With the increasing resistance of many Gram-negative bacteria to existing classes of antibiotics, identifying new paradigms in antimicrobial discovery is an important research priority. Of special interest are the proteins required for the biogenesis of the asymmetric Gram-negative bacterial outer membrane (OM). Seven Lpt proteins (LptA to LptG) associate in most Gram-negative bacteria to form a macromolecular complex spanning the entire envelope, which transports lipopolysaccharide (LPS) molecules from their site of assembly at the inner membrane to the cell surface, powered by adenosine 5′-triphosphate hydrolysis in the cytoplasm. The periplasmic protein LptA comprises the protein bridge across the periplasm, which connects LptB 2 FGC at the inner membrane to LptD/E anchored in the OM. We show here that the naturally occurring, insect-derived antimicrobial peptide thanatin targets LptA and LptD in the network of periplasmic protein-protein interactions required to assemble the Lpt complex, leading to the inhibition of LPS transport and OM biogenesis in Escherichia coli.

Thanatin was first isolated from the hemipteran insect Podisus maculiventris (spined soldier bug) in 1996 ( 17 ). The peptide contains 21 amino acids (GSKKPVPIIYCNRRTGKCQRM) with a disulfide bond between Cys 11 and Cys 18 . Antimicrobial activity for thanatin was reported against E. coli, Salmonella typhimurium, Klebsiella pneumoniae, and Enterobacter cloacae with minimal inhibitory concentrations (MICs) of <1.5 μM and with weaker activity against Erwinia carotovora and Pseudomonas aeruginosa (MICs of 10 to 12 μM). Although no activity was seen against Staphylococcus aureus, thanatin is active against some other Gram-positive bacteria with MICs of ≈1 to 5 μM. Of special interest is the observation that the enantiomeric form (d-thanatin) loses much of its activity against all the Gram-negative strains tested, indicating a likely chiral target.

The asymmetric outer membrane (OM) plays a critical role in protecting Gram-negative bacteria from extracellular cytotoxic molecules, including antibiotics. This unique bilayer comprises lipopolysaccharide (LPS) molecules in the outer leaflet and membrane glycerophospholipids in the inner leaflet. Integral OM proteins (OMPs) are crucial for the biogenesis of the OM, as well as for controlling the uptake and export of essential nutrients and signaling molecules across the OM. Of special interest are the seven proteins (LptA to LptG) needed to transport LPS molecules from their site of assembly at the inner membrane (IM), across the aqueous periplasm, to their final cell surface location during OM biogenesis ( 1 – 3 ). The LptA to LptG proteins associate to form a macromolecular complex that spans the entire envelope ( 4 , 5 ). The periplasmic protein LptA, likely as a head-to-tail oligomer, forms a protein bridge spanning the periplasm. LPS molecules are pushed across this bridge ( 6 – 10 ), from LptB 2 FGC anchored in the IM to the LptDE complex embedded in the OM ( Fig. 1A ) ( 11 – 16 ), powered by adenosine 5′-triphosphate (ATP) hydrolysis in the cytoplasm ( 4 ). We report here that the naturally occurring, insect-derived host-defense peptide thanatin ( Fig. 1B ) targets both LptA and LptD in Escherichia coli.

