Bovine Lactoferricin Research Articles

Linear and Polyvalent Peptides with Potent Antimicrobial Activity Against Sensitive and Multidrug-Resistant E. coli Clinical Isolates.

Peptides containing the sequences 20RRWQWR25 and 20RRWQWRMKKLG30 derived from Bovine lactoferricin (LfcinB) were synthesized and their antibacterial effect against reference strains and sensitive and resistant clinical isolates of E. coli was evaluated. Tetra-branched multiple antigen peptide (MAP) ((RRWQWR)2-K-Ahx-C)2 exhibited significant antibacterial activity against sensitive, resistant, and multidrug-resistant clinical isolates of E. coli. Peptide 3: RRWQWR-Nal-KKLG; MIC=16 µM, 26[F]: (RRWQWRFKKLG)2-K-Ahx; MIC=15 µM, 17: (RRWQWRFK)2-K-Ahx; MIC=9 µM, and LfcinB (20-25)2: (RRWQWR)2-K-Ahx; MIC=11 µM exhibited the highest antibacterial activity against E. coli strains, with bactericidal effect and haemolytic effect at MIC less than 5% and a therapeutic index >1. A synergistic effect of peptides 26[F] and 17 with ciprofloxacin (CIP) or ceftriaxone (CEF) was observed. Prolonged treatment of E. coli ATCC 25922 with sublethal concentrations of CIP induced resistance in this strain, whereas some peptides did not induce resistance. These peptides can be considered to be promising candidates for treating infections caused by resistant strains of E. coli.

Expression and antimicrobial activity of the recombinant bovine lactoferricin in Pichia pastoris

Lactoferricin, a multifunctional peptide located in the N-terminal region of lactoferrin, has a broad-spectrum bacteriostatic activity. It is a promising candidate as a food additive and immune fortification agent and does not have the risks associated with drug residues and drug resistance. First, we performed promoter and host cell screening to achieve the recombinant expression of lactoferricin in Pichia pastoris, showing an initial titer of 19.5 mg/L in P. pastoris X-33 using PAOX1 promoter. Second, we constructed a 0030-α hybrid signal peptide by fusing the 0030 signal peptide with the pro-sequence of α-factor secretory signal peptide. This further increased the production of lactoferricin, with a titer of 28.8 mg/L in the fermentation supernatant in the shaking flask. Next, we increased the expression of lactoferricin by fusing it with anionic antioxidant peptides. The neutralization of positive charges yielded a titer of 55.3 mg/L in the shaking flask, and a highest titer of 193.9 mg/L in a 3-L bioreactor. The antimicrobial activity analysis showed that recombinant-expressed lactoferricin exhibited potent antibacterial activity against Escherichia coli, Bacillus subtilis, and Staphylococcus aureus. This study provides a reference for the construction of microbial cell factories capable of efficiently synthesizing antimicrobial peptides.

The role of synthetic peptides derived from bovine lactoferricin against breast cancer cell lines: a mini-review

Abstract Breast cancer represents the most commonly diagnosed cancer worldwide, accounting for approximately one in eight cancers diagnosed. Despite significant advances in the diagnosis and detection of this disease, there is still a great need for more effective therapies to combat the invasive forms, especially those with a high incidence of metastasis. For that reason, bioactive molecules as peptides, including bovine lactoferricin (LfcinB), have been investigated. In this sense, there are reports that 20RRWQWR25 motif derivate from the LfcinB has shown activity against different cancer cell lines. Thus, current studies are being carried out with synthetic derivatives (linear, palindromic, dimer and tetrameric structures) that contain the 20RRWQWR25 motif in order to increase its activity against cancer cell lines by altering its hydrophobicity and net positive charge. In this regard, studies have focused on the use of LfcinB derivatives to combat breast cancer cell lines, with encouraging results. Therefore, in this mini-review, we present the state of the art regarding the activity of LfcinB and its analogs against breast cancer cell lines.

In Vitro Antifungal Activity of Chimeric Peptides Derived from Bovine Lactoferricin and Buforin II against Cryptococcus neoformans var. grubii.

