melittin hybrid, PR-39, three peptides derived from mitochondrial presequences and one from antithrombin 111. The natural forms of cecropin A and melittin are amidated at the Cterminus while cecropin P1 has a free carboxyl group. We have used an amidated form of magainin 2 despite the fact that the natural form has a free carboxyl group. In the prepro sequence for cecropin A, the processing sites are indicated by hyphens.

4

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4

5

3

m

LL

2 1

10

15

Peptide

pCOX IV(1.22): MLSLRQSIRF FKPATRTLCS SR

ATIII:

5

20

25

30

(FM)

Fig.3. Effects of cecropin A and three of its precursors on the respiration of potato tuber mitochondria. Respiration in mitochondria was measured as described in Materials and Methods. The difference between cecropin A and CA(1,38) is due to the inhibition of respiration by the former peptide. The primary translation product, preprocecropin A, CA(-26,38), (0) ; procecropin A, CA(-4,38), (X); the glycine-extended precursor CA(1,38), (0); and the mature cecropin A, CA(1,37)-NH,, (e).All peptides were added directly to the 0,-electrode chamber.

of potato tuber mitochondria. Fig. 2 shows that all five peptides stimulated state 2 respiration, acting as uncouplers at lower concentrations. The respiratory activity was not stimulated in the presence of uncouplers. However, the peptide concentration needed for maximal stimulation varied significantly. Melittin, magainin 2 and the hybrid peptide showed maximum stimulation at concentrations of 2-8 pM (530 nmol peptid/mg mitochondrial protein). The two cecropins, A from the cercropia moth and P1 from pig, acted as uncouplers only at much higher concentration, 13 pM and 23 pM (50 nmol/mg mitochondrial protein and 80 nmoVmg mitochondrial protein), respectively. With the peptide concentration giving maximal respiratory activity there was no further stimulation by CCCP, the standard uncoupler of the mitochondrial respiration. The porcine cecropin P1 together with pig heart mitochondria offered us a possibility to investigate a homologous system and determine whether the previously observed uncoupling was the result of a heterologous system. There was no significant difference between the two cecropins (data not shown) and it is therefore unlikely that the uncoupling is an artifact produced by the heterologous system used before. Since the processing of preprocecropin A, CA( -26,38), to mature cecropin A, CA(1,37)-NH2, is known (Boman et al., 1989a), we compared these peptides and the two intermediates, CA(-4,38) and CA(1,38) for their effect on respiration. Fig. 3 shows that the highest stimulation of the respiratory activity of potato tuber mitochondria was obtained with CA(1,38), the precursor of cecropin A with an additional C-terminal glycine. The difference seen between the mature CA(1,37)-NH2 and the glycine extended precursor, CA(1,38), is due to the stronger inhibition obtained with the former peptide. Procecropin A, CA( -4,38), was almost inert while the prepro form CA(-26,38) was clearly less active than the mature peptide CA(1,37)-NH2. Fig. 4 shows that PR-39 and pF,p(l,25) had almost no effect on respiration. Also peptide ATIII was without any activity (data not shown). The two peptides derived from the subunit IV of cytochrome-c oxidase stimulated respiration.

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30

100

120

Peptide (nmolhng protein)

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140

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Fig. 2. Uncoupling of respiration by five different peptides with antibacterial activties using potato tuber mitochondria. Respiration was measured as described in Materials and Methods. Peptides, cecropin A, (+) ; cecropin P1, (A) ; cecropin-melittin hybrid CA(1,13)-(1,13)-NH2 (CAM-NH,), ( X ) ; melittin, ( 0 ) ; and magainin, (+) were added directly to the 0,-electrode chamber. Uncoupling of respiration by antibacterial peptides and mitochondrial presequences

We have compared the concentration dependence for cecropins A and P1, magainin 2 amide, melittin and the hybrid CA(1,13) - (1,13)-NH, for their effects on the respiration

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Fig. 4. Uncoupling of respiration in potato tuber mitochondria by four peptides, three with sequences derived from mitochondrial presequences. The fourth is the proline + arginine-rich antibacterial peptide PR-39. Respiration was measured as described in Materials and Methods. Peptides, pC0X IV (1,23), (0);pCOX IV (1,22),(A)pF,P(1,25),( X ) ; and PR-39, (+), were added directly to the 0,-electrode chamber.

