Functional Characterization of SbmA,a Bacterial Inner Membrane Transporter Required for Importing the Antimicrobial Peptide Bac7(1-35)

SbmA is an inner membrane protein of Gram-negative bacteria that is involved in the internalization of glycopeptides and prokaryotic and eukaryotic antimicrobial peptides, as well as of peptide nucleic acid (PNA) oligomers. The SbmA homolog BacA is required for the development of Sinorhizobium meliloti bacteroids within plant cells and favors chronic infections with Brucella abortus and Mycobacterium tuberculosis in mice. Here, we investigated functional features of SbmA/BacA using the proline-rich antimicrobial peptide Bac7(1-35) as a substrate. Circular dichroism and affinity chromatography studies were used to investigate the ability of SbmA to bind the peptide, and a whole-cell transport assay with fluorescently labeled peptide allowed the determination of transport kinetic parameters with a calculated K_m value of 6.95 ± 0.89 μM peptide and a V_max of 53.91 ± 3.17 nmol/min/mg SbmA. Use of a bacterial two-hybrid system coupled to SEC-MALLS (size exclusion chromatography coupled with multiangle laser light scattering) analyses established that SbmA is a homodimer in the membrane, and treatment of the cells with arsenate or ionophores indicated that the peptide transport mediated by SbmA is driven by the electrochemical gradient. Overall, these results shed light on the SbmA-mediated internalization of peptide substrates and suggest that the transport of an unknown substrate(s) represents the function of this protein.     

Mechanistic and functional studies of the interaction of a proline-rich antimicrobial peptide with mammalian cells

Mammalian antimicrobial peptides provide rapid defense against infection by inactivating pathogens and by influencing the functions of cells involved in defense responses. Although the direct antibacterial properties of these peptides have been widely characterized, their multiple effects on host cells are only beginning to surface. Here we investigated the mechanistic and functional aspects of the interaction of the proline-rich antimicrobial peptide Bac7(1-35) with mammalian cells, as compared with a truncated analog, Bac7(5-35), lacking four critical N-terminal residues (RRIR) of the Bac7(1-35) sequence. By using confocal microscopy and flow cytometry, we showed that although the truncated analog Bac7(5-35) remains on the cell surface, Bac7(1-35) is rapidly taken up into 3T3 and U937 cells through a nontoxic energy- and temperature-dependent process. Cell biology-based assays using selective endocytosis inhibitors and spectroscopic and surface plasmon resonance studies of the interaction of Bac7(1-35) with phosphatidylcholine/cholesterol model membranes collectively suggest the concurrent contribution of macropinocytosis and direct membrane translocation. Structural studies with model membranes indicated that membrane-bound Bac7(5-35) is significantly more aggregated than Bac7(1-35) due to the absence of the N-terminal cationic cluster, thus providing an explanation for hampered cellular internalization of the truncated form. Further investigations aimed to reveal functional implications of intracellular uptake of Bac7(1-35) demonstrated that it correlates with enhanced S phase entry of 3T3 cells, indicating a novel function for this proline-rich peptide.     

Translocating proline-rich peptides from the antimicrobial peptide bactenecin 7

The intracellular delivery of most peptides, proteins, and nucleotides to the cytoplasm and nucleus is impeded by the cell membrane. To allow simplified, noninvasive delivery of attached cargo, cell-permeant peptides that are either highly cationic or hydrophobic have been utilized. Because cell-permeable peptides share half of the structural features of antimicrobial peptides containing clusters of charge and hydrophobic residues, we have explored antimicrobial peptides as templates for designing cell-permeant peptides. We prepared synthetic fragments of Bac 7, an antimicrobial peptide with four 14-residue repeats from the bactenecin family. The dual functions of cell permeability and antimicrobial activity of Bac 7 were colocalized at the N-terminal 24 residues of Bac 7. In general, long fragments of Bac(1-24) containing both regions were bactericidal and cell-permeable, whereas short fragments with only a cationic or hydrophobic region were cell-permeant without the attendant microbicidal activity when measured in a fluorescence quantitation assay and by confocal microscopy. In addition, the highly cationic fragments were capable of traversing the cell membrane and residing within the nucleus. A common characteristic shared by the cell-permeant Bac(1-24) fragments, irrespective of their number of charged cationic amino acids, is their high proline content. A 10-residue proline-rich peptide with two arginine residues was capable of delivering a noncovalently linked protein into cells. Thus, the proline-rich peptides represent a potentially new class of cell-permeant peptides for intracellular delivery of protein cargo. Furthermore, our results suggest that antimicrobial peptides may represent a rich source of templates for designing cell-permeant peptides.     