Cryptococcosis is associated with high rates of morbidity and mortality. The limited number of antifungal agents, their toxicity, and the difficulty of these molecules in crossing the blood-brain barrier have made the exploration of new therapeutic candidates against Cryptococcus neoformans a priority task. To optimize the antimicrobial functionality and improve the physicochemical properties of AMPs, chemical strategies include combinations of peptide fragments into one. This study aimed to evaluate the binding of the minimum activity motif of bovine lactoferricin (LfcinB) and buforin II (BFII) against C. neoformans var. grubii. The antifungal activity against these chimeras was evaluated against (i) the reference strain H99, (ii) three Colombian clinical strains, and (iii) eleven mutant strains, with the aim of evaluating the possible antifungal target. We found high activity against these strains, with a MIC between 6.25 and 12.5 µg/mL. Studies were carried out to evaluate the effect of the combination of fluconazole treatments, finding a synergistic effect. Finally, when fibroblast cells were treated with 12.5 µg/mL of the chimeras, a viability of more than 65% was found. The results obtained in this study identify these chimeras as potential antifungal molecules for future therapeutic applications against cryptococcosis.

Heterologous expression of antimicrobial peptides S-thanatin and bovine lactoferricin in the marine diatom Phaeodactylum tricornutum enhances native antimicrobial activity against Gram-negative bacteria

The increase in resistance of bacterial strains to existing antibiotics represents a major public health concern. Antibiotics are an invaluable tool still frequently used in agriculture as they can both improve feed/energy conversion ratios in livestock and treat pathogenic infections. However, the misuse of these compounds has contributed to escalating antimicrobial resistance (AMR). The rapid increase in drug-resistant infections has presented a serious challenge to antimicrobial therapies. Research is currently being carried out to identify alternative antimicrobial compounds to use in animal production although to date, no effective alternatives to production-purpose antibiotics (PPAs) have been developed. Recent reports indicate that lysate of marine diatom Phaeodactylum tricornutum was able to significantly lower the maximal growth rate of Gram-negative bacteria. We have engineered P. tricornutum to accumulate antimicrobial recombinant proteins, S-thanatin and bovine lactoferricin (bLFcin). Transgenic P. tricornutum expressing heterologous S-thanatin and bLFcin demonstrated significantly enhanced antimicrobial activity against Gram-negative bacteria Enterobacter cloacae ATCC 13047 and Vibrio natriegens ATCC 14048 respectively. The lysate of transgenic strain Pt_LFcin1.15, expressing bLFcin, lowered maximal growth rate (μmax) of V. natriegens by 33.2 %, which was a 15.5 % further reduction than by wild type (WT) lysate. The lysate of the optimized bLFcin-expressing transgenic strain Pt_LFcin2.3 lowered V. natriegens μmax by 38.5 %, which was a 27.4 % further reduction than by WT lysate. The S-thanatin-expressing transgenic cell lysate of strain Pt_S-Tha1.11 lowered E. cloacae μmax by 61.3 %, which was a 38.1 % greater reduction than by WT lysate. These statistically significant results (p < 0.01) demonstrate that microalgae can be engineered to express a range of naturally occurring antimicrobial and anti-inflammatory compounds which can substitute for PPAs and treat serious bacterial infections.

Sortase A Fusion Expression and mIFc2 Co-Expression of Bovine Lactoferricin and Analysis of Its Antibacterial Activity

