Antifungal Synergy: Mechanistic Insights into the R-1-R Peptide and Bidens pilosa Extract as Potent Therapeutics against Candida spp. through Proteomics.

Previous reports have demonstrated that the peptide derived from LfcinB, R-1-R, exhibits anti-Candida activity, which is enhanced when combined with an extract from the Bidens pilosa plant. However, the mechanism of action remains unexplored. In this research, a proteomic study was carried out, followed by a bioinformatic analysis and biological assays in both the SC5314 strain and a fluconazole-resistant isolate of Candida albicans after incubation with R-1-R. The proteomic data revealed that treatment with R-1-R led to the up-regulation of most differentially expressed proteins compared to the controls in both strains. These proteins are primarily involved in membrane and cell wall biosynthesis, membrane transport, oxidative stress response, the mitochondrial respiratory chain, and DNA damage response. Additionally, proteomic analysis of the C. albicans parental strain SC5314 treated with R-1-R combined with an ethanolic extract of B. pilosa was performed. The differentially expressed proteins following this combined treatment were involved in similar functional processes as those treated with the R-1-R peptide alone but were mostly down-regulated (data are available through ProteomeXchange with identifier PXD053558). Biological assays validated the proteomic results, evidencing cell surface damage, reactive oxygen species generation, and decreased mitochondrial membrane potential. These findings provide insights into the complex antifungal mechanisms of the R-1-R peptide and its combination with the B. pilosa extract, potentially informing future studies on natural product derivatives.

Combining the Peptide RWQWRWQWR and an Ethanolic Extract of Bidens pilosa Enhances the Activity against Sensitive and Resistant Candida albicans and C. auris Strains.

The antifungal activity of palindromic peptide RWQWRWQWR and its derivatives was evaluated against clinical isolates of Candida albicans and C. auris. Also, Bidens pilosa ethanolic extracts of leaves and stem were evaluated. Furthermore, combinations of peptide, extract, and/or fluconazole (FLC) were evaluated. The cytotoxicity of peptides and extracts in erythrocytes and fibroblasts was determined. The original palindromic peptide, some derivative peptides, and the ethanolic extract of leaves of B. pilosa exhibited the highest activity in some of the strains evaluated. Synergy was obtained between the peptide and the FLC against C. auris 435. The combination of the extract and the original palindromic peptide against C. albicans SC5314, C. auris 435, and C. auris 537 decreased the minimal inhibitory concentrations (MICs) by a factor of between 4 and 16. These mixtures induced changes in cell morphology, such as deformations on the cell surface. The results suggest that the combination of RWQWRWQWR and B. pilosa extract is an alternative for enhancing antifungal activity and decreasing cytotoxicity and costs and should be considered to be a promising strategy for treating diseases caused by Candida spp.

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.

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.

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.

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.

De novo expression and antibacterial potential of four lactoferricin peptides in cell-free protein synthesis system.

For the first time, we produced four lactoferricin (LFcin) peptides by a cell-free (in vitro) method. These short antimicrobial peptides were expressed in an E. coli cell-free protein synthesis (CFPS) system and the bioactivity of the produced peptides was demonstrated. Additionally, we designed a novel synthetic consensus peptide (ConLFcin). The genes of bovine Lfcin (bLFcin), human Lfcin (hLFcin), camel Lfcin (cLFcin), and ConLFcin were cloned into pET101/D-TOPO vector then peptides were synthesized in vitro by E. coli CFPS system. The antibacterial activity of these synthesized peptides was evaluated against Escherichia coli, Salmonella typhi, Pseudomonas aeruginosa, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus (MRSA). The four cell-free synthesized peptides showed significant antibacterial potency at minimum inhibitory concentration (MIC) values between 1.25 and 10 µg/mL. cLFcin and ConLFcin showed higher antibacterial effects than bLFcin and hLFcin. Thus, cell-free expression system is an ideal system for rapid expression of functionally active short bioactive peptides.

Peptides Derived from (RRWQWRMKKLG)2-K-Ahx Induce Selective Cellular Death in Breast Cancer Cell Lines through Apoptotic Pathway.

The effect on the cytotoxicity against breast cancer cell lines of the substitution of 26Met residue in the sequence of the Bovine Lactoferricin-derived dimeric peptide LfcinB (20-30)2: (20RRWQWRMKKLG30)2-K-Ahx with amino acids of different polarity was evaluated. The process of the synthesis of the LfcinB (20-30)2 analog peptides was similar to the original peptide. The cytotoxic assays showed that some analog peptides exhibited a significant cytotoxic effect against breast cancer cell lines HTB-132 and MCF-7, suggesting that the substitution of the Met with amino acids of a hydrophobic nature drastically enhances its cytotoxicity against HTB-132 and MCF-7 cells, reaching IC50 values up to 6 µM. In addition, these peptides have a selective effect, since they exhibit a lower cytotoxic effect on the non-tumorigenic cell line MCF-12. Interestingly, the cytotoxic effect is fast (90 min) and is maintained for up to 48 h. Additionally, through flow cytometry, it was found that the obtained dimeric peptides generate cell death through the apoptosis pathway and do not compromise the integrity of the cytoplasmic membrane, and there are intrinsic apoptotic events involved. These results show that the obtained peptides are extremely promising molecules for the future development of drugs for use against breast cancer.

