Lactoferrin Chimera Research Articles

Bovine lactoferrin and chimera lactoferrin prevent and destroy Salmonella Typhimurium biofilms in Caco-2 cells.

Salmonellosis is a common foodborne disease caused by Salmonella bacteria. The emergence of multidrug-resistant (MDR) Salmonella serotypes, such as Typhimurium, and Salmonella's ability to form biofilms contribute to their resistance and persistence in host and non-host environments. New strategies are needed to treat or prevent Salmonella infections. This work aimed to determine the effect of the bovine lactoferrin (bLF) and lactoferrin chimera (LFchimera) in preventing or disrupting biofilms formed on abiotic surfaces or Caco-2 cells by Salmonella Typhimurium ATCC 14028 or an MDR strain. The inhibitory activity of planktonic bacteria, prevention of biofilm formation, and destruction of biofilms of S. Typhimurium (ATCC 14028 or MDR strain) on the abiotic surface and Caco-2 cells of bLF and LFchimera were quantified by CFU/ml and visualized by microscopy using Giemsa-stained samples. bLF (75-1000µM) and LFchimera (1-20µM) inhibited more than 95% of S. Typhimurium planktonic growth cultures (ATCC 14028 and MDR). In addition, bLF (600, 800, and 1000 µM) and LFchimera (10 and 20µM) prevented more than 98% of S. Typhimurium adherence and biofilm formation on Caco-2 cells. Finally, bLF (600 and 1000 µM) and LFchimera (10 and 20µM) destroyed more than 80% of S. Typhimurium biofilms established on abiotic and Caco-2 cells. In conclusion, bLF and LF chimeras have the potential to inhibit and destroy S. Typhimurium biofilms.

Lactoferrin-derived chimeric peptide (LFch) strongly boosts TGFβ1-mediated inducible Treg differentiation possibly through downregulating TCR/CD28 signalling.

We recently reported that lactoferrin (LF) induces Foxp3+ Treg differentiation through binding to TGFβ receptor III (TβRIII), and this activity was further enhanced by TGFβ1. Generally, a low T-cell receptor (TCR) signal strength is favourable for Foxp3+ Treg differentiation. In the present study, we explored the effect of lactoferrin chimera (LFch, containing lactoferricin [aa 17-30] and lactoferrampin [aa 265-284]), along with TGFβ1 on Foxp3+ Treg differentiation. LFch alone did not induce Foxp3 expression, yet LFch dramatically enhanced TGFβ1-induced Foxp3 expression. LFch had little effect on the phosphorylation of Smad3, a canonical transcriptional factor of TGFβ1. Instead, LFch attenuated the phosphorylation of S6 (a target of mTOR), IκB and PI3K. These activities of LFch were completely abrogated by pretreatment of LFch with soluble TGFβ1 receptor III (sTβRIII). Consistent with this, the activity of LFch on TGFβ1-induced Foxp3 expression was also abrogated by treatment with sTβRIII. Finally, the TGFβ1/LFch-induced T cell population substantially suppressed the proliferation of responder CD4+ T cells. These results indicate that LFch robustly enhances TGFβ1-induced Foxp3+ Treg differentiation by diminishing TCR/CD28 signal intensity.

Lactoferrin-derived Chimeric Peptide (LFch) boosts TGFβ1-induced Treg Differentiation by diminishing of TCR Signal intensity via TβRIII