The coding region for the sortase A (SrtA) of Staphylococcus aureus was fused at the N-terminus of LfcinB. The SrtA-LfcinB fusion protein in E. coli C43(DE3) was expressed with the expected sizes of 21 kDa and 38 kDa by pET21b-SrtA-LfcinB and pET32-1SrtA-LfcinB constructs, respectively. Increased levels of the TrxA-His-SrtA-SrtA-LfcinB fusion protein were detected by the pET32-3SrtA-LfcinB construct having three expression cassettes. LfcinB is released from the expressed SrtA-LfcinB protein by SrtA self-cleavage which is induced in the presence of Ca2+. The antibacterial activity was detected after SrtA-mediated cleavage of LfcinB. Furthermore, to reduce the antimicrobial peptide toxicity to the E. coli host, the human interferon-γ (hIFN-γ) sequences were mutated into a negatively charged mIFc2 protein (7 kDa), which was co-expressed with LfcinB in an insoluble form. The yield of LfcinB was elevated while changing the gene order of LfcinB and mIFc2 (pET21b-fLfcinB-bmIFc2). Furthermore, increased levels of LfcinB were detected using the pET21b-(fLfcinB-bmIFc2)2 construct. To increase the dissolution rate of inclusion bodies, inclusion bodies treated with different temperatures and pH and resuspended in different volumes of 50 mM Tris-HCl were assayed. Our results reveal that heat-treated LfcinB/mIFc2 inclusion bodies at 90 °C, pH 10, and 16X resuspended volumes have the best resolubilization rate. This work suggests that the mIFc2 co-expression system shows higher efficiency for LfcinB production than the SrtA fusion system. The expressed LfcinB from the mIFc2 co-expression system exhibits excellent broad-spectrum antibacterial activities against thirteen Gram-negative and ten Gram-positive bacteria species with a range of minimum inhibitory concentrations (MIC) between 37–150 ug/mL.

Bovine lactoferricin on non-specific immunity of giant freshwater prawns, Macrobrachium rosenbergii

This study aimed to investigate the effects of dietary Bovine lactoferricin (LFcinB) on the growth performance and non-specific immunity in Macrobrachium rosenbergii. Five experimental diets were 1.0‰ Bovine lactoferricin (LCB1); 1.5‰ Bovine lactoferricin (LCB1.5); 2.0‰ Bovine lactoferricin (LCB2); 2.5‰ Bovine lactoferricin (LCB2.5); the control group, basal diet without Bovine lactoferricin. A total of 600 prawns were randomly assigned to 5 groups in triplicate in 15 tanks for an 8-week feeding trial. The results showed the final weight, weight gain rate, specific growth rate and survival rate of prawns in the treatment groups were significantly improved versus the control (P < 0.05). The feed conversion ratio was reduced significantly in treatment groups compared to the control (P < 0.05). Compared with the control, alkaline phosphatase (AKP), acid phosphatase (ACP), lysozyme (LZM), catalase (CAT), superoxide dismutase (SOD) activities in the hepatopancreas of the treatment groups were significantly enhanced, and malondialdehyde (MDA) content was reduced significantly (P < 0.05). Compared with the control, the relative expression levels of AKP, ACP, LZM, CAT, SOD, Hsp70, peroxiredoxin-5, Toll, dorsal and relish genes were significantly higher among treatment groups, except for the AKP gene in the LCB1 group and the Hsp70 gene in the LCB1.5 group (P < 0.05). Compared with the control, the relative expression levels of TOR, 4E-BP, eIF4E1α and eIF4E2 genes were significantly enhanced in the LCB1.5 group (P < 0.05). When resistance against Vibrio parahaemolyticus in prawn is considered, higher doses of Bovine lactoferricin show better antibacterial ability. The present study indicated that dietary Bovine lactoferricin could significantly improve the growth performance and improve the antioxidative status of M. rosenbergii. The suitable addition level is 1.5 g/kg. LFcinB has great potential as a new feed additive without the threat of drug resistance.

Chimeric Peptides Derived from Bovine Lactoferricin and Buforin II: Antifungal Activity against Reference Strains and Clinical Isolates of Candida spp.