Dual mode of action of Bac7, a proline-rich antibacterial peptide

Proline-rich peptides are a unique group of antimicrobial peptides that exert their activity selectively against Gram-negative bacteria through an apparently non-membranolytic mode of action that is not yet well understood. We have investigated the mechanism underlying the antibacterial activity of the proline-rich cathelicidin Bac7 against Salmonella enterica and Escherichia coli. The killing and membrane permeabilization kinetics as well as the cellular localization were assessed for the fully active N-terminal fragment Bac7(1-35), its all-D enantiomer and for differentially active shortened fragments. At sub-micromolar concentrations, Bac7(1-35) rapidly killed bacteria by a non-lytic, energy-dependent mechanism, whereas its D-enantiomer was inactive. Furthermore, while the L-enantiomer was rapidly internalized into bacterial cells, the D-enantiomer was virtually excluded. At higher concentrations (>or=64 microM), both L- and D-Bac7(1-35) were instead able to kill bacteria also via a lytic mechanism. Overall, these results suggest that Bac7 may inactivate bacteria via two different modes of action depending on its concentration: (i) at near-MIC concentrations via a mechanism based on a stereospecificity-dependent uptake that is likely followed by its binding to an intracellular target, and (ii) at concentrations several times the MIC value, via a non-stereoselective, membranolytic mechanism.     

Role of Cathelicidin Peptides in Bovine Host Defense and Healing

The in vitro antimicrobial activities and biological effects on host cells were compared for the bovine cathelicidins BMAP-28, an alpha-helical AMP, and Bac5 and Bac7, proline-rich AMPs. Our results confirm that the broad-spectrum activity of BMAP-28 correlates with a high capacity to interact with and permeabilize bacterial membranes, whereas the proline-rich AMPs selectively internalize into the cytoplasm of susceptible Gram-negative bacteria with a non-lytic mechanism. All peptides efficiently translocated into mammalian fibroblastic cells, but while Bac5 and Bac7(1–35) localized to nuclear structures and induced cellular proliferation, BMAP-28 associated with mitochondria and did not induce proliferation. Moreover, BMAP-28 was considerably more cytotoxic than the proline-rich peptides due to cytolytic and pro-apoptotic effects. Our results highlight important functional differences among the bovine cathelicidins and suggest that they contribute to an integrated response of the host to infection, with distinct but complementary activities.     

The Proline-rich Antibacterial Peptide Bac7 Binds to and Inhibits in vitro the Molecular Chaperone DnaK

Bac7, a cathelicidin peptide of the proline-rich group, inactivates bacteria in a stereospecific manner by entering target cells without any apparent membrane damage and by binding to as yet unknown intracellular targets. The present study was aimed at detecting these putative intracellular interactors, which might mediate the antibacterial action of this peptide. By using affinity resins functionalized with the N-terminal 1-35 fragment of Bac7, a single protein was specifically retained with high affinity from Escherichia coli cytoplasmic protein lysates. This ligand was identified as the heat shock protein DnaK, the Hsp70 homolog in E. coli. The interaction between the peptide and the chaperone is stereospecific, given that a resin prepared with the all- d enantiomer failed to retain the protein. In vitro, Bac7(1-35) formed a complex with DnaK with an affinity comparable to that of other known high-affinity peptide ligands. In addition, at 10–100 μM concentration, the peptide inhibited the protein refolding activity of the complete DnaK/DnaJ/GrpE/ATP molecular chaperone system in a dose-dependent manner. Despite these results, the in vitro sensitivity to the peptide, under growth permitting conditions, of DnaK-deficient E. coli strains was not significantly affected compared to the wild-type strain. This suggests that, apart from DnaK, other vital targets for the proline-rich AMPs are present in susceptible bacteria. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Marco Scocchi and Christine Lüthy contributed equally to this work.     