The effect of cecropins and its precursors on energizing of potato mitochondria measured as fluorescence quenching of Rhodamine 123. Cecropin A consisting of only D-amino acids showed similar efficiency as the natural cecropin A (data not shown). The order of efficiency of different derivatives of cecropin A as dissipators of the membrane potential correlated well with their capacity as uncouplers.

5

10

15

20

25

Peptide (FM)

Fig. 5. Effects of cecropin A and its precusors on the inhibition of respiration of potato tuber mitochondria. Respiration in mitochondria was measured as described in Materials and Methods and 100% respiration corresponds to the activity recorded in the presence of 1 pM CCCP. The primary translation product, preprocecropin A, CA(-26,38), (0);procecropin A, CA(-4,38),(X); the glycine-extended precursor CA(1,38), (0); and the mature cecropin A, CA(1,37)-NH2U. All peptides were added directly to the 0,-electrode chamber.

120 CCCP followed by cecropin A

Inhibition of mitochondrial respiration by cecropin A and its precursors Measurements of the respiratory activity in the presence of antibacterial peptides and CCCP (added after the peptide) revealed that CA( 1,37)-NH, inhibited respiration almost completely at a peptide concentration of 13 pM whereas the glycine-extended precursor. CA( 1,38) at the same concentration inhibited respiration only slightly. We therefore compared all the cecropin precursors for their inhibitory capacity on respiration. Fig. 5 shows that the mature cecropin A was a potent inhibitor of respiration while the glycin-extended precursor, CA(1,38), was clearly less effective (Fig. 3). Procecropin A, CA(-4,38) and the prepro form, CA(-26,38), gave only small effects. Fig. 6 shows an experiment with cecropin A added to mitochondria, either prior to or after the addition of CCCP. If CCCP was added first there was only a small inhibitory effect of cecropin A at 20 pM, while in the reverse experiment (when cecropin A was added first) almost complete inhibition was obtained at 10 pM peptide. This result indicates that cecropin A does preferentially act on mitochondria with intact membrane potential.

Cecropin A followed by CCCP

0

0

5

10

15

20

25

Peptide (pM)

Fig. 6. Effects of the order of addition of cecropin A and CCCP on the inhibition of respiration of potato tuber mitochondria. Respiration in mitochondria was measured as described in Materials and Methods. ( X ) Data with CCCP (final concentration 1 pM) added first and followed by different amounts of cecropin A (final concentration given). (*)Data with cecropin A added first (to different concentration) and followed by the addition of CCCP to a final concentration of 1 pM.

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Effects of cecropin A, its precursors and the D enantiomer on protein import into mitochondria

Fig. 7A shows that mature cecropin A was found to be the most active inhibitor of both binding and import of preF,P (Fig. 7A). The preproform, CA(-26,38) gave similar result while the proform, CA( -4,38) and the glycine-extended

molecule, CA(1,38) had hardly any effects. Fig. 7B shows a similar experiment with cecropin A, its glycine-extended precursor and the D enantiomer of cecropin A (Wade et al., 1990). It is evident that under conditions when the glycineextended precursor of cecropin A showed a slight stimulation of import, both cecropin A and its D enantiomer inhibited import completely. That there was no difference between the natural cecropin A and its D enantiomer shows that cecropin can act without any binding to a mitochondrial receptor with chiral properties.

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more, the peptide with highest antibacterial activity pF,P was almost inert in the uncoupling experiments (Fig. 4). Also a peptide derived from antithrombin I11 (ATIII) which resembles mitochondrial presequences (Glaser and Cumsky, 1990) showed moderate activity against B. megaterium. The synthetic peptides of Park et al. were found to be moderately antibacterial against Bacillus subtilis while there was essentially no activity towards Gram-negative bacteria (Park et al., 1989). In the case of these peptides there was no correlation between antibacterial activity and effects recorded on mitochondria.