Antimicrobial peptides (AMPs) are considered to be a valuable source for the identification and/or design of promising candidates for the development of antifungal treatments, since they have advantages such as lower tendency to induce resistance, ease of production, and high purity and safety. Bovine lactoferricin (LfcinB) and buforin II (BFII) are AMPs to which great antimicrobial potential has been attributed. The minimum motives with antimicrobial activity derived from LfcinB and BFII are RRWQWR and RLLR, respectively. Nine chimeras containing the minimum motives of both peptides were synthesized and their antifungal activity against fluconazole (FLC)-sensitive and resistant C. albicans, C. glabrata, and C. auris strains was evaluated. The results showed that peptides C9: (RRWQWR)2K-Ahx-RLLRRRLLR and C6: KKWQWK-Ahx-RLLRRLLR exhibited the greatest antifungal activity against two strains of C. albicans, a FLC-sensitive reference strain and a FLC-resistant clinical isolate; no medically significant results were observed with the other chimeras evaluated (MIC ~200 µg/mL). The chimera C6 was also active against sensitive and resistant strains of C. glabrata and C. auris. The combination of branched polyvalent chimeras together with FLC showed a synergistic effect against C. albicans. In addition to exhibiting antifungal activity against reference strains and clinical isolates of Candida spp., they also showed antibacterial activity against both Gram-positive and Gram-negative bacteria, suggesting that these chimeras exhibit a broad antimicrobial spectrum and can be considered to be promising molecules for therapeutic applications.

The hybrid oncolytic peptide NTP-385 potently inhibits adherent cancer cells by targeting the nucleus.

The use of oncolytic peptides with activity against a wide range of cancer entities as a new and promising cancer therapeutic strategy has drawn increasing attention. The oncolytic peptide LTX-315 derived from bovine lactoferricin (LfcinB) was found to be highly effective against suspension cancer cells, but not adherent cancer cells. In this study, we tactically fused LTX-315 with rhodamine B through a hybridization strategy to design and synthesize a series of nucleus-targeting hybrid peptides and evaluated their activity against adherent cancer cells. Thus, four hybrid peptides, NTP-212, NTP-217, NTP-223 and NTP-385, were synthesized. These hybrid peptides enhanced the anticancer activity of LTX-315 in a panel of adherent cancer cell lines by 2.4- to 37.5-fold. In model mice bearing B16-F10 melanoma xenografts, injection of NTP-385 (0.5 mg per mouse for 3 consecutive days) induced almost complete regression of melanoma, prolonged the median survival time and increased the overall survival. Notably, the administered dose of NTP-385 was only half the effective dose of LTX-315. We further revealed that unlike LTX-315, which targets the mitochondria, NTP-385 disrupted the nuclear membrane and accumulated in the nucleus, resulting in the transfer of a substantial amount of reactive oxygen species (ROS) from the cytoplasm to the nucleus through the fragmented nuclear membrane. This ultimately led to DNA double-strand break (DSB)-mediated intrinsic apoptosis. In conclusion, this study demonstrates that hybrid peptides obtained from the fusion of LTX-315 and rhodamine B enhance anti-adherent cancer cell activity by targeting the nucleus and triggering DNA DSB-mediated intrinsic apoptosis. This study also provides an advantageous reference for nucleus-targeting peptide modification.

Bovine lactoferricin ameliorates intestinal inflammation and mucosal barrier lesions in colitis through NF-κB/NLRP3 signaling pathways

• LfcinB ameliorates intestinal tissue morphology and distribution disorder of tight junction proteins in an established model of acute colitis. • LfcinB improves the production and distribution of mucin in colon of rats. • LfcinB alters the immune status of acute UC rats and inhibits the activities of inflammatory mediators. • NF-κB/NLRP3 signaling pathway involved the protection role of LfcinB on intestinal inflammation and mucosal barrier. Bovine lactoferricin (LfcinB) has biological properties such as anti-inflammatory and immunological activity. This study aimed to investigate the protective effects of LfcinB on mucosal injury during the pathological process of ulcerative colitis in rats. The thirty-two rats were randomly assigned to four groups: control (C group), colitis model (DSS group; oral intake of 5.0% dextran sodium sulfate), colitis model with LfcinB (LBD group; 5 mg/kg for 7 days, starting while DSS treatment) and LfcinB (LfcinB group; 5 mg/kg for 7 days) group. Treatment with LfcinB reduced the disease activity index and diamine oxidase concentration in rats with colitis. Histology analysis revealed a better preservation of the epithelium as well as attenuated inflammatory infiltration of the colon epithelium in LfcinB-treated rats. LfcinB treatment also downregulated the colonic expression of IL-1β and restored the expression levels of zonula occludens-1, occludin and mucin 2, by suppressing DSS-induced increases in inflammation and myeloperoxidase concentration in the rats of LBD group. During this process, the decreased expression levels of ASC, Caspase-1, NLRP3, and NF-κB in the rats of LBD group were characterized by quantitative RT-PCR. These results suggest LfcinB ameliorates DSS-induced changes in intestinal inflammation and mucosal barrier in colitis rats, possibly through NF-κB/NLRP3 signaling pathway.