Antimicrobial activity of Bac7 fragments against drug-resistant clinical isolates

Ten peptides from 13 to 35 residues in length and covering the whole sequence of the Pro-rich peptide Bac7 were synthesized to identify the domain responsible for its antimicrobial activity. At least 16 residues of the highly cationic N-terminal sequence were required to maintain the activity against Gram-negative bacteria. The fragments Bac7(1–35) and, to a lesser extent, Bac7(1–16) proved active against a panel of antibiotic-resistant clinical isolates of Gram-negative bacteria, with the notable exception of Burkholderia cepacia. In addition, when tested against fungi, the longer fragment was also active against collection strains and clinical isolates of Cryptococcus neoformans, but not towards clinical isolates of Candida albicans.     

Functional and Structural Study of the Dimeric Inner Membrane Protein SbmA

SbmA protein has been proposed as a dimeric secondary transporter. The protein is involved in the transport of microcins B17 and J25, bleomycin, proline-rich antimicrobial peptides, antisense peptide phosphorodiamidate morpholino oligomers, and peptide nucleic acids into the Escherichia coli cytoplasm. The sbmA homologue is found in a variety of bacteria, though the physiological role of the protein is hitherto unknown. In this work, we carried out a functional and structural analysis to determine which amino acids are critical for the transport properties of SbmA. We created a set of 15 site-directed sbmA mutants in which single conserved amino acids were replaced by glycine residues. Our work demonstrated that strains carrying the site-directed mutants V102G, F219G, and E276G had a null phenotype for SbmA transport functions. In contrast, strains carrying the single point mutants W19G, W53G, F60G, S69G, N155G, R190, L233G, A344G, T255G, N308G, and R385G showed transport capacities indistinguishable from those of strains harboring a wild-type sbmA. The strain carrying the Y116G mutant exhibited mixed phenotypic characteristics. We also demonstrated that those sbmA mutants with severely impaired transport capacity showed a dominant negative phenotype. Electron microscopy data and in silico three-dimensional (3D) homology modeling support the idea that SbmA forms a homodimeric complex, closely resembling the membrane-spanning region of the ATP-binding cassette transporter family. Direct mapping of the sbmA single point mutants on the protein surface allowed us to explain the observed phenotypic differences in transport ability.     

Structures of proline-rich peptides bound to the ribosome reveal a common mechanism of protein synthesis inhibition

With bacterial resistance becoming a serious threat to global public health, antimicrobial peptides (AMPs) have become a promising area of focus in antibiotic research. AMPs are derived from a diverse range of species, from prokaryotes to humans, with a mechanism of action that often involves disruption of the bacterial cell membrane. Proline-rich antimicrobial peptides (PrAMPs) are instead actively transported inside the bacterial cell where they bind and inactivate specific targets. Recently, it was reported that some PrAMPs, such as Bac7_1–35, oncocins and apidaecins, bind and inactivate the bacterial ribosome. Here we report the crystal structures of Bac7_1–35, Pyrrhocoricin, Metalnikowin and two oncocin derivatives, bound to the Thermus thermophilus 70S ribosome. Each of the PrAMPs blocks the peptide exit tunnel of the ribosome by simultaneously occupying three well characterized antibiotic-binding sites and interferes with the initiation step of translation, thereby revealing a common mechanism of action used by these PrAMPs to inactivate protein synthesis. Our study expands the repertoire of PrAMPs and provides a framework for designing new-generation therapeutics.     

牛抗菌肽Bac7-Bac5融合基因在大肠杆菌中的过表达，纯化及抑菌活性

牛抗菌肽Bac7和Bac5是一种线性阳离子小分子多肽,在机体天然免疫和获得性免疫中都发挥着重要作用。本研究根据Gen bank中公布的牛抗菌肽bac7和bac5成熟肽基因序列,人工合成了融合基因Bac7-Bac5片段,克隆于原核表达载体pET32(a+)中构建了重组表达载体（pET-B7-B5）,将其转化于E coli BL21(DE3) 中实现了重组蛋白B7-B5（rB7-B5）的过表达,表达的rB7-B5以包涵体形式存在,rB7-B5表达量约占细菌总蛋白的36.6％,分子量大小为33kD,与预测大小相符。以经Ni亲和层析柱纯化和多步透析法复性的rB7-B5,对猪胸膜肺炎放线杆菌和耐药性大肠杆菌具有很好的抑菌活性,本研究为新型抗菌制剂的研制和开发奠定了基础。     

