The lipid components of high-density lipoproteins (HDL) are essential for the binding and transportation of antimicrobial peptides in human serum.

Antimicrobial peptides (AMPs) have been developed for the treatment of bacterial infections, but their applications are limited to topical infections since they are sequestered and inhibited in serum. Here we have discovered that the inhibition of AMPs by human serum was mediated through high-density lipoproteins (HDL) which are known to remove cholesterol from peripheral tissues. The susceptibility of AMPs to HDL varied depending on the degree of hydrophobicity of AMPs and their binding affinities to HDL. The phospholipids, such as phosphatidylcholine, of HDL were essential for AMP-binding. The dynamic binding interactions between AMPs and HDL were mediated through the hydrophobic interactions rather than by ionic strength. Interestingly, some AMPs, such as SMAP29, dissociated from the AMP-HDL complex and translocated to bacteria upon contact, while some AMPs, such as LL37, remained in complex with HDL. These results suggest that HDL binds AMPs and facilitates the translocation of them to the bacteria.

Identification of potential therapeutic antimicrobial peptides against Acinetobacter baumannii in a mouse model of pneumonia.

Acinetobacter baumannii-induced nosocomial pneumonia has become a serious clinical problem because of high antibiotic resistance rates. Antimicrobial peptides (AMP) are an ideal alternative strategy due to their broad-spectrum of antimicrobial activity and low incidence of bacterial resistance. However, their application is limited by toxicity and stability in vivo. The present study used a mouse model to directly identify potential AMPs effective for treatment of A. baumannii-induced pneumonia. Fifty-eight AMPs were screened and two identified (SMAP-29 and TP4) to have prophylactic effects which prevented the death of mice with pneumonia. Furthermore, two TP4 derivatives (dN4 and dC4) were found to have therapeutic activity in pneumonia mouse models by peritoneal or intravenous administration. Both dN4 and dC4 also inhibited and/or eliminated A. baumannii biofilms at higher doses. Taken together, these data suggest the AMP derivatives dN4 and dC4 represent a potential treatment strategy for A. baumannii-induced pneumonia.

Fetal bovine serum albumin inhibits antimicrobial peptide activity and binds drug only in complex with α1-antitrypsin.

Several antimicrobial peptides (AMPs) have been developed for the treatment of infections caused by antibiotic-resistant microbes, but their applications are primarily limited to topical infections because in circulation they are bound and inhibited by serum proteins. Here we have found that some AMPs, such as TP4 from fish tilapia, and drugs, such as antipyretic ibuprofen, were bound by bovine serum albumin only in complex with α1-antitrypsin which is linked by disulfide bond. They existed in dimeric complex (2 albumin -2 α1-antitrypsin) in the bovine serum only at fetal stage, but not after birth. The hydrophobic residues of TP4 were responsible for its binding to the complex. Since bovine serum is a major supplement in most cell culture media, therefore the existence and depletion of active albumin/α1-antitrypsin complex are very important for the assay and production of biomolecules.

Inhibition of bacterial adherence to biomaterials by coating antimicrobial peptides with anionic surfactant.

Medical devices are widely used in modern medicine, but their utilities are often limited by the biofilm formation of bacteria that are tolerant to most antibiotics. In this report, antimicrobial peptides (AMPs) were coated onto biomaterials by the aid of surfactant through hydrophobic interactions. To increase the coating efficiency, stability of AMPs in body fluids and spectrum of antimicrobial activity, pairs of AMPs were coated simultaneously onto various substrates, such as silicone, polyurethane and titanium, which are commonly used components of biomedical devices. These coated AMPs exhibited very low cytotoxicity and hemolytic activities because they were gradually released into urine or serum. The AMP pairs, such as T9W + SAAP159 and T9W + RRIKA, coated onto the silicone discs were able to inhibit in vitro bacterial adherence in urine. Most importantly, AMP pairs coated onto the silicone tubing by surfactant SDBS could prevent bacterial adherence to mouse bladder and the silicone tubing implanted within it. These results provide a promising approach towards circumventing urinary catheter-associated infections caused by bacterial adherence.

Anionic Surfactant-Facilitated Coating of Antimicrobial Peptide and Antibiotic Reduces Biomaterial-Associated Infection.