Effect of bovine lactoferricin on the growth performance, digestive capacity, immune responses and disease resistance in Pacific white shrimp, Penaeus vannamei

The present study evaluated the growth performance, digestive enzyme activity, non-specific immunity, immunity and growth genes in Penaeus vannamei fed diets supplemented with Bovine lactoferricin (the basal diet without Bovine lactoferricin, the control; 1.0‰ Bovine lactoferricin，LCB1; 1.5‰ Bovine lactoferricin，LCB1.5; 2.0‰ Bovine lactoferricin, LCB2; 2.5‰ Bovine lactoferricin, LCB2.5) for 56 days. The feeding trial showed that the final weight, weight gain rate, and specific growth rate of the shrimp were improved significantly, while the feed conversion ratio was reduced significantly in the LCB1.5 group compared to the control (P < 0.05). The challenge test of Vibrio parahaemolyticus showed that the cumulative mortalities of shrimp in the LCB1.5, LCB2 and LCB2.5 groups were significantly lower than that in the control (P < 0.05). Compared with the control, Lipase and Trypsin activities in the hepatopancreas of LCB1.5 and LCB2 groups were significantly enhanced (P < 0.05). Compared with the control, alkaline phosphatase, acid phosphatase activities in the hepatopancreas and the relative expression levels of Relish, Toll, JAK, STAT, TOR, Raptor, 4E-BP, eIF4E1α, eIF4E2 genes in the hepatopancreas of LCB1.5, LCB2 and LCB2.5 groups were all significantly enhanced (P < 0.05). These results suggested that dietary Bovine lactoferricin could improve the growth performance, digestive capacity and immune responses of shrimp. When resistance against Vibrio parahaemolyticus in shrimp is considered, high dosage of Bovine lactoferricin showed a better effect than low dosage of Bovine lactoferricin. However, high dosage of Bovine lactoferricin can have a negative impact on the growth performance of shrimp. Considering collectively the above, Bovine lactoferricin could improve the growth performance, digestive enzymes activities, immune responses and disease resistance of P. vannamei.

Antimicrobial effect and mechanism of bovine lactoferrin against the potato common scab pathogen Streptomyces scabiei

Lactoferrin (LF) is a multifunctional protein with a broad spectrum of antimicrobial activities. In this study, we investigated the antimicrobial activity of LF against the potato common scab pathogen Streptomyces scabiei, which causes severe damage to potato tubers. LF derived from bovine (bLF) had much higher activity against S. scabiei than human LF. The minimal inhibitory concentration of bLF was 3.9 μM. The effects of both apo-bLF (iron-free) and holo-bLF (iron-saturated) on S. scabiei were not different. Bovine lactoferricin (LFcinB), a short peptide with a length of 25 amino acid residues located in the N-terminal region of bLF, showed antimicrobial activity against S. scabiei, similar to that of bLF. These results indicated that the antimicrobial activity of bLF against S. scabiei cannot be attributed to its iron-chelating effect but to the bioactivity of its peptides. When S. scabiei was treated with the fusion protein of mCherry-LFcinB (red fluorescent protein) expressed in Escherichia coli, the pseudohyphal cells instantly glowed, indicating that the peptide electrostatically binds to the surface of S. scabiei. An assay of synthetic peptides, with modified number of arginine (Arg) and tryptophan (Trp) residues based on the antimicrobial center (RRWQWR) of LFcinB showed that Trp residues are implicated in the antimicrobial activity against S. scabiei; however, Arg residues are also necessary to carry Trp residues to the cell surface to fully exert its activity. Although the single amino acid effect of Trp had low activity, Trp derivatives showed much higher activity against S. scabiei, suggesting that the derivatives effectively bind to the cell surface (cell membrane) by themselves without a carrier. Thus, amino acid derivatives might be considered effective and alternative antimicrobial substances.