RESULTS

Mechanism of action The mechanism of action of thanatin is so far unknown. The peptide is bactericidal against E. coli, shows only weak permeabilizing effects on the IM or OM, and does not cause hemolysis of blood erythrocytes even at 100× the MIC (17–20). We confirmed that thanatin has no membrane-permeabilizing effects on E. coli. The fluorescent dye SYTOX Green does not penetrate E. coli cells treated with thanatin even at 100 μg/ml (see section S3). Moreover, no release into the external medium of ß-lactamase expressed in the periplasm or of ß-galactosidase expressed in the cytoplasm of E. coli could be detected upon exposure to thanatin (see Supplementary Materials). Thus, even at concentrations much higher than the MIC, neither the IM nor the OM of E. coli is permeabilized by treatment with thanatin, unlike what is seen with some other cationic antimicrobial peptides, such as polymyxin B, colistin, or protegrin I (21, 22). The effects of thanatin on macromolecule biosynthesis were examined by monitoring the incorporation of appropriate [3H]-labeled precursors into macromolecules in E. coli American Type Culture Collection (ATCC) 25922 (see section S4). No inhibition of protein, RNA, DNA, or cell wall biosynthesis was observed. To analyze effects on morphology, we grew E. coli cells with thanatin at concentrations causing substantial growth inhibition and then examined them in thin sections by transmission electron microscopy (TEM). This revealed frequent defects in membrane architecture, with accumulations of membrane-like material inside cells (Fig. 2, A and B). These multilayered membrane folds inside the cell are typical of those reported for E. coli in which LPS transport is inhibited by down-regulation of LptA/B, LptC, or LptD (5, 16). The effects of thanatin on E. coli were also monitored by laser scanning stimulated emission depletion (STED) fluorescence microscopy, with staining of membranes by the membrane dye FM4-64, of nucleoids by 4′,6-diamidino-2-phenylindole (DAPI), and using SYTOX Green to detect permeabilized cells (Fig. 2, C and D). These studies revealed frequent accumulations of membrane-like material in the form of knobs stained bright red by FM4-64 and elongated cell assemblies, with neither effect visible in the absence of thanatin. Nucleoids stained with DAPI were not influenced significantly by thanatin, and again no significant staining was observed by SYTOX Green. These results reveal that thanatin causes defects in the membrane assembly in E. coli. Fig. 2 Electron and fluorescence microscopy studies. (A and B) TEM studies of E. coli ATCC 25922, before (A) and after (B) thanatin treatment (1.5 μg/ml), showing internal accumulations of membrane-like material. Scale bars, 500 nm. (C and D) Super-resolution fluorescence microscopy of E. coli ATCC 25922 without (C) or with thanatin (5 μg/ml) (D) and stained with FM4-64, SYTOX Green, or DAPI. Top: The FM4-64 channel (red staining). Bottom: Superimposition of all three channels [with DAPI (blue) and SYTOX Green (nondetected)]. (E) E. coli staining with thanatin-BDP-FL (8 μg/ml) for 2 hours at 30°C (both pictures). Cells were analyzed using a Leica CLSM SP8 gSTED microscope. Scale bars, 4 or 10 μm (bottom right). For experimental details, see section S5. A fluorescent derivative (thanatin-BDP-FL; MIC of ≈1 to 2 μg/ml; Fig. 1B) was used for STED fluorescence imaging. This probe stained the E. coli envelope, with marked focal accumulations (as green fluorescent knobs) across the cell and at the cell poles (Fig. 2E). This behavior is reminiscent of fluorescent labeling of OMPs that accumulate into membrane islands or clusters and at the poles in Gram-negative bacteria (23, 24). The interaction of thanatin with OMPs in E. coli was tested directly by photoaffinity labeling experiments. Two thanatin-derived photoprobes were synthesized containing photoproline (25, 26), in place of either Pro5 or Pro7, together with an N-terminal polyethylene glycol linker and biotin tag for pull-down assays (Fig. 1B and sections S2 and S5). Both probes (thanatin-PAL5 and thanatin-PAL7) maintained antimicrobial activity against E. coli ATCC 25922 (MICs of 2 to 4 μg/ml) and photolabeled the same membrane proteins in vivo, as shown in Western blotting for thanatin-PAL5 in Fig. 3A. In a competitive photolabeling experiment with native thanatin (200 μg/ml) as a competitor, thanatin-PAL5 (2 μg/ml) labeling of the ≈95-kDa band largely disappeared from the blot, whereas the other signals showed reduced labeling (Fig. 3A). When the in vivo photolabeled membrane protein extract was analyzed under nonreducing conditions to retain disulfide bonds, a shift of the ≈95-kDa band to ≈130 kDa was seen in the Western blot (Fig. 3B and the Supplementary Materials). This change in gel electrophoretic mobility is very characteristic of that reported for E. coli LptD in the disulfide-reduced and disulfide-oxidized forms (LptD ox ≈ 130 kDa and LptD red ≈ 90 kDa) (27, 28). Fig. 3 Photolabeling of E. coli ATCC 25922 with thanatin-PAL5. (A) Western blot (biotin detection) and corresponding SDS–polyacrylamide gel electrophoresis (SDS-PAGE) (Coomassie blue staining) of membrane protein fraction from: lane 1, control unlabeled cells; lanes 2 and 3, cells photolabeled with thanatin-PAL5 (10 and 2 μg/ml); and lane 4, cells photolabeled with thanatin-PAL5 (10 μg/ml) + competitor thanatin (200 μg/ml). (B) Western blot and SDS-PAGE after photolabeling with thanatin-PAL5 (2 μg/ml) with (+) or without (−) reduction of extracted membrane proteins with dithiothreitol (DTT). (C) Volcano plot showing relative abundance of E. coli proteins in thanatin-PAL5 labeled versus unlabeled control sample after streptavidin pulldown detected by MS-based proteomic analysis. Significantly enriched proteins (right/above dashed lines) are highlighted in green and represent PAL5-labeled proteins. It was technically not possible to identify photolabeled proteins directly from the Western blots. To identify thanatin interaction partners in a hypothesis-free, discovery-driven approach, we used the photoaffinity interaction mapping strategy outlined above, in combination with mass spectrometry (MS)–based proteomic analysis, which allows for the multiplexed and label-free quantification of E. coli interaction partners. Thanatin-PAL5–photolabeled and untreated control E. coli ATCC 25922 cells were lysed, and biotinylated proteins were purified using streptavidin-functionalized agarose resin. Enriched proteins were proteolytically digested and subsequently identified using high-performance liquid chromatography–tandem MS (HPLC-MS/MS). Four hundred proteins were identified at a false discovery rate (FDR) below 1%. Relative quantitative comparison revealed the specific and photolabeling-dependent enrichment of three proteins, namely, LptD, LptA, and BamB, of which LptD and LptA were the most significant (Fig. 3C). Whereas photolabeling of the membrane protein LptD was already suggested by the Western blotting experiments described above, photolabeling of LptA, a small (≈18 kDa) soluble periplasmic component of the LPS transport pathway (Fig. 1A), was unexpected.

