Factors contributing to the potency of antimicrobial cationic peptides from the N-terminal region of human lactoferrin

This study investigated the antimicrobial activities of peptides derived from the N-terminal region of human lactoferrin, and examined the contributions of individual residues to the activity of the most potent peptide. Two regions of antimicrobial activity were identified, the first corresponding to a weakly active peptide, HLP-9, comprising residues 1-9, and a second corresponding to a more potent peptide, HLP-10, comprising residues 18-26 and containing the hexapeptide motif, FQWQRN. Inhibitory studies on peptides from the first region confirm the importance of tryptophan residues in enhancing and broadening peptide activity. Inhibitory studies with glycine-substituted homologues of the more potent peptide showed that F21/G and R25/G substitutions resulted in a major reduction or complete loss of activity, while increased peptide cationicity or flexibility had little effect. Our findings demonstrate that F21 and R25 are critical determinants of potency for HLP-10, and that the second aromatic residue may act synergistically with W23 in developing and enhancing the activity of this cationic peptide.     

Novel lactoferrampin antimicrobial peptides derived from human lactoferrin

Human lactoferrampin is a novel antimicrobial peptide found in the cationic N-terminal lobe of the iron-binding human lactoferrin protein. The amino acid sequence that directly corresponds to the previously characterized bovine lactoferrin-derived lactoferrampin peptide is inactive on its own (WNLLRQAQEKFGKDKSP, residues 269-285). However, by increasing the net positive charge near the C-terminal end of human lactoferrampin, a significant increase in its antibacterial and Candidacidal activity was obtained. Conversely, the addition of an N-terminal helix cap (sequence DAI) did not have any appreciable effect on the antibacterial or antifungal activity of human lactoferrampin peptides, even though it markedly influenced that of bovine lactoferrampin. The solution structure of five human lactoferrampin variants was determined in SDS micelles and all of the structures display a well-defined amphipathic N-terminal helix and a flexible cationic C-terminus. Differential scanning calorimetry studies indicate that this peptide is capable of inserting into the hydrophobic core of a membrane, while fluorescence spectroscopy results suggest that a hydrophobic patch encompassing the single Trp and Phe residues as well as Leu, Ile and Ala side chains mediates the interaction between the peptide and the hydrophobic core of a phospholipid bilayer.     

Prophylactic effect of human lactoferrin against Streptococcus mutans bacteremia in lactoferrin knockout mice

Streptococcus mutans is the primary agent of dental caries, which is often detected in transient bacteremia. Lactoferrin is a multifunctional glycoprotein showing antibacterial activities against several Streptococcus species. We reported here the prophylactic effect of human lactoferrin (hLF) in a lactoferrin knockout mouse (LFKO^−/−) bacteremic model. The hLF treatment significantly cleared S. mutans from the blood and organs of bacteremic mice when compared to the non-hLF treated mice. Further, analysis of serum cytokines, spleen and liver cytokine mRNA levels revealed that hLF prophylaxis modulates their release differently when compared to the non-hLF treated group. C-reactive protein level (P = 0.003) also decreased following hLF prophylaxis in S. mutans induced bacteremic mice. Additional quantitative RT-PCR analysis revealed that hLF prophylaxis significantly decreased the expression level of IFN-γ, TNF-α, IL-1β, IL-6, MPO and iNOS in spleen and liver. These results suggested that the hLF protects the host against S. mutans-induced experimental bacteremia.     

Comparison of the effects of acylation and amidation on the antimicrobial and antiviral properties of lactoferrin

AIMS: To compare amidation and acylation of lactoferrin (LF) from bovine milk, as a means of enhancing its antimicrobial and antiviral properties. METHODS AND RESULTS: LF was chemically modified by amidation with a 1-ethyl-3-[3-(dimethylamino) propyl] carbodiimide (EDC) in the presence of ammonium ions or by acylation with either succinic or acetic anhydride. In the test systems used, amidation substantially enhanced the activity of LF against Pseudomonas fluorescens in comparison with native LF. However, increasing the net negative charge of LF by acylation had no effect on the activity of LF against P. fluorescens, and abrogated the antimicrobial activity of LF against Bacillus subtilis and Saccharomyces cerevisiae. Increasing the net negative charges of LF by acylation eliminated its antimicrobial and antiviral effects against poliovirus and feline calicivirus (nonenveloped viruses). CONCLUSIONS: The addition of positive charges to LF via amidation enhanced antimicrobial properties in contrast to increasing the negative charges by acylation, which abolished both the antimicrobial and antiviral properties of LF. SIGNIFICANCE AND IMPACT OF THE STUDY: The effects of charge alteration of LF determined in this study provides a basis for further development of LF formulations with enhanced antimicrobial effectiveness for use in food process hygiene, veterinary and health-care applications.     