Regulation of macrophage-associated inflammatory responses by species-specific lactoferricin peptides.

Inflammation is the body's response to injury or infection and is important for healing and eliminating pathogens; however, prolonged inflammation is damaging and may lead to the development of chronic inflammatory disorders. Recently, there has been interest in exploiting antimicrobial peptides (AMPs) that exhibit immunoregulatory activities to treat inflammatory diseases. In this study, we investigated the immunomodulatory effects of lactoferrin-derived lactoferricin AMPs from three different species (bovine, mouse, and human) with subtle differences in their amino acid sequences that alter their antimicrobial action; to our knowledge, no other studies have compared their immunomodulatory effects. Macrophages, key players in the induction and propagation of inflammation, were used to investigate the effects of species-specific lactoferricin peptides on inflammatory processes. Bovine lactoferricin was the only one of the three peptides studied that downregulated lipopolysaccharide (LPS)-induced pro-inflammatory cytokines, tumor necrosis factor (TNF)-α and interleukin (IL)-6, in both human and mouse macrophages. Lactoferricin regulated inflammation through targeting LPS-activated nuclear factor (NF)-κB and mitogen-activated protein kinase (MAPK) signaling pathways. Although the immunoregulatory role of lactoferricin during an inflammatory response in vivo is yet to be elucidated, further investigation with the use of animal models is warranted by the current findings. The ability of lactoferricin, especially that of bovine origin, to downregulate macrophage-mediated inflammatory responses suggests potential for the development of this peptide as a novel immunotherapeutic agent in the treatment of chronic inflammatory conditions.

Effects of Dietary Supplementation of Bovine Lactoferricin on Rumen Microbiota, Lactation, and Health in Dairy Goats.

This study aimed to investigate the biological effects of supplementation of bovine lactoferricin (BLFc) at the rate of 100 mg/kg/day (LF-1) or 200 mg/kg/day (LF-2) in lactating dairy goats. Dietary BLFc supplementation increased the concentration of lactoferrin (LF) in the milk and serum (p < 0.05) without affecting the feed intake. In the LF-1 group, serum Fe, total antioxidant (T-AOC), and immunoglobulin A (IgA) were increased (p < 0.05), while malondialdehyde (MDA) was decreased (p < 0.05). In the LF-2 group, ruminal fluid pH value was decreased (p < 0.05), and the composition of ruminal microflora on day 42 was more diversified. Firmicutes phylum in the LF-2 group was the most abundant phyla. In contrast, Bacteroidetes phylum in the control group and the LF-1 group were the most abundant. Lower milk somatic cell count and higher IgA were observed in the LF-1 group and the LF-2 group than those in the control group (p < 0.05). These results suggested beneficial effects of supplementation of 100 mg/kg/day BLFc on reducing the oxidative stress and altering diversity of ruminal microflora.

Bovine Lactoferricin Induces Intestinal Epithelial Cell Activation through Phosphorylation of FAK and Paxillin and Prevents Rotavirus Infection.

We investigated the effect of bovine lactoferricin (Lfcin-B), a peptide derived from bovine lactoferrin, on activation of intestinal epithelial cells in IEC-6 intestinal cell, and protection against in vivo rotavirus (RV) infection. Treatment with Lfcin-B significantly enhanced the growth of IEC-6 cells and increased their capacity for attachment and spreading in culture plates. Also, Lfcin-B synergistically augmented the binding of IEC-6 cells to laminin, a component of the extracellular matrix (ECM). In the analysis of the intracellular mechanism related to Lfcin-B-induced activation of IEC-6 cells, this peptide upregulated tyrosine-dependent phosphorylation of focal adhesion kinase (FAK) and paxillin, which are intracellular proteins associated with cell adhesion, spreading, and signal transduction during cell activation. An experiment using synthetic peptides with various sequences of amino acids revealed that a sequence of 9 amino acids (FKCRRWQWR) corresponding to 17-25 of the N-terminus of Lfcin-B is responsible for the epithelial cell activation. In an in vivo experiment, treatment with Lfcin-B one day before RV infection effectively prevented RV-induced diarrhea and significantly reduced RV titers in the bowels of infected mice. These results suggest that Lfcin-B plays meaningful roles in the maintenance and repair of intestinal mucosal tissues, as well as in protecting against intestinal infection by RV. Collectively, Lfcin-B is a promising candidate with potential applications in drugs or functional foods beneficial for intestinal health and mucosal immunity.