Abstract Regulatory T cells (Tregs) are a special subset of T cells to suppress excessive immune response and maintain immunologic tolerance. It is known that lesser TCR signal strength is favorable for Foxp3+ Treg differentiation. We recently reported that LF substantially promoted Foxp3 expression by activated CD4+ T cells and this activity was further enhanced by TGF-β1. In the present study, we explored the effect of lactoferrin chimera (LFch, synthetic LF-derived chimeric peptides) along with TGFβ1 on Foxp3+ Treg differentiation. LFch itself did not induce Foxp3 expression. However, LFch dramatically enhanced TGFβ1-induced Foxp3 expression. LFch little affected the phosphorylation of Smad3, which is a canonical TGF-β1 mediator. Instead, LFch decreased the phosphorylation of S6, IkB, and PI3 kinase (an activation hallmark of TCR signaling). These effects of LFch were completely abolished by pretreatment of LFch with soluble TβRIII. Consistently, the enhancement of TGFβ1-induced Foxp3 by LFch was also abrogated by treatment of soluble TβRIII. Finally, TGFβ1/LFch-induced T cell population substantially suppressed the proliferation of responder CD4+ T cells. These results indicate that LFch potently enhances TGFβ1-induced Foxp3+ Treg differentiation by diminishing TCR signal intensity through TβRIII.

Synergistic effects of LFchimera and antibiotic against planktonic and biofilm form of Aggregatibacter actinomycetemcomitans.

Adjunctive use of antibiotics in periodontal treatment have limitations and disadvantages including bacterial resistance. Antimicrobial peptides (AMPs) are potential new agents that can combat bacterial infection. In this study, antimicrobial activity of different concentrations of conventional antibiotics minocycline (MH), doxycycline (DOX), and antimicrobial peptides LL-37, LL-31, Lactoferrin chimera (LFchimera) and Innate Defense Regulator Peptide 1018 (IDR-1018) against Aggregatibacter actinomycetemcomitans ATCC 43718 were determined using colony culturing assay. Subsequently, in vitro activity of the most effective drug and peptide combination was evaluated by checkerboard technique. Impact of the drug and peptide co-administration on biofilm at different stages, i.e., during adhesion and 1-day old biofilm was compared to each of the agents used alone. Results revealed that the killing effects of all AMPs range from 13–100%. In contrast, MH and DOX at 1 and 5 μM showed no killing activity and instead stimulated growth of bacteria. DOX has better killing activity than MH. LFchimera displayed the strongest killing amongst the peptides. Checkerboard technique revealed that combining DOX and LFchimera yielded synergism. Confocal laser scanning microscopy further showed that the combination of DOX and LFchimera caused significant reduction of bacterial adhesion and reduction of biomass, average biofilm thickness and substratum biofilm coverage of 1-day old biofilm compared to DOX and LFchimera alone. In conclusion, LFchimera alone and in combination with DOX exhibited strong antibacterial and anti-biofilm property against A. actinomycetemcomitans. The findings suggest that LFchimera should be considered for development as a new potential therapeutic agent that may be used as an adjunctive treatment for periodontitis.

Killing activity of LFchimera on periodontopathic bacteria and multispecies oral biofilm formation in vitro.

Lactoferrin chimera (LFchimera), a heterodimeric peptide containing lactoferrampin (LFampin265-284) and a part of lactoferricin (LFcin17-30), possesses a broad spectrum of antimicrobial activity. However, there is no report on the inhibitory effects of LFchimera against multispecies oral biofilms. This study aimed to determine the effects of LFchimera in comparison to chlorhexidine digluconate (CHX) and minocycline hydrochloride (MH), on in vitro multispecies biofilms derived from subgingival plaque of periodontitis patients harboring Aggregatibacter actinomycetemcomitans. First the effects of LFchimera against planktonic and an 1-day old biofilm of the periodontopathic bacteria, A. actinomycetemcomitans ATCC 43718 were established. Then, the effects on biofilm formation and bacterial viability in the multispecies biofilm were determined by crystal violet staining and LIVE/DEAD BacLight Bacterial Viability kit, respectively. The results revealed that a significant reduction (P < 0.05) in biofilm formation occurred after 15min exposure to 20µM of LFchimera or CHX compared to control. In contrast, MH at concentration up to 100µM did not inhibit biofilm formation. The ratio of live/dead bacteria in biofilm was also significantly lower after 15min exposure to 20µM of LFchimera compared to control and 20-50µM of CHX and MH. Altogether, the results obtained indicate that LFchimera is able to inhibit in vitro subgingival biofilm formation and reduce viability of multispecies bacteria in biofilm better than CHX and MH.