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L-CA

D-CA

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Okada and Natori (1984) reported early that a flesh-fly cecropin released the membrane potential in E. coli. Then Westerhoff et al. (1989a) suggested that the effects they recorded on mitochondria with the two frog peptides, magainin and PGLa were a likely mechanism for the antimicrobial effects of these compounds. Romeo’s group have reported that a proline-rich antibacterial peptide, Bac5, affected the energy metabolism of susceptible bacteria (Skerlavaj et al., 1990). Many investigators have claimed that channel formation and membrane disruption are the primary mechanisms of action of antibacterial peptides (for reviews see Boman, 1991 ; Zasloff, 1992). In the present work we have demonstrated that the structural similarities between certain antibacterial peptides and mitochondrial presequences also result in certain functional similarities. All peptide antibiotics, except PR-39, can act as uncouplers of oxidative phosphorylation and the mitochondrial presequences did show a moderate capacity to lull B. megaterium. We have confirmed the finding of Westerhoff et al. (1989a) that magainin 2 amide is an effective uncoupler of mitochondria. However, we have extended the work to include, comparisons of several antimicrobial peptides from different species with peptides derived from mitochondrial presequences, and for cecropin A we have compared the mature peptide with some of its precursors. The first comparison shows that melittin and the hybrid CA( 1,13) -M( 1,13)-NH, were more active uncouplers than magainin 2 amide, while the two cecropins were considerably less potent than magainin 2 amide. The effectivity of different derivatives of cecropin A as uncouplers correlates well with their capacity to release membrane potential measured as fluorescence quenching of Rhodamine 123. Magainin 2 amide has optimum uncoupling around 5 p M (20 nmol peptide/mg mitochondrial protein) while the corresponding value for cecropin A is around 13 pM (60 nmol peptide/mg mitochondrial protein ; Fig. 2). Since the lethal concentration for these two peptides on E. coli is 4 pM and 0.2 pM (Table l),respectively, there cannot be a simple correlation between the uncoupling in mitochondria and the killing of bacteria. Another lack of correlation is seen when comparing our results with the two peptides derived from the prosequence of cytochrome-c oxidase. Finally, when we tested PR-39 on mitochondria, virtually no effects could be recorded (Fig. 4), nevertheless, this peptide is highly active against E. coli and some other Gram-negative bacteria (Agerberth et al., 1991, and Table 1). This is in agreement with the fact that PR-39 does not lyse susceptible bacteria, it being more likely that it affects chromosome replication (Boman et al., 1993). For peptides which rapidly lyse bacteria, like the cecropins, it is impossible by experiment to demonstrate any

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Fig. 7. Effect of cecropin A, its precursors and D-enantiomer of ecropin A on in vitro import of 35S-labeledpre-F,/?into spinach leaf mitochondria. Synthesis of Neurospora F,P and import conditions were described in Material and Methods. Proteinase K (PK) was used to determine if the proteins were outside or inside the mitochondria. Cecropins and derivatives were added to a final concentration of 3 pM. (A) Lane 1, translation product of pre-F,P; lanes 2 and 3, in vitro import of pre-F, into spinach leaf mitochondria prior to and after proteinase K treatment. In vitro import experiments as described in lanes 2 and 3 with spinach leaf mitochondria in the presence of CA(-4,38) (lanes 4 and 3,CA(-26,38) (lanes 6 and 7), CA(1,38)(lanes 8 and 9) and CA(1,37)-NH2 (lanes 10 and 11). (B) Lane 1, translation product of pre-F,P; lanes 2 and 3, in vitro import of pre-F,P into spinach leaf mitochondria prior to and after proteinase K treatment. Lanes 4 and 5, Gly-extended C(1-38). Lanes 6 and 7, L-enantiomer of cecropin A (L-CA). Lanes 8 and 9, D-enantiomer of cecropin A (D-CA), all at 3 pM.

Antibacterial activities of different peptides Table 1 shows the antibacterial activities, as LC values, of the peptides used here. Some of these data have been published previously. Only one peptide is totally without antibacterial activity, namely preprocecropin A. However, preprocecropin A acts at higher concentration as uncoupler. The proform showed an activity that was about 30-times less than the mature peptide both on Escherichia coli and Pseudomonns aeruginosa. The natural cecropin P1 is clearly a more potent agent against Gram-negative bacteria than magainin (Table l), but the reverse is true of the uncoupling activity (Fig. 2). Finally, PR-39 is highly potent against several Gram-negative bacteria but does not uncouple mitochondrial respiration. We have tested synthetic peptides derived from other mitochondrial presequences for antibacterial activity (Table 2). Two of these peptides pF,P(1-25) and COX IV(122) show moderate activity against B. megaterium; the first one also has some activity against E. coli. It is noticeable that the addition of a single residue (a tyrosine) to the Cterminus of the second peptide reduced the antibacterial activity (Table 2 ) while it increased the uncoupling. Further-

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Table 1. Lethal concentrations of antibacterial peptides. Lethal concentrations calculated from inhibition zones on thin agarose plates seeded with the respective organisms. D21, E. coli; OT97, f! aeruginosa; B s l l , B. subtilis; Staph, S. uureus, Cowan I; Strep, S. pyogenes; SRC, Sheep red cells. The date for preprocecropin A and procecropin A are from (Boman et al., 1989), those for PR-39 are from (Agerberth et al., 1991). Compound

