INFECTION AND IMMUNITY, Sept. 1988, p. 2324-2329 Vol. 56, No. 90019-9567/88/092324-06$02.00/0Copyright © 1988, American Society for Microbiology

Effect of Small Cationic Leukocyte Peptides (Defensins) on thePermeability Barrier of the Outer Membrane

P. VILJANEN,l P. KOSKI,' AND M. VAARAl 2*

National Public Health Institute, SF-00280 Helsinki,' and Department of Bacteriology and Immunology,University of Helsinki, SF-00290 Helsinki,2 Finland

Received 4 January 1988/Accepted 26 May 1988

Defensins are small cationic antibacterial peptides that are abundant in polymorphonuclear leukocytes fromhuman and other sources (T. Ganz, M. Selsted, D. Szklarek, S. Harwig, K. Daher, D. F. Bainton, and R. J.Lehrer, J. Clin. Invest. 76:1427-1435, 1985). We studied whether subinhibitory concentrations of defensinsincrease the outer membrane (OM) permeability of Escherichia coli, Salmonella typhimurium, and Pseudomo-nas aeruginosa to hydrophobic probes, as do many other polycations that have been studied previously.Throughout the study, we used polymyxin B nonapeptide (PMBN) as a reference peptide. PMBN has a knownpotent OM permeability-increasing action. As a sharp contrast to PMBN, subinhibitory concentrations ofdefensins did not permeabilize (or, under some test conditions, permeabilized very slightly) the OM to theprobes that were used (rifampin and Triton X-100). At bacteriostatic or bactericidal defensin concentrations,some degree of synergism with rifampin was seen.

Gram-negative bacteria are more or less sensitive tovarious strongly cationic peptides from practically anysource. Such peptides include the antibiotics of the poly-myxin series (38), cationic polyamino acids (4, 14, 20, 40, 44,47), protamines (14, 40, 44, 47), histones (14), platelet-derived basic peptide (6), seminal plasmin from bull semen(31, 39), cecropins from the giant silk moth (2), the anti-lipopolysaccharide (LPS) factor from Limulus crab he-mocytes (1, 25), and the cationic proteins (BPI, CAP37, andcathepsin G) from the granules of polymorphonuclear leu-kocytes (PMNs) (8, 11, 37).

Several independent lines of evidence indicate that thefirst target of cationic peptides on the surface of gram-negative bacteria is the polyanionic LPS molecules locatedon the outer surface of the outer membrane (OM) (5, 26, 28,30, 32, 35, 37, 40, 42, 46, 48, 53). Furthermore, many of thelarger cationic peptides (BPI, CAP37, and anti-LPS factor[9, 25, 36, 51, 52]) are mainly active on mutants which haveonly the proximal part of the LPS remaining (the anionicdeep core part and lipid A) but lack the more distal polysac-charides.One of the most striking characteristics of the intact

bacterial OM is its remarkable impermeability to hydropho-bic molecules such as hydrophobic antibiotics (27). Thebinding of a polycation to LPS in vivo often causes the OMto lose this effective permeability barrier function. Thisaction of a polycation is first observed at concentrationsclose to the lethal concentration of the polycation (BPI) (8,52, 53), at a slightly lower concentration (polymyxin, prota-mine, polylysines, and compound 48/80 oligomers [19, 20,40, 44, 45]), or at even 100-fold lower concentrations (poly-myxin nonapeptides and Lys-20 [7, 17, 22, 44, 45, 47, 49, 50])than the lethal concentration of the particular polycation.Accordingly, measurement of this loss of the barrier againsthydrophobic probes is often one of the most sensitive assaysto study the effects of certain polycations on the OM.