Medical devices are widely used in modern medicine, but the high prevalence of biomaterial-associated infections still presents a major problem. Especially problematic is the formation of biofilms that are tolerant to most antibiotics. In this report, antimicrobial peptides (AMPs) were driven into an amphipathic structure by anionic surfactant. To increase the coating efficacy and spectrum of antimicrobial activity, the AMPs were coated simultaneously with antibiotic, Polymyxin B, by surfactant onto polystyrene, silicone, polyurethane, and titanium which are commonly used with biomedical devices. These coated antimicrobials stably adhered to the substrate and were gradually released into urine and serum. They exhibited high bactericidal activity, but low cytotoxicity and hemolytic activity. Most importantly, the antimicrobials coated onto silicone tubing inhibited the planktonic growth of E. coli in mouse urine and also markedly prevented bacterial adherence to the bladder and the silicone tubing implanted in the bladder. These results provide a promising approach to circumvent catheter-associated infections due to bacterial adherence.

Oligomerization and insertion of antimicrobial peptide TP4 on bacterial membrane and membrane-mimicking surfactant sarkosyl.

Antimicrobial peptides (AMPs) are important components of the host innate defense mechanism against invading microorganisms. Although AMPs are known to act on bacterial membranes and increase membrane permeability, the action mechanism of most AMPs still remains unclear. In this report, we found that the TP4 peptides from Nile tilapia anchored on E. coli cells and enabled them permeable to SYTOX Green in few minutes after TP4 addition. TP4 peptides existed in small dots either on live or glutaraldehyde-fixed cells. TP4 peptides were driven into oligomers either in soluble or insoluble form by a membrane-mimicking anionic surfactant, sarkosyl, depending on the concentrations employed. The binding forces among TP4 components were mediated through hydrophobic interaction. The soluble oligomers were negatively charged on surface, while the insoluble oligomers could be fused with each other or piled on existing particles to form larger particles with diameters 0.1 to 20 µm by hydrophobic interactions. Interestingly, the morphology and solubility of TP4 particles changed with the concentration of exogenous sarkosyl or trifluoroethanol. The TP4 peptides were assembled into oligomers on or in bacterial membrane. This study provides direct evidence and a model for the oligomerization and insertion of AMPs into bacterial membrane before entering into cytosol.

Hydrophobic residues are critical for the helix-forming, hemolytic and bactericidal activities of amphipathic antimicrobial peptide TP4.

Antimicrobial peptides are important components of the host innate defense mechanism against invading pathogens, especially for drug-resistant bacteria. In addition to bactericidal activity, the 25 residue peptide TP4 isolated from Nile tilapia also stimulates cell proliferation and regulates the innate immune system in mice. In this report, TP4 hyperpolarized and depolarized the membrane potential of Pseudomonas aeruginosa at sub-lethal and lethal concentrations. It also inhibited and eradicated biofilm formation. The in vitro binding of TP4 to bacterial outer membrane target protein, OprI, was markedly enhanced by a membrane-like surfactant sarkosyl and lipopolysaccharide, which converted TP4 into an α-helix. The solution structure of TP4 in dodecylphosphocholine was solved by NMR analyses. It contained a typical α-helix at residues Phe10-Arg22 and a distorted helical segment at Ile6-Phe10, as well as a hydrophobic core at the N-terminus and a cationic patch at the C-terminus. Residues Ile16, Leu19 and Ile20 in the hydrophobic face of the main helix were critical for the integrity of amphipathic structure, other hydrophobic residues played important roles in hemolytic and bactericidal activities. A model for the assembly of helical TP4 embedded in sarkosyl vesicle is proposed. This study may provide valuable insight for engineering AMPs to have potent bactericidal activity but low hemolytic activity.

Sarkosyl-Induced Helical Structure of an Antimicrobial Peptide GW-Q6 Plays an Essential Role in the Binding of Surface Receptor OprI in Pseudomonas aeruginosa.