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Cecropin A-NH, (CA) Cecropin A-NH, Cecropin CA( 1,13)-M( 1 ,I 3) CA(l ,I 3)-M( 1 ,I 3)-NH, Cecropin A-Gly Cecropin PI Cecropin P1-NH, Melittin-NH, Magainin 2-NH2 Preprocecropin A Procecropin A PR-39

Number and form of residues

Lethal concentration for

D21

OT97

37L 37D 37L 261 2 6 3 8L 3 1 311 26L 2 3 641 4 2 39L

0.2 0.3 0.5 0.6 0.5 0.5 0.4 0.4 0.8 4 70 13 0.3

1 1 8 3 1 3 13 6 3 30 140 80 200

______.

~

~

~

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Table 2. Antibacterial activity as lethal concentration for three peptides derived from mitochondria1 presequences. Lethal concentration was determined as in Table 1. pF,P(1,25) is derived from ATP synthase, the next two from cytochrome c oxidase while ATIII is a perfect n helix designed from antithrombin 111. ~

Presequences

~~

Lethal concentration for B. megaterium

E. coli

w pFiB(1,25) p c o x IV(1,22) pCOX IV( 1.23) ATIII

4.7 6.1 15 3.1

27 210 390 44

convincing effects on the bacterial energy metabolism. Our results show that there cannot be a simple relationship between the bacterial killing by peptide antibiotics and effects recorded on mitochondria. However, if we consider mitochondria to have evolved from bacterial endosymbionts, then it is conceivable that the effects here recorded for some antibacterial peptides on mitochondria reflect a very ancient mechanism of antibacterial activity. During evolution the peptides would have been modified to become lytic and specific for the membranes of present-day, free-living bacteria. Cecropin A is the only antibacterial peptide for which the biosynthesis has been elucidated and where the preproform and the intermediates have been chemically synthesized (Boman et al., 1989). We have used this set of peptides for a comparison of three different mitochondrial functions, coupling, respiration and protein import. In all cases the three precursors (Figs 3, 5 and 7) of cecropin A were less aggressive on mitochondria than the mature peptide CA(1,37)-NH2. Preprocecropin A is the primary translation product and it is presumably delivered directly from the ribosome to the endoplasm reticulum (or possibly to a transport vesicle with its own signal peptidase) where the signal is removed. The proform CA(-4,38) generated by the removal of the signal,

Bsll

Staph

Strep

SRC

3 3

>300 >300

5 2

>200 >300

14 2 >200 >500 >500 0.2 300

0.6

20 44 29 0.5 4

500 600 >200 >400 >700

200

2

0.3 0.7 8 10 5 0.2 3

15

1

4

300

was almost inert on mitochondria and its antibacterial activity is also low (Table 1).Cecropin A and its glycin-extended precursor CA(1,38) were about equally potent as uncouplers, but the mature peptide did inhibit respiration at much lower concentration than the precursors. We have also found significant differences (Figs 3 and 6) between CA(1,37), the mature amidated cecropin A and its glycin-extended precursor CA(1,38). Thus, to have C-terminal amidation as a last processing step may be a way for nature to minimize the intracellular action of a potentially harmful peptide made for export from the synthesizing organ. Taken together, the relative inertness of the proform and the other cecropin precursors indicates that one force behind the evolution of the cecropin pathway may have been to reduce unwanted exposure of the different cellular membranes to the end product, cecropin A, a highly membrane-active peptide. We have here compared five naturally occurring peptides from two insects (cecropin A from hemolymph of the cecropia moth and melittin from bee venom), from one frog (magainin) and from pig intestine (cecropin PI and PR-39). The overall conclusion from this comparison of different antimicrobial peptides is that peptides produced for delivery outside of the frog skin (magainin) or outside of the exoskeleton of the insect (melittin) are much more aggressive to mitochondria than the two cecropins which are delivered inside the respective animals (the two cecropins and PR-39). Thus evolution may have selected peptides for external use to have a highly deterrent effect on predators while those for internal use were selected to have a potent antibacterial activity combined with a minimum of self-destruction. The work was supported by grants from The Swedish Natural Science Research Council (BU1595 to E. G. and BU2453 to H. G. B.) and from Carl Trygger Foundation (to E. G.) and from Comision Interministerial de Ciencia y Tecnologia (PTR93-00032, to D. A,),

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