Antibacterial and cell penetrating effects of LFcin17-30, LFampin265-284, and LF chimera on enteroaggregative Escherichia coli.

Lactoferrin (LF) is a protein with antimicrobial activity, which is conferred in part by 2 regions contained in its N-terminal lobe. These regions have been used to develop the following synthetic peptides: lactoferricin17-30, lactoferrampin265-284, and LF chimera (a fusion of lactoferricin17-30 and lactoferrampin265-284). We have reported that these LF peptides have antibacterial activity against several pathogenic bacteria; however, the exact mechanism of action has not been established. Here, we report the effects of LF peptides on the viability of enteroaggregative Escherichia coli (EAEC) and the ability of these peptides to penetrate into the bacteria cytoplasm. The viability of EAEC treated with LF peptides was determined via enumeration of colony-forming units, and the binding and internalization of the LF peptides was followed via immunogold labeling and electron microscopy. Treatment of EAEC with 20 and 40 μmol/L LF peptides reduced bacterial growth compared with untreated bacteria. Initially the peptides associated with the plasma membrane, but after 5 to 30 min of incubation, the peptides were found in the cytoplasm. Remarkably, bacteria treated with LF chimera developed cytosolic electron-dense structures that contained the antimicrobial peptide. Our results suggest that the antibacterial mechanism of LF peptides on EAEC involves their interaction with and penetration into the bacteria.

Bactericidal effect of bovine lactoferrin and synthetic peptide lactoferrin chimera in Streptococcus pneumoniae and the decrease in luxS gene expression by lactoferrin.

Streptococcus pneumoniae (pneumococcus) is responsible for nearly one million child deaths annually. Pneumococcus causes infections such as pneumonia, otitis media, meningitis, and sepsis. The human immune system includes antibacterial peptides and proteins such as lactoferrin (LF), but its activity against pneumococcus is not fully understood. The aim of this work was to evaluate the bactericidal effect of bovine lactoferrin (bLF) and the synthetic LF-peptides lactoferricin (LFcin17-30), lactoferrampin (LFampin265-284), and LFchimera against S. pneumoniae planktonic cells. The mechanism of damage was also investigated, as well as the impact of these peptides on the transcription levels of genes known to encode important virulence factors. S. pneumoniae planktonic cells were treated with bLF, LFcin17-30, LFampin265-284 and LFchimera at different time points. The viability of treated planktonic cells was assessed by dilution and plating (in CFU/ml). The interaction between LF and LF-peptides coupled to fluorescein was visualized using a confocal microscope and flow cytometry, whereas the damage at structural levels was observed by electron microscopy. Damage to bacterial membranes was further evaluated by membrane permeabilization by use of propidium iodide and flow cytometry, and finally, the expression of pneumococcal genes was evaluated by qRT-PCR. bLF and LFchimera were the best bactericidal agents. bLF and peptides interacted with bacteria causing changes in the shape and size of the cell and membrane permeabilization. Moreover, the luxS gene was down-regulated in bacteria treated with LF. In conclusion, LF and LFchimera have a bactericidal effect, and LF down-regulates genes involved in the pathogenicity of pneumococcus, thus demonstrating potential as new agents for the treatment of pneumococcal infections.

Design and activity of novel lactoferrampin analogues against O157:H7 enterohemorrhagic escherichia coli

Lactoferrampin 265-284 (LFampin 265-284) is a peptide consisting of residues 265-284 of N1-domain of bovine Lactoferrin (LF). This peptide has several cationic groups in the C-terminal lobe, exhibiting an antibacterial activity against a wide range of microorganisms. However, LFampin 265-284 exhibits low antimicrobial activity against the O157:H7 enterohaemorrhagic Escherichia coli (EHEC O157:H7) when compared with Lactoferrin chimera and Lactoferricin. Here, we have designed three analogues of LFampin 265-284 based on the distribution of cationic groups, hydrophobicity, size, and sequence. Analogues were synthesized by solid phase chemistry using Fmoc methodology obtaining peptides with 95% purity. All peptides maintain the ability to adopt helical conformations (checked by circular dichroism spectra and molecular simulations). Some of these analogues exhibited a significant increase in antimicrobial activity by counting colony forming units against EHEC O157:H7 compared to native LFampin 265-284, with MIC of 10 and 40 µM for 264G-D265K and 264G-D265K/S272R, respectively. The incorporation of a GKLI sequence in the N-terminal lobe increased dramatically its antibacterial activity, an effect which has been attributed to the addition of cationic groups in the N-terminal side that may stabilize the helical conformation of the new designed peptides.