Antibacterial Activity and Mechanism of Action of Bovine Lactoferricin Derivatives with Symmetrical Amino Acid Sequences.

In recent years, the overuse of antibiotics has become very serious. Many pathogenic bacteria have become resistant to them, with serious potential health consequences. Thus, it is urgent that we develop new antibiotic drugs. Antimicrobial peptides (AMPs) are important endogenous antibacterial molecules that contribute to immunity. Most have spectral antibacterial properties and do not confer drug resistance. In this paper, an 11-residue peptide (LFcinB18⁻28) with a sequence of KCRRWQWRMKK was modified by amino acid substitution to form a symmetrical amino acid sequence. The antibacterial activities and mechanisms of action of engineered peptides including KW-WK (KWRRWQWRRWK), FP-PF (FPRRWQWRRPF), FW-WF (FWRRWQWRRWF), and KK-KK (KKRRWQWRRKK) were investigated. The four engineered peptides could more effectively inhibit bacteria than the original peptide, LFcinB18⁻28. This suggested that a symmetrical amino acid sequence might enhance the antibacterial activity of AMPs. However, only peptides KW-WK, FP-PF, and KK-KK were safe; FW-WF displayed hemolytic activity. The engineered peptides shared cationic and amphipathic characteristics that facilitated interactions with the anionic microbial membranes, leading to disruption of membrane integrity and permeabilizing microbial membranes, resulting in cell death. Therefore, a symmetrical amino acid sequence and related structural parameters offer an alternative approach to the design of AMPs. This will provide a scientific basis for the design and synthesis of new AMPs.

A PiggyBac mediated approach for lactoferricin gene transfer in bovine mammary epithelial stem cells for management of bovine mastitis.

The antibacterial and anti-inflammatory properties of lactoferricin have been ascribed to its ability to sequester essential iron. The objective of the study was to clone bovine lactoferricin (LFcinB) gene into PiggyBac Transposon vector, expression study in the bovine mammary epithelial stem cells (bMESCs) and also to determine the antimicrobial property of recombinant LFcinB against bovine mastitis-causing organisms. The PiggyBac-LFcinB was transfected into bMESCs by electroporation and a three fold of LFcinB secretion was observed in the transfected bMESCs medium by ELISA assay. Furthermore, the assessment of antimicrobial activity against mastitis causing pathogens Staphylococcus aureus and Escherichia coli demonstrated convincing evidence to prove strong antibacterial activity of LFcinB with 14.0±1.0 mm and 18.0±1.5 mm zone of inhibition against both organisms, respectively. The present study provides the convincing evidence to suggest the potential of PiggyBac transposon system to transfer antibacterial peptide into bMESCs or cow mammary gland and also pave the way to use bovine mammary gland as the bioreactors. Simultaneously, it also suggest toward commercial utilization of LFcinB bioreactor system in pharmaceutical industry.

Antimicrobial Activity of Truncated and Polyvalent Peptides Derived from the FKCRRQWQWRMKKGLA Sequence against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923.

Peptides derived from LfcinB were designed and synthesized, and their antibacterial activity was tested against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923. Specifically, a peptide library was constructed by systemically removing the flanking residues (N or C-terminal) of Lfcin 17-31 (17FKCRRWQWRMKKLGA31), maintaining in all peptides the 20RRWQWR25 sequence that corresponds to the minimal antimicrobial motif. For this research, also included were (i) a peptide containing an Ala instead of Cys ([Ala19]-LfcinB 17-31) and (ii) polyvalent peptides containing the RRWQWR sequence and a non-natural amino acid (aminocaproic acid). We established that the lineal peptides LfcinB 17-25 and LfcinB 17-26 exhibited the greatest activity against E. coli ATCC 25922 and S. aureus ATCC 25923, respectively. On the other hand, polyvalent peptides, a dimer and a tetramer, exhibited the greatest antibacterial activity, indicating that multiple copies of the sequence increase the activity. Our results suggest that the dimeric and tetrameric sequence forms potentiate the antibacterial activity of lineal sequences that have exhibited moderate antibacterial activity.

Bovine lactoferrin and lactoferricin exert antitumor activities on human colorectal cancer cells (HT-29) by activating various signaling pathways.

Lactoferrin (Lf) is an iron-binding glycoprotein that is present at high concentrations in milk. Bovine lactoferricin (LfcinB) is a peptide fragment generated by pepsin proteolysis of bovine lactoferrin (bLf). LfcinB consists of amino acid residues 17-41 proximal to the N-terminus of bLf and a disulfide bond between residues 19 and 36, forming a loop. Both bLf and LfcinB have been demonstrated to have antitumor activities. Colorectal cancer is the second most common cause of cancer death in developed countries. We hypothesized that bLf and LfcinB exert antitumor activities on colon cancer cells (HT-29) by triggering various signaling pathways. bLf and LfcinB significantly induced apoptosis in HT-29 cells but not in normal human intestinal epithelial cells, as revealed by the ApoTox-Glo Triplex Assay. The LIVE/DEAD cell viability assay showed that both bLf and LfcinB reduced the viability of HT-29 cells. Transcriptome analysis indicated that bLf, cyclic LfcinB, and linear LfcinB exerted antitumor activities by differentially activating diverse signaling pathways, including p53, apoptosis, and angiopoietin signaling. Immunoblotting results confirmed that both bLf and LfcinBs increased expression of caspase-8, p53, and p21, critical proteins in tumor suppression. These results provide valuable information regarding bLf and LfcinB for potential clinical applications in colon cancer therapy.