Computational study,synthesis and evaluation of active peptides derived from Parasporin-2 and spike protein from Alphacoronavirus against colorectal cancer cells

Parasporin-2Aa1 (PS2Aa1) is a toxic protein of 37 KDa (30 kDa, activated form produced by proteolysis) that was shown to be cytotoxic against specific human cancer cells, although its mechanism of action has not been elucidated yet. In order to study the role of some native peptide fragments of proteins on anticancer activity, here we investigated the cytotoxic effect of peptide fragments from domain-1 of PS2Aa1 and one of the loops present in the binding region of the virus spike protein from Alphacoronavirus (HCoV-229E), the latter according to scientific reports, who showed interaction with the human APN (h-APN) receptor, evidence corroborated through computational simulations, and thus being possible active against colon cancer cells. Peptides namely P264-G274, Loop1-PS2Aa, and Loop2-PS2Aa were synthesized using the Fmoc solid-phase synthesis and characterized by mass spectrometry (MS). Additionally, one region from loop 1 of HCoV-229E, Loop1-HCoV-229E, was also synthesized and characterized. The A4W-GGN5 anticancer peptide and 5-fluorouracil (5-FU) were taken as a control in all experiments. Circular dichroism revealed an α-helix structure for the peptides derived from PS2Aa1 (P264-G274, Loop1-PS2Aa, and Loop2-PS2Aa) and β-laminar structure for the peptide derived from Alphacoronavirus spike protein Loop1-HCoV-229E. Peptides showed a hemolysis percentage of less than 20% at 100 µM concentration. Besides, peptides exhibited stronger anticancer activity against SW480 and SW620 cells after exposure for 48 h. Likewise, these compounds showed significantly lower toxicity against normal cells CHO-K1. The results suggest that native peptide fragments from Ps2Aa1 may be optimized as a novel potential cancer-therapeutic agents.     

Natural antimicrobial peptides from bacteria: characteristics and potential applications to fight against antibiotic resistance

Because of the emergence of antibiotic‐resistant pathogens worldwide, a number of infectious diseases have become difficult to treat. This threatening situation is worsened by the fact that very limited progress has been made in developing new and potent antibiotics in recent years. However, a group of antimicrobials, the so‐called bacteriocins, have been much studied lately because they hold a great potential in controlling antibiotic‐resistant pathogens. Bacteriocins are small antimicrobial peptides (AMPs) produced by numerous bacteria. They often act toward species related to the producer with a very high potency (at pico‐ to nanomolar concentration) and specificity. The common mechanisms of killing by bacteriocins are destruction of target cells by pore formation and/or inhibition of cell wall synthesis. Several studies have revealed that bacteriocins display great potential in the medical sector as bacteriocinogenic probiotics and in the clinic as therapeutic agents. In this review, we discuss the emerging antibiotic resistance and strategies to control its dissemination, before we highlight the potential of AMPs from bacteria as a new genre of antimicrobial agents.     

Insect antimicrobial peptides show potentiating functional interactions against Gram-negative bacteria

Antimicrobial peptides (AMPs) and proteins are important components of innateimmunity against pathogens in insects. The production of AMPs is costly owing toresource-based trade-offs, and strategies maximizing the efficacy of AMPs at lowconcentrations are therefore likely to be advantageous. Here, we show thepotentiating functional interaction of co-occurring insect AMPs (the bumblebeelinear peptides hymenoptaecin and abaecin) resulting in more potentantimicrobial effects at low concentrations. Abaecin displayed no detectableactivity against Escherichia coli when tested alone atconcentrations of up to 200 μM, whereas hymenoptaecin affected bacterialcell growth and viability but only at concentrations greater than 2 μM.In combination, as little as 1.25 μM abaecin enhanced the bactericidaleffects of hymenoptaecin. To understand these potentiating functionalinteractions, we investigated their mechanisms of action using atomic forcemicroscopy and fluorescence resonance energy transfer-based quenching assays.Abaecin was found to reduce the minimal inhibitory concentration ofhymenoptaecin and to interact with the bacterial chaperone DnaK (anevolutionarily conserved central organizer of the bacterial chaperone network)when the membrane was compromised by hymenoptaecin. These naturally occurringpotentiating interactions suggest that combinations of AMPs could be usedtherapeutically against Gram-negative bacterial pathogens that have acquiredresistance to common antibiotics.     