Protective effects of lactoferrin chimera and bovine lactoferrin in a mouse model of enterohaemorrhagic Escherichia coli O157:H7 infection1This article is part of a Special Issue entitled Lactoferrin and has undergone the Journal's usual peer review process.

Mice orally infected with enterohaemorrhagic Escherichia coli (EHEC) O157:H7 were used to evaluate the activity of bovine lactoferrin (bLF) and the synthetic peptide LFchimera. Groups of BALB/c mice inoculated intragastrically with EHEC O157:H7 showed chronic intestinal infection with the pathogen that persisted over 6 days and resulted in a high mortality rate (90%). LFchimera and kanamycin significantly decreased (40%) this mortality rate (P = 0.028). On the other hand, although mice administered with bLF showed an important reduction in mortality (50%), this was not statistically significant (P = 0.070). In infected and untreated mice, severe tubular necrosis, glomerular lesions, and moderate intratubular hyaline casts were found in the kidney. However, in the bLF and LFchimera groups we found a reduction in the damage and a substantial decrease in the bacterial concentration excreted in feces 48 h after infection. Furthermore, sepsis caused by EHEC was reduced by the treatments, evidenced by the fact that bacteria were not detected in the kidney or liver 72 h after infection. The results suggest the bLF and LFchimera could have potential as therapeutics in EHEC infections.

Lactoferrin and lactoferrin chimera inhibit damage caused by enteropathogenic Escherichia coli in HEp-2 cells

Enteropathogenic Escherichia coli (EPEC) is an important cause of infant diarrhea in developing countries. It produces a characteristic intestinal histopathological lesion on enterocytes known as 'attaching and effacing' (A/E), and these two steps are mediated by a type-III secretory system. In the present study, we evaluated the effect on the initial host cell attachment step produced by bovine lactoferrin (bLF) and three synthetic peptides: lactoferricin (LFcin), lactoferrampin (LFampin) and LFchimera. A special focus was given to the hemolytic activity and EPEC-induced actin polymerization in HEp-2 cells, as well as to the espA gene expression, which produces the protein responsible for primary contact with the host cells. Results show that EPEC attachment to HEp-2 cells was significantly suppressed by bLF and LFchimera at 125 and 40μM, respectively. EPEC-mediated actin polymerization was blocked by bLF and LFchimera at 88 and 99%, respectively. LFchimera inhibited the attachment and A/E lesion caused by EPEC in a dose-dependent manner. In the presence of 125μM bLF, the expression level of the espA gene was decreased by 50% compared to the untreated control. LFchimera at concentrations of 20μM and 40μM diminished the level of espA gene expression 100 and 1000 fold, respectively (P<0.001). Although bLF, LFchimera, LFcin, and LFampin all significantly blocked the hemolysis produced by EPEC (P<0.001), the two former compounds produced this effect at lower concentrations. These two compounds, bLF and LFchimera, were able to inhibit the first steps of the mechanism of the damage used by EPEC. This data suggests that LFchimera could provide protection against enteropathogens that share this mechanism.