Enteric YaiW Is a Surface-Exposed Outer Membrane Lipoprotein That Affects Sensitivity to an Antimicrobial Peptide

yaiW is a previously uncharacterized gene found in enteric bacteria that is of particular interest because it is located adjacent to the sbmA gene, whose bacA ortholog is required for Sinorhizobium meliloti symbiosis and Brucella abortus pathogenesis. We show that yaiW is cotranscribed with sbmA in Escherichia coli and Salmonella enterica serovar Typhi and Typhimurium strains. We present evidence that the YaiW is a palmitate-modified surface exposed outer membrane lipoprotein. Since BacA function affects the very-long-chain fatty acid (VLCFA) modification of S. meliloti and B. abortus lipid A, we tested whether SbmA function might affect either the fatty acid modification of the YaiW lipoprotein or the fatty acid modification of enteric lipid A but found that it did not. Interestingly, we did observe that E. coli SbmA suppresses deficiencies in the VLCFA modification of the lipopolysaccharide of an S. meliloti bacA mutant despite the absence of VLCFA in E. coli. Finally, we found that both YaiW and SbmA positively affect the uptake of proline-rich Bac7 peptides, suggesting a possible connection between their cellular functions.     

The proline-rich peptide Bac7(1-35) reduces mortality from <Emphasis Type="Italic">Salmonella typhimurium</Emphasis> in a mouse model of infection

Background  

Bac7 is a proline-rich peptide with a potent in vitro antimicrobial activity against Gram-negative bacteria. Here we investigated its activity in biological fluids and in vivo using a mouse model of S. typhimurium infection.     

Effects of antimicrobial peptides of neutrophils on tumor and normal cells in culture

We performed a comparative study of effects of two structurally different cationic antimicrobial peptides of cathelicidin family, porcine protegrin 1 (PG1) and caprine bactenecin 5 (Bac5) on selected tumor and normal mammalian cells in vitro. Protegrins are amphiphilic beta-hairpin molecules having broad-spectrum antimicrobial activity due to their marked membranolytic properties. Bac5 belongs to the group of proline-rich peptides, which adopt a polyproline type II extended helix and kill microorganisms rather by a non-lytic mechanism. We have shown that while PG1 exerts distinct and fast cytotoxic effects on most of used tumor cells being slightly less toxic for nontransformed host cell, the proline-rich Bac5 is much less cytotoxic for all the cells tested. The toxic effects of PG1 were partially declined in the presence of 10% fetal calf serum. It was revealed that PG1 was able to interact with proteins of serpin family (as had been previously established for human defensins by Panyutich et al., 1995). Pre-incubation of PG1 with alpha1-antitrypsin caused the decrease of the cytotoxic activity of the peptide and, on the other hand, the antiprotease activity of alpha1-antitrypsin was reduced after interaction of the serpin with PG1 (not with Bac5). Confocal microscopy experiments allowed to monitor the internalization of fluorescent labeled (by BODIPY FL) peptides into target cells and their intracellular distribution. Bac5-BODIPY (at 5 microM) was rapidly taken into the cells. PG1-BODIPY at non-toxic concentrations was also able to enter the cells without significant damage to them. The comparative study of the kinetics of the peptides uptake into the target cells and the influence of low temperature, energy-depletion and endocytosis inhibitors on the process of the internalization of the peptides into the cells was carried out using flow cytometry.     

Effects of antimicrobial peptides of neutrophils on tumor and normal host cells in culture