Activity and mode of action against fungal phytopathogens of bovine lactoferricin-derived peptides

AIM: To evaluate the activity against fungal phytopathogens of two synthetic peptides derived from the protein bovine lactoferricin: the antibacterial active core of six amino acid residues (LfcinB(20-25)) and an extension of 15 amino acids (LfcinB(17-31)). METHODS AND RESULTS: In vitro activity against fungal pathogens was determined and compared with that against model micro-organisms. Activity was demonstrated against fungi of agronomic relevance. Distinct antimicrobial properties in vitro were found for the two peptides. LfcinB(17-31) had growth inhibitory activity higher than LfcinB(20-25). However, LfcinB(17-31) was not fungicidal to quiescent conidia of Penicillium digitatum at the concentrations assayed, while LfcinB(20-25) killed conidia more efficiently. Microscopical observations showed that the mycelium of P. digitatum treated with LfcinB(17-31) developed alterations of growth, sporulation and chitin deposition, and permeation of hyphal cells. In experimental inoculations of mandarins, both peptides showed limited protective effect against the disease caused by P. digitatum. CONCLUSIONS: LfcinB(20-25) and LfcinB(17-31) peptides were shown to have antimicrobial activity against plant pathogenic filamentous fungi, with distinct properties and mode of action. SIGNIFICANCE AND IMPACT OF THE STUDY: LfcinB(20-25) and LfcinB(17-31) peptides offer novel alternatives to develop resistant plants by molecular breeding.     

Histatin and lactoferrin derived peptides: antimicrobial properties and effects on mammalian cells

In order to analyze the clinical potential of two antimicrobial peptides, human lactoferrin 1-11 (hLF1-11) and synthetic histatin analogue Dhvar-5, we measured the killing effect on bacteria, and the potential toxicity on erythrocytes and bone cells. The antimicrobial activity was determined in a killing assay on six strains, including methicillin resistant Staphylococcus Aureus. The effect on human erythrocytes and MC3T3 mouse bone cells was measured with a hemolysis assay and a viability assay, respectively. Both hLF1-11 and Dhvar-5 dose-dependently killed all bacterial strains, starting at concentrations of 6 μg/mL. hLF1-11 had no effect on mammalian cells at concentrations up to 400 μg/mL, but Dhvar-5 induced significant hemolysis (37% at 200 μg/mL) and bone cell death (70% at 400 μg/mL). This indicates that both peptides are able to kill various resistant and non-resistant bacteria, but Dhvar-5 may exert a cytotoxic effect on host cells at higher concentrations.     

Antimicrobial peptides and proteins, exercise and innate mucosal immunity

This review examines the question of whether exercise can be used as an experimental model to further our understanding of innate antimicrobial peptides and proteins (AMPs) and their role in susceptibility to infection at mucosal surfaces. There is strong evidence to suggest that AMPs, in combination with cellular and physical factors, play an important role in preventing infection. Although AMPs act directly on microorganisms, there is increasing recognition that they also exert their protective effect via immunomodulatory mechanisms, especially in noninflammatory conditions. Further studies that manipulate physiologically relevant concentrations of AMPs are required to shed light on the role they play in reducing susceptibility to infection. Evidence shows that in various form prolonged and/or exhaustive exercise is a potent modulator of the immune system, which can either sharpen or blunt the immune response to pathogens. The intensity and duration of exercise can be readily controlled in experimental settings to manipulate the degree of physical stress. This would allow for an investigation into a potential dose-response effect between exercise and AMPs. In addition, the use of controlled exercise could provide an experimental model by which to examine whether changes in the concentration of AMPs alters susceptibility to illness.     