The emergence of antibiotic-resistant microbial strains has become a public health issue and there is an urgent need to develop new anti-infective molecules. Although natural antimicrobial peptides (AMPs) can exert bactericidal activities, they have not shown clinical efficacy. The limitations of native peptides may be overcome with rational design and synthesis. Here, we provide evidence that the bactericidal activity of a synthetic peptide, GW-Q6, against Pseudomonas aeruginosa is mediated through outer membrane protein OprI. Hyperpolarization/depolarization of membrane potential and increase of membrane permeability were observed after GW-Q6 treatment. Helical structure as well as hydrophobicity was induced by an amphipathic surfactant, sarkosyl, for binding to OprI and possible to membrane. NMR studies demonstrated GW-Q6 is an amphipathic α-helical structure in DPC micelles. The paramagnetic relaxation enhancement (PRE) approach revealed that GW-Q6 orients its α-helix segment (K7-K17) into DPC micelles. Additionally, this α-helix segment is critical for membrane permeabilization and antimicrobial activity. Moreover, residues K3, K7, and K14 could be critical for helical formation and membrane binding while residues Y19 and W20 for directing the C-terminus of the peptide to the surface of micelle. Taken together, our study provides mechanistic insights into the mode of action of the GW-Q6 peptide and suggests its applicability in modifying and developing potent AMPs as therapeutic agents.

Antimicrobial Properties of an Immunomodulator - 15 kDa Human Granulysin.

Granulysin, a cationic protein expressed by human natural killer cells and cytotoxic T lymphocytes, is a mediator for drug-induced Stevens-Johnson syndrome and graft-versus-host disease. Some 15 kDa granulysin are processed into 9 kDa forms and sequestered in cytolytic granules, while others are constitutively secreted into body fluids. Both 9 and 15 kDa granulysin have been shown to be a serum marker for cell-mediated immunity. Furthermore, 15 kDa is able to activate monocyte differentiation. However, its antimicrobial properties have not been clearly addressed. Here, we report a novel method to prepare both the soluble 9 and 15 kDa granulysin and show that the 15 kDa form is more effective than the 9 kDa form in exerting specific antimicrobial activity against Pseudomonas aeruginosa within a range of few micromolars. We also show that the 15 kDa granulysin is able to hyperpolarize the membrane potential and increase membrane permeability of treated bacteria. Interestingly, the bactericidal activity and membrane permeability of the granulysins were markedly reduced at lower pH (pH 5.4) as a result of probable increase in hydrophobicity of the granulysins. Additionally, we've also shown the granulysin to inhibit biofilm formation by P. aeruginosa. These results suggest that the 15 kDa granulysin exhibits a novel mechanism in bacteria killing in a way that's different from most antimicrobial peptides. Our novel granulysin preparation methodology will be useful for further study of action mechanisms of other antimicrobial, cytotoxic and immunomodulating properties in granulysin-mediated diseases.

Solution structure and base specificity of cytotoxic RC-RNase 2 from Rana catesbeiana.

Cytotoxic ribonucleases found in the oocytes and early embryos of frogs with antitumor activity are well-documented. RC-RNase 2, a cytotoxic ribonuclease isolated from oocytes of bullfrog Rana catesbeiana, consists of 105 residues linked with 4 disulfide bridges and belongs to the bovine pancreatic ribonuclease (RNase A) superfamily. Among the RC-RNases, the base preference for RNase 2 is UpG but CpG for RC-RNase 4; while RC-RNase possesses the base specificity of both UpG and CpG. Interestingly, RC-RNase 2 or 4 has much lower catalytic activity but only three-fold less cytotoxicity than RC-RNase. Here, we report the NMR solution structure of rRC-RNase 2, comprising three alpha-helices and two sets of antiparallel beta-sheets. The differences of side-chain conformations of subsite residues among RNase A, RC-RNase, RC-RNase 4 and rRNase 2 are related to their distinct catalytic activities and base preferences. Furthermore, the substrate-related residues in the base specificity among native RC-RNases are derived using the chemical shift perturbation on ligand binding.

Key Residues of Outer Membrane Protein OprI Involved in Hexamer Formation and Bacterial Susceptibility to Cationic Antimicrobial Peptides.