Recently, Lehrer and co-workers (10, 11, 33, 34) foundand characterized a family of small (3,000 to 4,000 daltons)cationic peptides from the granules of PMNs that are rich in

* Corresponding author.

arginine, cysteine, and hydrophobic amino acids. Whentested in vitro at neutral pH in a low-ionic-strength medium(0.01 M sodium phosphate buffer), those peptides displayedantimicrobial activity against a wide variety of bacteria,fungi, and even some enveloped viruses (10, 33, 34) (hence,the proposed name of defensins for those peptides). How-ever, the microbicidal activity against gram-negative bacte-ria, for example, was seen only at relatively high defensin(.50 pug/ml) and physiological salt (0.14 M NaCl) concentra-tions, and acidic pH abolished the activity.We studied whether sublethal concentrations of defensins

increase OM permeability to hydrophobic probes, as domany other polycations (as mentioned above). We used thehuman leukocyte-derived defensins as a pool of the threedescribed peptides (33), each of which contains four arginineresidues and a total of five basic charges (33). Throughoutthe study we used polymyxin B nonapeptide (PMBN) as thereference peptide. It is a cyclic nonapeptide containing fivebasic charges and has a very weak bactericidal action but apotent OM permeability-increasing property (43-45, 49, 50).

MATERIALS AND METHODSIsolation of small cationic leukocyte peptides (defensins).

Human PMNs were prepared from fresh buffy coats of unitsof whole blood (from healthy blood donors) either by themethod of Boyum (3), who used a Ficoll-Paque (PharmaciaFine Chemicals, Piscataway, N.J.) gradient, or by themethod of Hjorth et al. (16), who used a Percoll gradient.Before gradient centrifugation, most of the erythrocyteswere removed by dextran precipitation, as described previ-ously (16), and from the final PMN preparation, by hypoto-nic lysis (10).The PMNs were broken by using a glass pestle homoge-

nizer, and the PMN granules were isolated and extractedwith 10% acetic acid exactly as described by Ganz et al. (10).The extract was then ultrafiltrated (nominal cutoff limit,5,000 daltons; YM 5 filter; Amicon Corp., Danvers, Mass.),concentrated on a filter (nominal cutoff, 1,000 daltons; YM 2;Amicon), lyophilized, dissolved in 0.01% acetic acid in waterto yield a stock solution of 1 mg/ml (micro-Coomassie blueassay; Bio-Rad Laboratories, Richmond, Calif.), and stored

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DEFENSINS AND OM PERMEABILITY 2325

at -20°C. All dilutions of this defensin stock solution (whichwere needed in the bactericidal assays) were made similarlyin 0.01% acetic acid (to avoid absorption of the peptides onplastic).Methods used in purity analysis of defensin preparation.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(PAGE) was carried out in 15% acrylamide (thickness, 0.8mm) by the method of Laemmli (21). The chromatogram wasstained with Coomassie blue. Acid-urea PAGE was per-formed as described previously (29) with 15% acrylamide,2.5 M urea, and 5% acetic acid and by staining with amidoblack.A reversed-phase high-pressure liquid chromatography

(RP-HPLC) system consisted of a liquid chromatograph(5000; Varian Instruments, Inc., Palo Alto, Calif.), a loopinjector (Rheodyne, Cotati, Calif.), a C18 column (4.6 mm by25 cm; particle size, 5 ,.am; pore size , 30 nm; Vydac 218 TP;Separations Group, Hesperia, Calif.), and a variable wave-length detector (UV-100; Varian). Chromatography solventswere 0.1% (vol/vol) trifluoroacetic acid in water (solvent A)and 0.075% (vol/vol) trifluoroacetic acid in acetonitrile (sol-vent B). The solvent gradient was linear from 20% solvent Bto 60% solvent B in 20 min. The flow rate was 1.2 ml/min.Effluent was monitored at an A215 at a sensitivity setting of0.05 absorbance units per mV.

Bacterial strains. Escherichia coli IH3080 (018:KI:H7)and Salmonella typhimurium LT2 SL696 were smoothstrains that we used in our earlier studies (43-45, 48, 50). Thesource of these strains has been described previously (44).The smooth strain Pseudomonas aeruginosa PA01 (12) wasa kind gift from H. Nikaido.