We carried out a study of the effects of two structurally different cationic antimicrobial peptides of cathelicidin family, porcine protegrin 1 (PG1) and caprine Bac5 on selected tumor and normal mammalian cells in vitro. Protegrins are amphiphilic β-hairpin molecules having broad-spectrum antimicrobial activity due to their marked membranolytic effects. Bac5 belongs to a group of proline-rich peptides, which adopt a polyproline type II extended helix and kill microorganisms rather by a nonlytic mechanism. We have shown that while PG1 exerted distinct and fast cytotoxic effects towards most of used tumor cells being in a lesser degree toxic for nontransformed host cells; the proline-rich peptide Bac5 possessed modest cytotoxic activity for all tested cells. The toxic effects of PG1 were partially declined in the presence of 10% fetal calf serum. It was revealed that PG1 was able to interact with proteins of serpin family (as was previously established for human defensins by Panyutich at al., 1995). Pre-incubation of PG1 with α1-antitrypsin caused the decrease of the cytotoxic activity of the peptide and, on the other hand, the antiprotease activity of α1-antitrypsin was reduced after the interaction of the serpin with PG1 (while Bac5 did not affect the antiprotease activity of α1-antitrypsin). We used BODIPY FL-tagged PG1 and Bac5 to study the internalization of the labeled peptides into target cells and their intracellular distribution by confocal microscopy. Bac5-BODIPY (at 5 μ M) was rapidly taken into the cells. PG1-BODIPY at non-toxic concentrations (1—3 μM) was also able to enter the cells without their damaging. By using flow cytometry we showed that lowering a temperature to 4°C caused a significant decrease in the uptake into K562 and U937 cells for both Bac5-BODIPY and PG1-BODIPY. A decline of target cells metabolism also diminished the process of both peptides internalization but for a lesser degree. In the presence of endocytosis inhibitors the penetration of Bac5-BODIPY and PG1-BODIPY into K562 cells was also reduced, but not completely abolished, suggesting that along with endocytosis process some direct penetration of the peptides across cell membranes takes place. The ability of the peptides to internalize into eukaryotic cells may contribute to the idea of participation of AMPs in varied intracellular events, occurring in normal or malignant host cells, for instance, in the modulation of intracellular serpins activity.     

A peptidomics study reveals the impressive antimicrobial peptide arsenal of the wax moth Galleria mellonella

The complete antimicrobial peptide repertoire of Galleria mellonella was investigated for the first time by LC/MS. Combining data from separate trypsin, Glu-C and Asp-N digests of immune hemolymph allowed detection of 18 known or putative G. mellonella antimicrobial peptides or proteins, namely lysozyme, moricin-like peptides (5), cecropins (2), gloverin, Gm proline-rich peptide 1, Gm proline-rich peptide 2, Gm anionic peptide 1 (P1-like), Gm anionic peptide 2, galiomicin, gallerimycin, inducible serine protease inhibitor 2, 6tox and heliocin-like peptide. Six of these were previously known only as nucleotide sequences, so this study provides the first evidence for expression of these genes. LC/MS data also provided insight into the expression and processing of the antimicrobial Gm proline-rich peptide 1. The gene for this peptide was isolated and shown to be unique to moths and to have an unusually long precursor region (495 bp). The precursor region contained other proline-rich peptides and LC/MS data suggested that these were being specifically processed and were present in hemolymph at very high levels. This study shows that G. mellonella can concurrently release an impressive array of at least 18 known or putative antimicrobial peptides from 10 families to defend itself against invading microbes.     

抗菌肽Bactenecin7重组质粒构建及其在乳酸菌的分泌表达和活性鉴定

构建抗菌肽Bactenecin7分泌表达载体,鉴定表达产物及检测其生物活性。采用重叠延伸PCR方法拼接合成Bactenecin7基因及其相关调控元件,将目的基因克隆到穿梭载体 pMG36e中,经鉴定后电击转化乳酸菌。通过RT PCR,Western blot检测目的基因表达情况,采用琼脂扩散法对表达产物的生物活性进行初步检测。结果显示成功构建了携Bactenecin7基因的重组质粒,将重组质粒转化至乳酸菌后,该乳酸菌能有效分泌表达Bactenecin7,经体外抑菌实验证实表达产物Bactenecin7具有抑菌活性。实验表明携Bactenecin7基因的乳酸菌能有效分泌表达具有生物活性的抗菌肽Bactenecin7,为进一步研究口服重组乳酸菌进行肠道细菌感染的治疗奠定了基础。     

Enhancement of the antimicrobial activity and selectivity of GNU7 against Gram-negative bacteria by fusion with LPS-targeting peptide

Antimicrobial peptides (AMPs) provide a potential source of new antimicrobial therapeutics for the treatment of multidrug-resistant pathogens. To develop Gram-negative selective AMPs that can inhibit the effects of lipopolysaccharide (LPS)-induced sepsis, we added various rationally designed LPS-targeting peptides [amino acids 28–34 of lactoferrin (Lf28–34), amino acids 84–99 of bactericidal/permeability increasing protein (BPI84–99), and de novo peptide (Syn)] to the potent AMP, GNU7 (RLLRPLLQLLKQKLR). Compared to our original starting peptide GNU7, hybrid peptides had an 8- to 32-fold improvement in antimicrobial activity against Gram-negative bacteria, such as Escherichia coli and Salmonella typhimurium. Among them, Syn-GNU7 showed the strongest LPS-binding and -neutralizing activities, thus allowing it to selectively eliminate Gram-negative bacteria from within mixed cultures. Our results suggest that LPS-targeting peptides would be useful to increase the antimicrobial activity and selectivity of other AMPs against Gram-negative bacteria.     