Bovine lactoferricin P13 triggers ROS-mediated caspase-dependent apoptosis in SMMC7721 cells.

Bovine lactoferricin P13 (LfcinB-P13) is a peptide derived from LfcinB. In the present study, the effect of LfcinB-P13 on the human liver cancer cell line SMMC7721 was investigated in vitro and in vivo. The results of the present study indicate that LfcinB-P13 significantly decreased SMMC7721 cell viability in vitro (P=0.032 vs. untreated cells), while exhibiting low cytotoxicity in the wild-type liver cell line L02. In addition, the rate of apoptosis in SMMC7721 cells was significantly increased following treatment with 40 and 60 µg/ml LfcinB-P13 (P=0.0053 vs. the control group), which was associated with an increase in the level of reactive oxygen species (ROS) and the activation of caspase-3 and -9. Furthermore, ROS chelation led to the suppression of LfcinB-P13-mediated caspase-3 and -9 activation in SMMC7721 cells. LfcinB-P13 was demonstrated to markedly inhibit tumor growth in an SMMC7721-xenograft nude mouse model. The results of the present study indicate that LfcinB-P13 is a novel candidate therapeutic agent for the treatment of liver cancer.

Bovine lactoferricin B induces apoptosis of human gastric cancer cell line AGS by inhibition of autophagy at a late stage.

Gastric cancer is one of the most common malignant cancers, with poor prognosis and high mortality rates worldwide. Therefore, development of an effective therapeutic method without side effects is an urgent need. It has been reported that cationic antimicrobial peptides can selectively bind to negatively charged prokaryotic and cancer cell membranes and exert cytotoxicity without causing severe drug resistance. In the current study, we prepared a series of peptide fragments derived from bovine lactoferrin and evaluated their anticancer potency toward the gastric cancer cell line AGS. Cell viability assay revealed that a 25-AA peptide fragment, lactoferricin B25 (LFcinB25), exhibited the most potent anticancer capability against AGS cells. Lactoferricin B25 selectively inhibited AGS cell growth in a dose-dependent manner, exhibiting a half-maximal inhibitory concentration (IC50) value of 64 µM. Flow cytometry showed a notable increment of the sub-G1 populations of the cell cycle, indicating the induction of apoptosis by LFcinB25. Western blot analysis further revealed that upon LFcinB25 treatment for 2 to 6h, apoptosis-related caspases-3, 7, 8, 9, and poly(ADP-ribose) polymerase (PARP) were cleaved and activated, whereas autophagy-related LC3-II and beclin-1 were concomitantly increased. Thus, both apoptosis and autophagy are involved in the early stage of LFcinB25-induced cell death of AGS cells. However, upon treatment with LFcinB25 for 12 to 24h, LC3-II began to decrease, whereas cleaved beclin-1 increased in a time-dependent manner, suggesting that consecutive activation of caspases cleaved beclin-1 to inhibit autophagy, thus enhancing apoptosis at the final stage. These findings provide support for future application of LFcinB25 as a potential therapeutic agent for gastric cancer.

Generation of a hematologic malignancy-selective membranolytic peptide from the antimicrobial core (RRWQWR) of bovine lactoferricin.

Cationic antimicrobial peptides such as bovine lactoferricin (LfcinB) constitute an important innate defense mechanism against many microbial pathogens. LfcinB also binds to and selectively kills human cancer cells via a mechanism that involves reactive oxygen species (ROS) generation and caspase activation. The antimicrobial core of LfcinB consists of only six amino acids (RRWQWR), referred to in this study as LfcinB6. Although free LfcinB6 is devoid of cytotoxic activity against cancer cells, we show here that adding a cell-penetrating hepta-arginine sequence via a glycine-glycine linker to LfcinB6 generates a peptide (MPLfcinB6) that is selectively cytotoxic for human T-leukemia and B-lymphoma cells. Flow cytometric analysis of propidium iodide and fluorescein isothiocyanate-dextran uptake by MPLfcinB6-treated cancer cells revealed extensive damage to the cell membrane, which was confirmed by scanning electron microscopy. MPLfcinB6-induced cytotoxicity was also associated with sequential ROS production and mitochondrial membrane permeabilization; however, neither ROS nor caspase activation caused by the loss of mitochondrial membrane integrity was essential for peptide-mediated cell death. We conclude that MPLfcinB6 selectively kills human T-leukemia and B-lymphoma cells by causing extensive and irreparable damage to the cell membrane.