Cultivation of bacteria. Bacteria were grown in L broth (10g of tryptone [Oxoid Ltd., Hampshire, United Kingdom], 5g of yeast extract [Difco Laboratories, Detroit, Mich.], and5 g of NaCl per liter [pH 7.0]) on a rotary shaker at 220 rpmand 37°C to the early logarithmic phase of growth (60 Klettunits, as determined with a Klett-Summerson colorimeterequipped with a red filter). The grown bacteria were usedimmediately (without chilling).

Assay media. The following media were used in the assayfor the OM permeability-increasing activity described be-low: A, 0.01 M NaH2PO4-Na2HPO4 buffer (PB; pH 7.4)containing 300 ,g of solid tryptic soy broth (TSB) (Difco) perml, as described previously (10); B, like medium A, but alsocontaining 150 mM NaCl; C, PB containing 300 jig of TSBper ml and set to a final pH of 5.7; D, like medium C, but alsocontaining 150 mM NaCl; E, Davis minimal medium, withglucose (1 g/liter), Casamino Acids (1 g/liter; Difco), andtryptophan (20 mg/liter), as described previously (44).

Assay for OM permeability-increasing activity. In the assayfor OM permeability-increasing activity, bactericidal probes(the antibiotic rifampin and the nonionic detergent TritonX-100), which do not permeate the intact OM but whichtraverse the damaged OM, were used (23, 27). The assay wasperformed as a bactericidal synergy assay by using checker-board dilutions.The bacteria were grown as described above and were

used to inoculate the assay medium with 5 x 104 cells per ml(see above). The assay medium also contained increasingconcentrations of rifampin or Triton X-100. Portions (100 ,ul)of these inoculated media were pipetted into wells of amicrotiter plate (Nunclon Delta; catalog no. 167008; Nunc,Roskilde, Denmark), after which increasing amounts of thecationic peptides (defensins or PMBN) in 10 ,ul of 0.01%acetic acid were added to each well. The control wells lackedany cationic peptide but contained an equal amount (10 pl) of

0.01% acetic acid. This addition of acetic acid did not changethe pH of media A, B, and E; but it decreased the pH ofmedia C and D by 0.2 units, to pH 5.5. The microtiter plateswere incubated at 37°C for 2 h (E. coli), 3 h (S. typhimurium),or 5 h (P. aeruginosa); and the viable counts in each wellwere determined by transferring the whole contents of eachwell to separate vials, followed by serial dilution of thecontents (in 150 mM saline) and plating of duplicate portionsonto L agar (L broth with 1.5% agar). The colonies wereallowed to grow for 24 h at 37°C.When applicable, the fractional inhibitory concentration

(FIC) index (24) was calculated as follows: [(A)/(MICA)] +[(B)/(MICB)] = FICA + FICB = FIC index, where (A) and(B) are the concentrations of drugs A and B in a checker-board tube which were inhibitory to the test organism andwhere synergism with drug A and B was suspected and(MICA) and (MICB) are the corresponding inhibitory con-centrations of drug A and drug B alone, respectively.

Source of chemicals. The papain-cleaved derivative ofpolymyxin (PMBN) was prepared essentially as describedby Viljanen and Vaara (50) and was a kind gift from FarmosGroup, Ltd. (Turku, Finland). Its residual content of poly-myxin B was found to be approx. 0.1%, as determined byRP-HPLC with a end-capped, large-pore C18 column(Vydac; Separations Group), a water-acetonitrile solventgradient system, and trifluoroacetic acid as the ion-pairingagent (42a). All stock solutions of PMBN and the dilutionsthereof were made in 0.01% acetic acid, because that vehiclewas also used for all defensin preparations.

Rifampin was obtained from Sigma Chemical Co. (St.Louis, Mo.). Rifampin stock solution (1 mg/ml) was made in10% methanol (50); all subsequent dilutions were made in theassay medium. Triton X-100 was from BDH Chemicals Ltd.(Poole, England). Histone VII S was from Sigma. Hen eggwhite lysozyme was from Boehringer GmbH (Mannheim,Federal Republic of Germany).