Antimicrobial peptides (AMPs) are important components of the host innate defense mechanism against invading pathogens. Our previous studies have shown that the outer membrane protein, OprI from Pseudomonas aeruginosa or its homologue, plays a vital role in the susceptibility of Gram-negative bacteria to cationic α-helical AMPs (Y. M. Lin, S. J. Wu, T. W. Chang, C. F. Wang, C. S. Suen, M. J. Hwang, M. D. Chang, Y. T. Chen, Y. D. Liao, J Biol Chem 285:8985-8994, 2010, http://dx.doi.org/10.1074/jbc.M109.078725; T. W. Chang, Y. M. Lin, C. F. Wang, Y. D. Liao, J Biol Chem 287:418-428, 2012, http://dx.doi.org/10.1074/jbc.M111.290361). Here, we obtained two forms of recombinant OprI: rOprI-F, a hexamer composed of three disulfide-bridged dimers, was active in AMP binding, while rOprI-R, a trimer, was not. All the subunits predominantly consisted of α-helices and exhibited rigid structures with a melting point centered around 76°C. Interestingly, OprI tagged with Escherichia coli signal peptide was expressed in a hexamer, which was anchored on the surface of E. coli, possibly through lipid acids added at the N terminus of OprI and involved in the binding and susceptibility to AMP as native P. aeruginosa OprI. Deletion and mutation studies showed that Cys1 and Asp27 played a key role in hexamer formation and AMP binding, respectively. The increase of OprI hydrophobicity upon AMP binding revealed that it undergoes conformational changes for membrane fusion. Our results showed that OprI on bacterial surfaces is responsible for the recruitment and susceptibility to amphipathic α-helical AMPs and may be used to screen antimicrobials.

Identification of a novel antimicrobial peptide from human hepatitis B virus core protein arginine-rich domain (ARD).

The rise of multidrug-resistant (MDR) pathogens causes an increasing challenge to public health. Antimicrobial peptides are considered a possible solution to this problem. HBV core protein (HBc) contains an arginine-rich domain (ARD) at its C-terminus, which consists of 16 arginine residues separated into four clusters (ARD I to IV). In this study, we demonstrated that the peptide containing the full-length ARD I-IV (HBc147-183) has a broad-spectrum antimicrobial activity at micro-molar concentrations, including some MDR and colistin (polymyxin E)-resistant Acinetobacter baumannii. Furthermore, confocal fluorescence microscopy and SYTOX Green uptake assay indicated that this peptide killed Gram-negative and Gram-positive bacteria by membrane permeabilization or DNA binding. In addition, peptide ARD II-IV (HBc153-176) and ARD I-III (HBc147-167) were found to be necessary and sufficient for the activity against P. aeruginosa and K. peumoniae. The antimicrobial activity of HBc ARD peptides can be attenuated by the addition of LPS. HBc ARD peptide was shown to be capable of direct binding to the Lipid A of lipopolysaccharide (LPS) in several in vitro binding assays. Peptide ARD I-IV (HBc147-183) had no detectable cytotoxicity in various tissue culture systems and a mouse animal model. In the mouse model by intraperitoneal (i.p.) inoculation with Staphylococcus aureus, timely treatment by i.p. injection with ARD peptide resulted in 100-fold reduction of bacteria load in blood, liver and spleen, as well as 100% protection of inoculated animals from death. If peptide was injected when bacterial load in the blood reached its peak, the protection rate dropped to 40%. Similar results were observed in K. peumoniae using an IVIS imaging system. The finding of anti-microbial HBc ARD is discussed in the context of commensal gut microbiota, development of intrahepatic anti-viral immunity and establishment of chronic infection with HBV. Our current results suggested that HBc ARD could be a new promising antimicrobial peptide.

Differential influences of gastric bypass and sleeve gastrectomy on plasma nesfatin-1 and obestatin levels in patients with type 2 diabetes mellitus.

OBJECTIVE: The mechanisms by which bariatric surgeries, including gastric bypass (GB) and sleeve gastrectomy (SG), achieve remission of type 2 diabetes mellitus (T2DM) and sustained weight reduction are unknown. We hypothesized that the novel anorexic hormone nesfatin-1 and another new hormone obestatin might contribute to the marked improvement in glycemic homeostasis and weight loss in diabetics after GB and SG. METHODS: A hospital-based, prospective study was conducted. Overnight fasting plasma concentrations of nesfatin-1 and obestatin were analyzed in T2DM patients before surgery, and at 3 and 12 months after laparoscopic GB (n =12) and SG (n = 6). RESULTS: At 12 months, reductions of body mass index (BMI), fasting blood glucose, and glycated hemoglobin were similar between GB and SG groups (P all > 0.05). Plasma nesfatin-1 levels in patients undergoing GB or SG significantly decreased after surgeries (P both < 0.05). In contrast, plasma obestatin concentrations significantly increased in patients after SG (P < 0.05) but without any alteration after GB. The alterations of plasma nesfatin-1 were significantly and negatively associated with the reduction of fasting blood glucose (P <0.05) at 12 months after GB and SG. In the SG group, the reduction of nesfatin-1 significantly and positively correlated with the decrease of BMI (P < 0.05). CONCLUSIONS: GB and SG produce differential influences with regards to circulating nesfatin-1 and obestatin levels in non-morbidly obese, T2DM patients. Circulating nesfatin-1 may modulate glucose homeostasis in two surgical procedures, and participate in regulating body weight in SG.