Fatty acid modified-antimicrobial peptide analogues with potent antimicrobial activity and topical therapeutic efficacy against Staphylococcus hyicus.

There is an urgent need to explore new antimicrobial agents due to the looming threat of bacteria resistance. Bovine lactoferricin (LfcinB), as a multifunctional peptide, has the potential to be a new active drug in the future. In this study, it aims to investigate the effect of fatty acid conjugation on antimicrobial peptide activity and topical therapeutic efficacy in a mouse model infected with Staphylococcus hyicus. Both Lfcin4 and Lfcin5 were conjugated with the unsaturated fatty acid linoleic acid (18-C) at their N-terminus and modified by acylation at the C-terminus. The derived peptides of Lin-Lf4NH2 and Lin-Lf5NH2 showed better antibacterial activity (MICs of 3.27 to 6.64 μM) than their parent peptides (MICs of 1.83 to 59.57 μM). Lin-Lf4NH2 (63.2%, 5 min) and Lin-Lf5NH2 (35.8%, 5 min) could more rapidly penetrate bacterial membrane than Lf4NH2 (2.34%, 5 min) and Lf5NH2 (1.94%, 5 min), which further confirmed by the laser scanning confocal microscopy (LSCM). Electron microscopy observations showed Lin-Lf4NH2 and Lin-Lf5NH2 disrupted S. hyicus cell membranes and led to the leakage of contents. Furthermore, after treatment with Lin-Lf4NH2 and Lin-Lf5NH2, the abscess symptoms of mice were significantly alleviated; the recovery rate of abscesses scope of Lin-Lf4NH2 (73.25%) and Lin-Lf5NH2 (71.71%) were 38.8 and 37.9-fold higher than that of untreated group (1.89%), respectively, and superior to Lf4NH2 (46.87%) and Lf5NH2 (58.75%). They significantly reduced the bacterial load and the levels of the pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) and chemokine (MCP-1) in S. hyicus skin lesions. This study provides evidence that conjugation of a fatty acid to antimicrobial peptides can improve the activity and have potential for topical therapeutic of S. hyicus skin infections. KEY POINTS: • Lin-Lfcin4NH2/Lfcin5NH2 showed stronger antimicrobial activity than parent peptides. • Lin-Lfcin4NH2/Lfcin5NH2 had a more effective ability to destroy bacterial membranes. • Lin-Lfcin4NH2/Lfcin5NH2 showed a topically higher efficacy than parent peptides.

Effects of Substituting Arginine by Lysine in Bovine Lactoferricin Derived Peptides: Pursuing Production Lower Costs, Lower Hemolysis, and Sustained Antimicrobial Activity

Monomeric, dimeric, palindromic, and tetrameric peptides derived from bovine lactoferricin (LfcinB) containing the RRWQWR or the KKWQWK sequence were synthesized via SPPS- Fmoc/tBu strategy, and Lys-peptides showed higher synthesis efficiency. The antibacterial activity of the peptides against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus ATCC reference strains and a multidrug resistant clinical isolate was evaluated. Lys peptides exhibited similar antibacterial activity to the Arg peptides, suggesting that the Arg for Lys substitution does not significantly affect the antibacterial activity. The synergy assays showed that the covalent dimeric Lys peptide decreased the MIC value of the ciprofloxacin up to 32 times, and LfcinB (20-25), and one of the Lys-peptides [K]-LfcinB (20-25)Pal presented a synergistic effect. Additionally, Lys peptides lowered the hemolytic activity by 2 to 7 times. These results suggest that short peptides derived from Lys-LfcinB could be a cost-effective alternative for the development of drugs or combinatory treatments against bacterial infections.