RESULTS

Isolation, purity analysis, and direct antibacterial activity ofthe defensin preparation. The defensins were isolated essen-tially as described earlier by Ganz et al. (10). When thepreparation was analyzed by sodium dodecyl sulfate-PAGE(Fig. 1A), only one band was visible; it had a molecularweight of approx. 3,000 to 4,000 (corresponding to that ofdefensins). In acid-urea PAGE (Fig. 1B), where the migra-tion of peptides partially depends on the peptide charge, ourpreparation ran as a cationic double spot that was fairlysimilar to that reported by Ganz et al. (10) for the naturallyoccurring mixture of the three defensins. RP-HPLC (Fig.1C) revealed, besides the main peak, two minor impurities.They made up approx. 2% of the total protein (if quantifiedon the basis of the A215).The maximal antibacterial action of defensins has been

shown by Lehrer and co-workers (10, 34) to be manifested ata neutral pH (7.4) in a low-ionic-strength medium. Perhapssurprisingly, the activity is weaker or absent at the pH whichprobably prevails inside the polymorphonuclear phagolyso-some, as well as in the medium with a physiological ionicstrength (10, 34). In accordance with those results, we foundthat when our defensin preparation was tested under optimalconditions (in growth-permitting, low-ionic-strength medium[pH 7.4; medium A; see above] identical to the medium usedby Ganz et al. [10]), approx. 30 pug of defensin per ml was

required to decrease the viable count of E. coli by 2 log1ounits from the original count. The activity was weaker at pH

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2326 VILJANEN ET AL.

A

CNp

I,._,w-

.q1_--

144K 6

aim

B C

5 10 15

RETENTION TIME (MIN.)

A B C D A B CFIG. 1. Electrophoretic and HPLC analysis of the purified small cationic leukocyte peptide (defensin) preparation. (A) Sodium dodecyl

sulfate-PAGE (slab; Coomassie blue staining). Lane A, molecular weight standards; lanes B and C, two independently isolated batches ofdefensins (3 jig per lane); lane D, hen egg white lysozyme (1 ,ug). (B) Acid-urea PAGE (slab; amido black staining). Lane A, basic proteinstandards, histone VII S (upper band; 5 ,ug) and lysozyme (lower band; 5 jig); lanes B and C, two batches of defensins (20 ,ug per lane). (C)Chromatogram obtained by RP-HPLC of the defensin preparation (4 ,ug; detection at 215 nm). A linear gradient from 20% solvent B to 60%solvent B in 20 min was used. See text for HPLC conditions. The peak eluting at 2.5 min is acetic acid. Bar, 0.025 absorbance units.

5.5 (medium C). Even 100 ,ug of defensin per ml wascompletely ineffective at physiological ionic strength both atpH 7.4 (medium B) and pH 5.5 (medium D).

Sensitization assay. Rifampin is a hydrophobic bactericidalantibiotic which does not effectively permeate the intact OM

PMBM (pg/mi)

ILC.ae

SCLP(pg/ml)

RIFAMP.(hAnI) RIFAMP. (pgAnl)

of enteric bacteria but which traverses through the OM ofcertain OM-defective mutants as well as through the OMdamaged by polycations (27). Results of preliminary exper-iments indicated that approx. 10 ,ug of rifampin per ml wasrequired to kill E. coli IH3080 during a 2-h incubation periodin a growth-permitting medium (e.g., medium A or B),whereas 30- to 100-fold less was as effective if the OM wassimultaneously damaged by PMBN (1 ,uglml), the knownOM-damaging agent. Accordingly, this assay with rifampinwas chosen for most of the subsequent studies.