Impact of intracerebroventricular obestatin on plasma acyl ghrelin, des-acyl ghrelin and nesfatin-1 levels, and on gastric emptying in rats.

Obestatin, which is a putative 23-amino-acid peptide, is derived from the C-terminal part of the mammalian preproghrelin gene. Nesfatin-1 mRNA is co-expressed with ghrelin in gastric endocrine X/A-like cells; therefore, nesfatin-1 may also interact with preproghrelin gene products in the stomach. In this study, we investigated the impact of obestatin on the plasma levels of acyl ghrelin, des-acyl ghrelin and nesfatin-1, and on the gastric emptying of a solid nutrient meal 2 h after an intracerebroventricular (ICV) injection in conscious, fasted rats. The rats were implanted with ICV catheters. Plasma levels of acyl ghrelin, des-acyl ghrelin and nesfatin-1, expected to be co-expressed with obestatin, were measured, whereas the human/rat corticotropin-releasing factor (h/rCRF) was applied as an inhibitor of gastric emptying. The ICV administration of obestatin (0.1, 0.3 and 1.0 nmol/rat) did not modify the plasma acyl ghrelin and des-acyl ghrelin levels, the acyl ghrelin/des-acyl ghrelin ratio and nesfatin-1 concentrations. The ICV acute administration of obestatin had no influence on the 2-h rate of gastric emptying of a solid nutrient meal, but the ICV h/rCRF injection delayed it. The weight of food ingested 1 h before ICV injection significantly, but negatively correlated with the gastric emptying of a solid nutrient meal. Our study indicates that the ICV injection of obestatin does not change the 2-h rate of gastric emptying of a solid nutrient meal and the relatively weak interrelationships between ghrelin gene products and nesfatin-1. However, the weight of the ingested food negatively affects the gastric emptying of a solid nutrient meal in conscious, fasted rats.

Outer membrane lipoprotein Lpp is Gram-negative bacterial cell surface receptor for cationic antimicrobial peptides.

Antimicrobial peptides/proteins (AMPs) are important components of the host innate defense mechanisms. Here we demonstrate that the outer membrane lipoprotein, Lpp, of Enterobacteriaceae interacts with and promotes susceptibility to the bactericidal activities of AMPs. The oligomeric Lpp was specifically recognized by several cationic α-helical AMPs, including SMAP-29, CAP-18, and LL-37; AMP-mediated bactericidal activities were blocked by anti-Lpp antibody blocking. Blebbing of the outer membrane and increase in membrane permeability occurred in association with the coordinate internalization of Lpp and AMP. Interestingly, the specific binding of AMP to Lpp was resistant to divalent cations and salts, which were able to inhibit the bactericidal activities of some AMPs. Furthermore, using His-tagged Lpp as a ligand, we retrieved several characterized AMPs, including SMAP-29 and hRNase 7, from a peptide library containing crude mammalian cell lysates. Overall, this study explores a new mechanism and target of antimicrobial activity and provides a novel method for screening of antimicrobials for use against drug-resistant bacteria.

NMR and biophysical elucidation of structural effects on extra N-terminal methionine residue of recombinant amphibian RNases from Rana catesbeiana.

The stability, structures and steric hindrances of recombinant RNases 2 and 4 expressed in bacteria were studied by circular dichroism (CD) and NMR techniques, and the results were compared with those of their authentic RNases extracted from oocytes of Rana catesbeiana. Although the overall structures of the recombinant and authentic proteins are almost identical, the extra N-terminal Met residue of the recombinant protein remarkably affects catalytic activity and stability. NMR chemical shift comparison of recombinant RNases and the authentic proteins indicated that the structural differences are mainly confined to the N-terminal helical and S2 anti-parallel beta-sheet regions. Significant shift changes for the residues located on the S2 region indicate that the major influences on the structure around the N terminus is due to the loss of the hydrogen bond between Pyr(1) and Val(95(96)) in recombinant RNases 2 and 4. We concluded the apparent steric hindrances of the extra Met to the binding pocket. As well, the affected conformational changes of active residues are attributed to the reduced activities of recombinant RNases. The structural integrity exerted by the N-terminal Pyr(1) residue may be crucial for amphibian RNases and the greatest structural differences occur on the network of the Pyr(1) residue and S2 beta-sheet region.

