Ability of Cecropin B To Penetrate the Enterobacterial Outer Membrane

2 downloads 0 Views 741KB Size Report
certain gram-positive bacteria but also againstgram-negative bacteria. Many other ... Hans Bennich (Uppsala,Sweden) and Yukio Kimura (Nishi- nomiya, Japan) ...
Vol. 38, No. 10

ANTIMICROBiAL AGENTS AND CHEMOTHERAPY, OCt. 1994, p. 2498-2501

0066-4804/94/$04.00+0 Copyright © 1994, American Society for Microbiology

Ability of Cecropin B To Penetrate the Enterobacterial Outer Membrane MAR1TI VAARA* AND TIMO VAARA Department of Bacteriology and Immunology, University of Helsinki, 00014 Helsinki, Finland Received 20 April 1994/Returned for modification 6 July 1994/Accepted 9 August 1994

The cationic amphipathic insect peptide cecropin B was almost as active on wild-type enteric bacteria as it their lipopolysaccharide and lipid A mutants that have very defective outer membrane. The polymyxin-resistant strains, which elaborate altered, less anionic lipopolysaccharide, were completely susceptible to cecropin B. No synergism was found between cecropin B and hydrophobic antibiotics. Throughout the study, the activity of cecropin B resembled that of quaternary detergents.

was on

present communication, we have used a collection of wellcharacterized OM mutants of E. coli and Salmonella typhimurium to elucidate this question and show that cecropin B, unlike many other cationic peptides (23), resembles cationic detergents in its ability to cross the OM. Bacterial strains. E. coli strains defective in lipid A biosynthesis were SM101 (lpxA2) and CDH23-213 (lpxD [the gene formerly known as firA and ssc]). Detailed analyses of these strains, as well as their isogenic control strains SM105 (lpxA4+) and CDH23-210 (lpxD+), have previously been published (24,

Cecropins are powerful antimicrobial compounds present in insect hemolymph and are also found in pig intestines (2, 4, 13). They are strongly cationic, amphipathic, a-helical peptides with 35 to 39 residues and are notably active not only against certain gram-positive bacteria but also against gram-negative bacteria. Many other cationic peptides, such as defensins from leukocyte granules and magainins from frog skin, have evoked considerable interest in recent years (5, 8, 14, 16, 18, 29, 30). However, cecropins are in molar terms 10 to 30 times more active than defensins and magainins against Escherichia coli and Pseudomonas aeruginosa (2, 28). Other groups of intensely studied antibacterial peptides are the synthetic fragments of cationic leukocyte proteins CAP37 (azurocidin) and the serine protease cathepsin G (1, 19, 20). These fragments, too, are rather weakly antibacterial. On a molar basis, they are approximately 2 or 3 orders of magnitude less active than cecropins. Cecropins cause instantaneous lysis of bacterial cells, through disintegration of their cytoplasmic membrane (2-4, 21). They also lyse artificial negatively charged and neutral liposomes, but not erythrocytes or positively charged liposomes containing phosphatidylcholine, stearylamine, and cholesterol. The strongly cationic N-terminal amphipathic helix of cecropins is necessary for effective binding to bacterial membranes. Cecropins form ion channels in artificial membranes (7), and cecropin dimers can be predicted by computer modeling to form channel-containing regular lattice structures on the membrane surface (9). However, such channels probably develop only when cecropin density is high and disintegration of the membrane takes place (7, 9). Accordingly, the lethal target of cecropins is the bacterial cytoplasmic membrane. However, to reach it in gram-negative bacteria, cecropins must first penetrate the outermost surface structure, the outer membrane (OM). The OM of E. coli and other enterobacteria is known as an effective permeability barrier to a wide variety of noxious agents (17). The diffusion of hydrophobic compounds through the OM is very restricted because of the lack of glycerophospholipid bilayers, the effective pathway for hydrophobic diffusion. In addition, most of the hydrophilic compounds are excluded; they cannot diffuse through the narrow water-filled porin pores, the channels for simple sugars and other small hydrophilic nutrients (17). How do the cecropins then penetrate the OM? In the

27). Strains with defective lipopolysaccharide (LPS) inner core parts were E. coli D21f2 and Salmonella typhimurium SL1102. Both elaborate Re-type LPS and have previously been well characterized (24). D21 is the parent of D21f2 and has normal E. coli K-12 LPS. IH3080 (018:K1 [25]) was used as a representative of smooth, encapsulated E. coli. Polymyxin-resistant pmrA mutants SH6497 (pmrA125) and SH7426 (pmrA163) of S. typhimurium, as well as their corresponding parent-type controls SH6482 and SH9178, have been previously well characterized (11, 22, 23). SC9252 and SC9253 are polymyxin-resistant E. coli K-12 mutants, and SC9251 is their parent (15). Polymyxin-susceptible mutants IH1301 and IH1304 derive from the inherently polymyxin-resistant Proteus mirabilis R45 (LPS chemotype Re). Antimicrobial agents. Synthetic cecropin B of Hyalophora cecropiae (2) and deacylpolymyxin B (25) were kind gifts from Hans Bennich (Uppsala, Sweden) and Yukio Kimura (Nishinomiya, Japan), respectively. Polymyxin B nonapeptide (26) originated from Farmos Group Ltd. (Turku, Finland). Polymyxin B sulfate, sodium fusidate, erythromycin base, bacitracin, rifampin, novobiocin, and cetylpyridinium chloride were from Sigma Chemical Co. (St. Louis, Mo.), and benzalkonium chloride was from Oy Medica Ab (Helsinki, Finland). Susceptibility determinations. The MICs were determined in L broth as described previously (25) at 28°C for SM101 and CDH23-213 and at 37°C for the other strains. The lowest concentration of a drug which reduced the bacterial growth by .95% was interpreted as the MIC. Assay for OM permeability-increasing activity. Hydrophobic antibiotics (rifampin, fusidic acid, novobiocin, and erythromycin), which penetrate very poorly the intact OM of E. coli IH3080 but which traverse the damaged OM, were used. The assay was performed as a synergistic growth inhibition assay by using checkerboard dilutions as described previously (25). Fractional inhibitory concentration (FIC) indices (10) were calculated as follows: [(A)/(MICA)] + [(B)I(MICB)I = FICA +

* Corresponding author. Mailing address: Department of Bacteriology and Immunology, University of Helsinki, P.O.B. 21, 00014 Hel-

sinki, Finland. Fax: 358-0-4346382. 2498

NOTES

VOL. 38, 1994

TABLE 1. Susceptibility of lipid A mutants and LPS deep core mutants to cecropin B, polymyxin B, cationic detergents, hydrophobic antibiotics, and bacitracin Susceptibility index' for: Compound

Cecropin B Polymyxin B Benzalkonium-Cl

Cetylpyrimidinium-CI Rifampin Fusidic acid Erythromycin Bacitracin

E. coli

E. coli

SM101 (IpxA2)

CDH23-213 (IpxD)

D21f2 (rfa)b

2-4 4

8 4 512 128 512

2-4 8 8 2 256 256 128

.512

.32

1-2 2 4 4 64 64 64 4

E. coli

S. typhimurium SL1102

(rfaE)b 2 2 4 4 32 256 64