Sensitizing effect of defensins. (i) E. coli IH3080. Thesensitization assays were made in four media (A, B, C, andD) that differed on the basis of their pHs and ionic strengths(see above). Throughout the assays, PMBN was used as anactive reference peptide. The results are given in Fig. 2 and3.As a sharp contrast to PMBN, subinhibitory concentra-

tions of defensins did not sensitize (or sensitized veryslightly) the target E. coli to the bactericidal action ofrifampin. This lack of effect was seen in all the assay media(Fig. 2 and 3). However, at bacteriostatic or bactericidal

U.

be

RIFAMP.(Mti)

FIG. 2. Combined effect of the small cationic leukocyte peptide(SCLP) preparation (defensins) and rifampin against E. coli Kl:018IH3080 in low-ionic-strength media (medium A [top panels; pH 7.4]and medium C [bottom panels; pH 5.5]; see text). CFU weredetermined after an incubation period of 2 h at 37°C and comparedwith the CFU present at the beginning of the experiment. Thesynergism of PMBN and rifampin (under identical conditions) isshown for comparison. Each panel displays the results of a singleexperiment. Each datum point is the geometric mean of duplicatesamples; the standard deviation was <0.1 log1o unit.

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DEFENSINS AND OM PERMEABILITY 2327

PMBN(pIgml) SCLP (pg/ml)

EXPMT 1.

RIFAMP. (0gn)

EXPMT 2.

RIFAMP.("M) RIFAMP.(")

IL-U.

RIFAMPA(t/mil) RIFAMP.(h"AI)0.3 1 3

RIPAMP.(")FIG. 3. Combined effect of the small cationic leukocyte peptide (SCLP) preparation (defensins) and rifampin against E. coli IH3080 in

media containing 150 mM NaCl (medium B [top panels; pH 7.4] and medium D [bottom panels; pH 5.5]; see text). Other experimentalconditions were as described in the legend to Fig. 2. The results of two independent experiments are shown. The synergistic effect ofPMBNand rifampin (under identical conditions) is shown for comparison. Each panel displays the results of a single experiment. Each datum pointis the geometric mean of duplicate samples; the standard deviation was s0.1 log1o unit.

defensin concentrations, some degree of synergisnm (betweendefensins and rifampin) was seen. Thus, in medium A a FICindex (24) of approx. 0.37 was achieved (a FIC index of .0.5indicates synergism). Similarly, in medium C the FIC indexwas approx. 0.38.

(ii) P. aeruginosa PAO1. Only a rich growth medium(medium E) was used to study P. aeruginosa PAQ1. Inmedium FE defensins (tested up to 100 pxg/ml) did not have

PMBN (pg/mi)

any antibacterial activity and did not sensitize the bacteria torifampin. In contrast, 1 ,ug of PMBN per ml was able todecrease the MIC of rifampin by a factor of approx. 100 (Fig.4).

(iii) S. typhimurium SL696. For S. typhimurium SL696 weused, besides rifampin, a neutral bactericidal detergent,Triton X-100, as another probe for OM-damaging action.While PMBN effectively sensitized the target bacteria to

SCLP (pg/mi)

IL

0

I I

0 0.01 0.03 0.1 0.3 1

RIfmp.(pg/mI)

1000-

100

10-

3 10

SCLP 30

SCLP 100

* CLP 0

0.01 0.03 0.1 0.3 1 3 10

RHabmp.(Pg/mIFIG. 4. Combined effect of the small cationic leukocyte peptide (SCLP) preparation (defensins) and rifampin against P. aeruginosa PA01

in a neutral growth medium (medium E; see text). Incubation was for 5 h at 37°C. Each panel displays the results of a single experiment. Eachdatum point is the geometric mean of duplicate samples; the standard deviation was '0.1 log1o unit.