Outer membrane protein I of Pseudomonas aeruginosa is a target of cationic antimicrobial peptide/protein.

Cationic antimicrobial peptides/proteins (AMPs) are important components of the host innate defense mechanisms against invading microorganisms. Here we demonstrate that OprI (outer membrane protein I) of Pseudomonas aeruginosa is responsible for its susceptibility to human ribonuclease 7 (hRNase 7) and alpha-helical cationic AMPs, instead of surface lipopolysaccharide, which is the initial binding site of cationic AMPs. The antimicrobial activities of hRNase 7 and alpha-helical cationic AMPs against P. aeruginosa were inhibited by the addition of exogenous OprI or anti-OprI antibody. On modification and internalization of OprI by hRNase 7 into cytosol, the bacterial membrane became permeable to metabolites. The lipoprotein was predicted to consist of an extended loop at the N terminus for hRNase 7/lipopolysaccharide binding, a trimeric alpha-helix, and a lysine residue at the C terminus for cell wall anchoring. Our findings highlight a novel mechanism of antimicrobial activity and document a previously unexplored target of alpha-helical cationic AMPs, which may be used for screening drugs to treat antibiotic-resistant bacterial infection.

Granulysin is a key mediator for disseminated keratinocyte death in Stevens-Johnson syndrome and toxic epidermal necrolysis.

Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are life-threatening adverse drug reactions characterized by massive epidermal necrosis, in which the specific danger signals involved remain unclear. Here we show that blister cells from skin lesions of SJS-TEN primarily consist of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, and both blister fluids and cells were cytotoxic. Gene expression profiling identified granulysin as the most highly expressed cytotoxic molecule, confirmed by quantitative PCR and immunohistochemistry. Granulysin concentrations in the blister fluids were two to four orders of magnitude higher than perforin, granzyme B or soluble Fas ligand concentrations, and depleting granulysin reduced the cytotoxicity. Granulysin in the blister fluids was a 15-kDa secretory form, and injection of it into mouse skin resulted in features mimicking SJS-TEN. Our findings demonstrate that secretory granulysin is a key molecule responsible for the disseminated keratinocyte death in SJS-TEN and highlight a mechanism for CTL- or NK cell--mediated cytotoxicity that does not require direct cellular contact.

The flexible and clustered lysine residues of human ribonuclease 7 are critical for membrane permeability and antimicrobial activity.

The ubiquitous ribonucleases (RNases) play important roles in RNA metabolism, angiogenesis, neurotoxicity, and antitumor or antimicrobial activity. Only the antimicrobial RNases possess high positively charged residues, although their mechanisms of action remain unclear. Here, we report on the role of cationic residues of human RNase7 (hRNase7) in its antimicrobial activity. It exerted antimicrobial activity against bacteria and yeast, even at 4 degrees C. The bacterial membrane became permeable to the DNA-binding dye SYTOX(R) Green in only a few minutes after bactericidal RNase treatment. NMR studies showed that the 22 positively charged residues (Lys(18) and Arg(4)) are distributed into three clusters on the surface of hRNase7. The first cluster, K(1),K(3),K(111),K(112), was located at the flexible coil near the N terminus, whereas the other two, K(32),K(35) and K(96),R(97),K(100), were located on rigid secondary structures. Mutagenesis studies showed that the flexible cluster K(1),K(3),K(111),K(112), rather than the catalytic residues His(15), Lys(38), and His(123) or other clusters such as K(32),K(35) and K(96),R(97),K(100), is critical for the bactericidal activity. We suggest that the hRNase7 binds to bacterial membrane and renders the membrane permeable through the flexible and clustered Lys residues K(1),K(3),K(111),K(112). The conformation of hRNase7 can be adapted for pore formation or disruption of bacterial membrane even at 4 degrees C.