Improved diabetic wound healing by LFcinB is associated with relevant changes in the skin immune response and microbiota

Bovine lactoferricin (LFcinB) has antimicrobial and immunomodulatory properties; however, the effects on diabetic wound healing remain poorly understood. The wound healing potential of LFcinB was investigated with in vitro, ex vivo, and in vivo models. Cell migration and proliferation were tested on keratinocytes and on porcine ears. A type 1 diabetic mouse model was also used to evaluate wound healing kinetics, bacterial diversity patterns, and the effect of LFcinB on oxidative stress, macrophage phenotype, angiogenesis, and collagen deposition. LFcinB increased keratinocyte migration in vitro (p < 0.05) and ex vivo (p < 0.001) and improved wound healing in diabetic mice (p < 0.05), though not in normoglycemic control mice. In diabetic mouse wounds, LFcinB treatment led to the eradication of Bacillus pumilus, a decrease in Staphylococcus aureus, and an increase in the Staphylococcus xylosus prevalence. LFcinB increased angiogenesis in diabetic mice (p < 0.01), but this was decreased in control mice (p < 0.05). LFcinB improved collagen deposition in both diabetic and control mice (p < 0.05). Both oxidative stress and the M1-to-M2 macrophage ratios were decreased in LFcinB-treated wounds of diabetic animals (p < 0.001 and p < 0.05, respectively) compared with saline, suggesting a downregulation of inflammation in diabetic wounds. In conclusion, LFcinB treatment demonstrated noticeable positive effects on diabetic wound healing.

Designing Chimeric Peptides: A Powerful Tool for Enhancing Antibacterial Activity.

Chimeric peptides containing short sequences derived from bovine Lactoferricin (LfcinB) and Buforin II (BFII) were synthetized using solid-phase peptide synthesis (SPPS) and characterized via reversed-phase liquid chromatography and mass spectrometry. The chimeras were obtained with high purity, demonstrating their synthetic viability. The chimeras' antibacterial activity against Gram-positive and Gram-negative strains was evaluated. Our results showed that all the chimeras exhibited greater antibacterial activity against the evaluated strains than the individual sequences, suggesting that chemical binding of short sequences derived from AMPs significantly increased the antibacterial activity. For each strain, the chimera with the best antibacterial activity exerted a bacteriostatic and/or bactericidal effect, which was dependent on the concentration. It was found that: (i) the antibacterial activity of a chimera is mainly influenced by the linked sequences, the palindromic motif RLLRRLLR being the most relevant one; (ii) the inclusion of a spacer between the short sequences did not significantly affect the chimera's synthesis process; however, it enhanced its antibacterial activity against Gram-negative and Gram-positive strains; on the other hand, (iii) the replacement of Arg with Lys in the LfcinB or BFII sequences improved the chimeras' synthesis process without significantly affecting their antibacterial activity. These results illustrate the great importance of the synthesis of chimeric peptides for the generation of promising antibacterial peptides.

Lactoferricin-Derived L5a Cell-Penetrating Peptide for Delivery of DNA into Cells.

Cell-penetrating peptides (CPPs) are small peptides which help intracellular delivery of functional macromolecules, including DNAs, RNAs, and proteins, across the cell membrane and into the cytosol, and even into the nucleus in some cases. Delivery of macromolecules can facilitate transfection, aid in gene therapy and transgenesis, and alter gene expression. L5a (RRWQW), originally derived from bovine lactoferricin, is one kind of CPPs which can promote cellular uptake of plasmid DNA and enters cells via direct membrane translocation. The peptide complexes noncovalently with DNA over a short incubation period. DNA plasmid and L5a complex stability is confirmed by a decrease in mobility in a gel retardation assay, and successful transfection is proven by the detection of a reporter gene in cells using fluorescent microscopy. Here, we describe methods to study noncovalent interactions between L5a and plasmid DNA, and the delivery of L5a/DNA complexes into cells. L5a is the one of the smallest CPPs discovered to date, providing a small delivery vehicle for macromolecules in mammalian cells. A small vehicle which can enter the nucleus is ideal for efficient gene uptake, transfer, and therapy. It is simple to complex with DNA plasmids, and its nature allows mammalian cells to be easily transfected.