La.0e

IL.0

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TABLE 1. Combined effect of the small cationic leukocyte peptide preparation (defensins) with rifampin or Triton X-100 againstS. typhimurium SL696 in a neutral growth medium (medium E)a

Viable countsb in the presence of:

Medium SCLP (,ug/ml)' PMBN

0 6 20 60 200 (6 Fig/ml)Medium E 1.3 x 103 1.3 x 103 1.3 x 103 1.3 x 103 1.3 x 103 1.3 x 103Medium E + rifampin (0.6 ,ug/ml) 1.3 x 103 1.3 x 103 1.2 x 103 5.7 x 102 5.9 x 102 2Medium E + Triton X-100 (300 ,ug/ml) 1.3 x 10" 1.3 x 103 1.3 x 103 1.3 x 103 1.3 x 103 <1

a Incubation was for 3 h at 37°C. Other experimental conditions were as described in the text. The values are based on the results of one experiment and aremeans of duplicate samples.

b Values are related to an arbitrary value of 100 at the beginning of the experiment.c SCLP, Small cationic leukocyte peptide.

both probes, no clear sensitization was achieved by defen-sins (when tested up to 200 ,ug/ml) (Table 1).

DISCUSSIONAs reviewed earlier in this report, the first measurable

action of probably all antibacterial polycations against gram-negative bacteria is a damage to their OM. The lethal actionis secondary to that of the OM-damaging action and ismanifested, depending on the particular polycation, at poly-cation concentrations that are not much higher, or even100-fold higher, than that required for OM damage. Becausethe small cationic peptides from leukocyte granules (defen-sins) are only weakly bactericidal, we thought it would beinteresting to see whether subinhibitory concentrations ofdefensins could, however, damage the OM. We showed thatdefensins practically lacked such an action.

Accordingly, neither bactericidal nor OM-damaging actionwas found against smooth strains of E. coli, S. typhimurium,or P. aeruginosa, even at defensin concentrations as high as100 to 200 ,ug/ml, when the assays were performed in neutralmedium at physiological ionic strength. For comparison, 1,ug of PMBN per ml effectively damaged the OM. In acorresponding acidic medium (pH 5.5, possibly mimickingthe pH inside the phagolysosome), very slight OM damageprobably took place. In low-ionic-strength medium, defen-sins appeared to be bactericidal at relatively low concentra-tions; subinhibitory concentrations did not damage the OM,but inhibitory concentrations had a slight synergistic actionwith the OM probe antibiotic.OM damage is usually most sensitively detected as an OM

permeability increase to hydrophobic probes (13, 27), likethose used in this study. Other probes such as lysozyme orphospholipases are considered to be less sensitive or insen-sitive (13, 18; M. Vaara, unpublished data).Human defensins are not the only relatively cationic

agents which lack any potent OM-damaging action. Nisin,tetralysine, and colistin heptapeptide belong in this category(15, 17, 44). Recently, we studied lysine oligomers in moredetail and found that pentalysine, but not the shorter oligo-mers, decreased the viable count of P. aeruginosa andenteric bacteria and, at subinhibitory concentrations, per-meabilized their OMs. Those effects were, however, ob-served only in the low-ionic-strength medium; the additionof 150 mM NaCl to the medium abolished both activities (M.Vaara, unpublished data). Thus, the behavior of pentalysineis somewhat reminiscent of that of defensins and probablyindicates that its binding to LPS is of low affinity andsensitive to the competition of buffer ions (see also reference41).

Defensins from other sources, such as the rabbit, were notincluded in this study. Even though they appear, in general,

to have approximately similar antibacterial action as thehuman defensins, they are more cationic and, consequently,might be more active against the OM.

Finally, as pointed out by Ganz et al. (11), it should benoted that even though defensins are (on a weight ormolarity basis) weak antibacterial agents against gram-neg-ative organisms, their role as a part of the defense againsteven these pathogens might still be considerable, becauseapparently very high concentrations (milligrams per millili-ter) of defensins exist in the phagocytic granule (11).

ACKNOWLEDGMENTS

We thank P. H. Makela and T. Vaara for many useful discussions.This study was supported by grants from the Emil Aaltonen

Foundation (to P.V.) and the Academy of Finland (to M.V.).

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