Design, synthesis and antibacterial activity studies of model peptidess of proline/arginine-rich region in bactenecin7

Bactenecin 7 (Bac7), a cationic antibacterial peptide, contains a repeating region of Xaa-Pro-Arg-Pro (Xaa = hydrophobic residue). To investigate the structure and property of a Pro/Arg-rich region, e synthesized a series of peptides, Xaa-Pro-Arg-Pro (Xaa = Gly, Arg, Leu, Ile, and Phe) as models and characterized . The conformational preferences of these peptides in water and trifluoroethanol were examined by circular dichroism. The results suggest the presence of largely poly(Pro)-II helical conformation in aqueous and trifluoroethanol solutions. Their antibacterial activity against gram-negative bacteria such as Escherichia coli, Klebsiella Pneumoniae, Pseudomonas aeruginosa, and Escherichia coliHB101, and gram-positive bacteria such as Staphylococcus aureus were measured at various peptide concentrations. Two of our synthetic tetrapeptide fragments containing Gly and Arg were efficiently killed with gram-positive bacteria, Staphylococcus aureus, at the concentration level of 200 microg/mL.     

Identification of crucial residues for the antibacterial activity of the proline-rich peptide, pyrrhocoricin.

Members of the proline-rich antibacterial peptide family, pyrrhocoricin, apidaecin and drosocin appear to kill responsive bacterial species by binding to the multihelical lid region of the bacterial DnaK protein. Pyrrhocoricin, the most potent among these peptides, is nontoxic to healthy mice, and can protect these animals from bacterial challenge. A structure-antibacterial activity study of pyrrhocoricin against Escherichia coli and Agrobacterium tumefaciens identified the N-terminal half, residues 2-10, the region responsible for inhibition of the ATPase activity, as the fragment that contains the active segment. While fluorescein-labeled versions of the native peptides entered E. coli cells, deletion of the C-terminal half of pyrrhocoricin significantly reduced the peptide's ability to enter bacterial or mammalian cells. These findings highlighted pyrrhocoricin's suitability for combating intracellular pathogens and raised the possibility that the proline-rich antibacterial peptides can deliver drug leads into mammalian cells. By observing strong relationships between the binding to a synthetic fragment of the target protein and antibacterial activities of pyrrhocoricin analogs modified at strategic positions, we further verified that DnaK was the bacterial target macromolecule. Inaddition, the antimicrobial activity spectrum of native pyrrhocoricin against 11 bacterial and fungal strains and the binding of labeled pyrrhocoricin to synthetic DnaK D-E helix fragments of the appropriate species could be correlated. Mutational analysis on a synthetic E. coli DnaK fragment identified a possible binding surface for pyrrhocoricin.     

ChBac3.4: A Novel Proline-Rich Antimicrobial Peptide from Goat Leukocytes

We isolated a new proline-rich peptide, ChBac3.4, from leukocytes of the goat (Capra hirca) and determined its amino acid sequence by Edman degradation and mass spectrometry. ChBac3.4 (RFRLPFRRPPIRIHPPPFYPPFRRFL–NH2) had over 50% sequence identity to the Bac5 peptides found in the leukocytes of goats, sheep and cattle. ChBac3.4 exhibited broadspectrum antimicrobial activity, especially under low salt conditions. Since E. coli ML35p treated with ChBac3.4 manifested increased outer and inner membrane permeability and a rapid and extensive loss of cytoplasmic potassium, the antimicrobial properties of this peptide may depend, in part, on its ability to damage microbial membranes. Nevertheless, even high concentrations of ChBac3.4 were not significantly hemolytic for human erythrocytes. In vitro, ChBac3.4 was selectively cytotoxic, damaging human K562 erythroleukemia cells and human U937 hystiocytic lymphoma cells, but not other human target cells. ChBac3.4 appears to differ from other proline-rich cathelicidins in virtue of its increased ability to damage microbial membranes. This novel antimicrobial peptide warrants further study, especially with respect to its various effects on microbial and mammalian cells. An erratum to this article can be found at