Synergistic effects of short peptides and antibiotics against bacterial and fungal strains

There is a rising tide of concern about the antibiotic resistance issue. To reduce the possibility of antibiotic-resistant infections, a new generation of antimicrobials must be developed. Antimicrobial peptides are potential alternatives to antibiotics that can be used alone or together with conventional antibiotics to combat antimicrobial resistance. In this work, lead compounds LP-23, DP-23, SA4, and SPO from previously published studies were synthesized by solid-phase peptide synthesis and their antimicrobial evaluation was carried out against various bacterial and fungal strains. Peptide combinations with antibiotics were evaluated by using the checkerboard method and their minimal inhibitory concentration (MIC) in combination was calculated by using the fractional inhibitory concentration (FIC) index. Cytotoxicity evaluations of these peptides further confirmed their selectivity toward microbial cells. Based on the FIC values, LP-23, DP-23, and SPO demonstrated synergy in combination with gentamicin against a gentamicin-resistant clinical isolate of Escherichia coli. For Staphylococcus aureus, Escherichia coli, and Salmonella typhimurium, seven combinations exhibited synergistic effects between peptide/peptoids and the tested antibiotics. Additionally, almost all the combinations of peptides/peptoids with amphotericin B and fluconazole also showed effective synergy against Aspergillus niger and Aspergillus flavus. The synergy found between LP-23, DP-23, SA4, and SPO with the selected antibiotics may have the potential to be used as a combination therapy against various microbial infections.     

Enhancing the antimicrobial effects of bovine lactoferrin against Escherichia coli O157:H7 by cation chelation, NaCl and temperature

AIM: To evaluate the effect of NaCl, growth medium and temperature on the antimicrobial activity of bovine lactoferrin (LF) against Escherichia coli O157:H7 in the presence of different chelating agents. METHODS AND RESULTS: LF (32 mg ml(-1)) was tested against E. coli O157:H7 strain 3081 in Luria broth (LB) and All Purpose Tween (APT) broth with metal ion chelators sodium bicarbonate (SB), sodium lactate (SL), sodium hexametaphosphate (SHMP), ethylene diamine tetraacetic acid (EDTA) or quercetin at 0.5 and 2.5% NaCl at 10 and 37 degrees C. LF and the chelators were tested against four other E. coli O157:H7 strains in LB at 2.5% NaCl and 10 degrees C. LF alone was bacteriostatic against strains 3081 and LCDC 7283 but other strains grew. Antimicrobial effectiveness of LF was reduced in APT broth but enhanced by SB at 2.5% NaCl and 10 degrees C where 4.0 log(10) CFU ml(-1) inoculated cells were killed. EDTA enhanced antimicrobial action of the LF-SB combination. SL alone was effective against E. coli O157:H7 but a reduction in its activity at 2.5% NaCl and 10 degrees C was reversed by LF. The combinations LF-SHMP and LF-quercetin were more effective at 37 degrees C and NaCl effects varied. CONCLUSIONS: LF plus SB or SL were bactericidal toward the same 3/5 E. coli O157:H7 strains and inhibited growth of the others at 2.5% NaCl and 10 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: The combination of LF with either SL or SB shows potential for reducing viability of E. coli O157:H7 in food systems containing NaCl at reduced, but growth permissive temperature.     

Production of recombinant bovine lactoferrin N-lobe in insect cells and its antimicrobial activity

Lactoferrin is a multifunctional, iron-binding glycoprotein found in physiological fluids of mammals. In the present study, a gene encoding the N-terminal half (N-lobe) of bovine lactoferrin was cloned and expressed in cultured insect cells using a baculovirus expression system. One mutation was found in the lactoferrin N-lobe gene, but it resulted in no amino acid substitution. The recombinant lactoferrin N-lobe was secreted into the culture medium and partially purified by means of an immobilized heparin column. The recombinant lactoferrin N-lobe secreted was not glycosylated, but it possessed antimicrobial activity toward Escherichia coli O111. The recombinant product synthesized and accumulated in the host cells exhibited greater electrophoretic mobility on SDS-PAGE than the secreted product and showed no potency to inhibit the growth of bacteria. It is thought that the product accumulated intracellularly lacks antimicrobial ability due to its degradation in the host cells or due to disruption of the active conformation.     