Resistant mutants Spontaneous thanatin-resistant (ThanR) mutants of E. coli ATCC 25922 were sought for genetic analysis. ThanR mutants could be isolated at low frequency (≈1 in 108 colony-forming units (CFU); 10−6%) by passaging on Mueller-Hinton II (MH-II) agar containing thanatin (10 to 50 μg/ml). Five ThanR mutants were selected that remained stable over at least four passages on thanatin-free agar (see section S6). Three mutants showed no difference in growth behavior compared to the wild-type (WT) in MH-II media and no increased sensitivity on MH-II agar supplemented with 0.5% SDS and 1 mM EDTA. Also, the susceptibility of all mutants to a series of standard antibiotics was unchanged (see section S6). Whole-genome sequencing of the three ThanR mutants revealed several mutations compared to the WT genome, including lptA as the only mutated gene common to all three. Moreover, one resistant mutant (ThanR-8) contained only a single point mutation in the entire genome, corresponding to a change of glutamine to leucine at position 62 (Q62L) in LptA. We also tested whether the identified mutations Q62L-LptA and D31N-LptA would confer resistance to thanatin when introduced into a sensitive E. coli strain. For this, genes encoding LptA with a His6 tag at the C terminus (LptA-His6), as well as the corresponding mutated variants (Q62L-LptA-His6 and D31N-LptA-His6), were introduced into E. coli. Introduction of the mutant alleles led to a markedly higher MIC for thanatin, whereas introduction of the WT sequence (LptA-His6) gave no significant change in MIC (see section S6). In summary, the genetic and photolabeling results reveal a link between the antimicrobial activity of thanatin and LptA and point to LptA as an interaction partner for thanatin in E. coli. On the other hand, no mutations in lptD were detected in the genomes of the three analyzed ThanR mutants.

In vitro binding studies To date, no small molecules (apart from LPS) are known to interact with LptA, so we tested whether thanatin can directly bind to LptA in vitro. A recombinant full-length LptA [with a His6 tag fused to the C terminus (LptA-His6)] was produced in E. coli BL21(DE3) and purified to apparent homogeneity by SDS-PAGE after Ni–nitrilotriacetic acid affinity and anion exchange chromatography (see section S7). The binding of thanatin-BDP-FL (Fig. 1B) to LptA-His6 was then studied by fluorescence polarization (FP) and of thanatin binding to LptA labeled with DyLight650 by thermophoresis. Using both biophysical methods, fitting the binding isotherm to a 1:1 Langmuir binding model (see section S8) gave by FP a dissociation constant (K d ) of 12 ± 3 nM and by thermophoresis a K d of 20 ± 1 nM. In control experiments using the enantiomeric form of thanatin (comprising all d-amino acids), no interaction of d-thanatin with LptA was observed by FP. The binding of thanatin to LptD was also measured in vitro using a recombinant His-tagged LptD/E complex purified from E. coli (see section S7.2). Using FP, thanatin-BDP-FL binds to LptD/E His with a K d of 34 ± 5 nM, whereas thermophoresis binding assays gave a K d of 44 ± 27 nM (see section S9). Thanatin therefore binds in vitro to both LptA and LptD/E in the low nanomolar range.