乳腺生物反应器表达的重组人乳铁蛋白的分离纯化及生物活性鉴定

以国产高交联度的快流速琼脂糖为基质,合成了不同配基密度的SP(Sulfopropyl,磺酸基)离子交换介质,建立了乳腺生物反应器表达重组人乳铁蛋白(Recombinant Human Lactoferrin,rHLF)的纯化方法。以溶菌酶为模型蛋白考察了不同配基密度离子交换介质的静态和动态吸附行为,结果表明介质具有良好的吸附性能。不同配基密度离子交换介质均可纯化得到rHLF,其中,高配基密度(0.24mol/L)的离子交换介质每毫升可以处理50mL rHLF牛乳,rHLF收率为86.5%,纯度为98.5%。圆二色谱的测定结果表明纯化的rHLF二级结构与天然人乳铁蛋白一致。生物学功能实验结果表明,rHLF的铁结合与释放活性与天然人乳铁蛋白相似,浓度为5g/L的rHLF对大肠杆菌的生长具有明显的抑制作用。     

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.     

The protein structure of recombinant human lactoferrin produced in the milk of transgenic cows closely matches the structure of human milk-derived lactoferrin

Human lactoferrin (hLF) is an iron-binding glycoprotein involved in the host defence against infection and excessive inflammation. As the availability of (human milk-derived) natural hLF is limited, alternative means of production of this biopharmaceutical are extensively researched. Here we report the crystal structure of recombinant hLF (rhLF) expressed in the milk of transgenic cows at a resolution of 2.4 Å. To our knowledge, the first reported structure of a recombinant protein produced in milk of transgenic livestock. Even though rhLF contains oligomannose- and hybrid-type N-linked glycans next to complex-type glycans, which are the only glycans found on natural hLF, the structures are identical within the experimental error (r.m.s. deviation of only 0.28 Å for the main-chain atoms). Of the differences in polymorphic amino acids between the natural and rhLF variant used, only the side-chain of Asp⁵⁶¹ could be modeled into the rhLF electron density map. Taken together, the results confirm the structural integrity of the rhLF variant used in this study. It also confirms the validity of the transgenic cow mammary gland as a vehicle to produce recombinant human proteins.Ellen A. J. Thomassen, Harrie A. van Veen - These authors have contributed equally to this paper.The PDB-code of recombinant human lactoferrin is 2BJJ     

The synergistic effect of nisin and lactoferrin on the inhibition of Listeria monocytogenes and Escherichia coli O157:H7

AIMS: The goal of this study was to determine whether nisin and lactoferrin would act synergistically to inhibit the growth of Listeria monocytogenes and Escherichia coli O157:H7. METHODS AND RESULTS: Lactoferrin and nisin separately or in combination were suspended in peptone yeast glucose broth and following inoculation with L. monocytogenes or E. coli O157:H7 growth inhibition of each pathogen was determined. At 1000 microg ml(-1) lactoferrin L. monocytogenes was effectively inhibited. However, E. coli O157:H7 initially was inhibited and then grew to cell density similar to the control. A combination of 500 microg ml(-1) of lactoferrin and 250 IU ml(-1) of nisin effectively inhibited the growth of E. coli O157:H7, whereas, 250 microg ml(-1) of lactoferrin and 10 IU ml(-1) of nisin were inhibitory to L. monocytogenes. CONCLUSIONS: The results suggest that lactoferrin and nisin act synergistically to inhibit the growth of L. monocytogenes and E. coli O157:H7. SIGNIFICANCE AND IMPACT OF THE STUDY: Natural preservatives that are active against gram-positive and gram-negative pathogens are desirable to the food industry and consumers. This study demonstrates that lactoferrin and nisin work synergistically reducing the levels required independently inhibiting growth of two major foodborne pathogens. Previous reported results indicated a low level of antimicrobial activity; however, this work was not performed in low divalent cation concentration media. It has been suggested that nondivalent cation-limiting medium such as trypticase soy broth (TSB), can reduce or completely eliminate the inhibitory activity. Further knowledge of these interactions can increase the understanding of the antimicrobial activity of lactoferrin. This should make the use of these compounds by industry more attractive.