Bactericidal effect of bovine lactoferrin, LFcin, LFampin and LFchimera on antibiotic-resistant Staphylococcus aureus and Escherichia coli

Increased prevalence of antibiotic-resistant bacteria has become a major threat to the health sector worldwide due to their virulence, limited therapeutic options and distribution in both hospital and community settings. Discovery and development of new agents to combat antibiotic-resistant bacteria is thus needed. This study therefore aimed to evaluate the ability of bovine lactoferrin (LF), peptides from two antimicrobial domains lactoferricin B (LFcin17-30) and lactoferrampin (LFampin265-284) and a chimeric construct (LFchimera) containing both peptides, as potential bactericidal agents against clinical isolates of antibiotic-resistant Staphylococcus aureus and Escherichia coli. Results in kinetics of growth show that LF chimera and peptides inhibited the growth of both bacterial species. By confocal microscopy and flow cytometry it was observed that LF and FITC-labeled peptides are able to interact with these bacteria and cause membrane permeabilization, as monitored by propidium iodide staining, these effects were decreased by preincubation with lipopolysaccharide in E. coli. By electron microscopy, a clear cellular damage was observed in bacteria after treatments with LFchimera and peptides, suggesting that interaction and membrane disruption are probably involved as a mechanism of action. In conclusion, results show that LFchimera, LF and peptides have potential as bactericidal agents in the antibiotic-resistant strains of S. aureus and E. coli and also the work strongly suggest that LFcin17-30 and LFampin265-284 acts synergistically with antibiotics against multidrug resistant EPEC and MRSA in vitro.

Microbicidal effect of the lactoferrin peptides Lactoferricin17–30, Lactoferrampin265–284, and Lactoferrin chimera on the parasite Entamoeba histolytica

Entamoeba histolytica is a parasitic protozoan that produces amoebiasis, an intestinal disease characterized by ulcerative colitis and dysentery. In some cases, trophozoites can travel to the liver leading to hepatic abscesses and death. Recently, lactoferrin and lactoferricin B have been shown to be amoebicidal in axenic cultures. The aim of this work was to determine whether the lactoferrin-peptides lactoferricin amino acids 17-30, lactoferrampin amino acids 265-284, and lactoferrin chimera which is a fusion product of the two peptides, are capable of producing a microbicidal effect to trophozoites of E. histolytica. We evaluated the killing effect of these peptides in growth kinetics carried out in axenic culture medium to which different concentrations of peptides were added. At 50 muM of peptide concentration, lactoferricin and lactoferrampin had a moderate amoebicidal effect, since a 45-50% of trophozoites remained viable at 24 h culture. However, at 50 microM of the lactoferrin chimera 75% amoeba were killed whereas at 100 microM all cells died. These data indicate that of lactoferrin-peptides mainly the chimera have amoebicidal activity in a time- and concentration-dependent manner. The lactoferrin-peptides might be useful as therapeutic agents against amoebiasis and thereby diminish the use of metronidazole, which is extremely toxic for the host.

Bactericidal effect of lactoferrin and lactoferrin chimera against halophilic Vibrio parahaemolyticus

Infections caused by Vibrio parahaemolyticus, an halophilic member of the genus Vibrio, have increased globally in the last 5 years. Diarrhea caused by V. parahaemolyticus results from eating raw or undercooked seafood. The aim of this work was to investigate whether lactoferrin and some lactoferrin-peptides have bactericidal activity against Vibrio parahaemolyticus ATCC 17802, the pandemic strain O3:K6, and the multidrug resistant isolate 727, as well as against Vibrio cholerae strains O1 and non-O1. Whereas both peptides lactoferricin (17–30) and lactoferrampin (265–284) did not have bactericidal activity, 40 μM of lactoferrin chimera (a fusion of the two peptides) inhibited the growth of all Vibrio tested to the same extent as the antibiotic gentamicin. The cidal effect of LFchimera showed a clear concentration response in contrast to bovine lactoferrin which showed higher inhibition at 10 μM than at 40 μM. FITC-labeled LFchimera bound to the bacterial membranes. Moreover LFchimera permeabilized bacterial cells and membranes were seriously damaged. Finally, in experiments with the multidrug resistant isolate 727, sub-lethal doses of LFchimera strongly reduced the concentrations of ampicillin, gentamicin or kanamicin needed to reach more than 95% growth inhibition, suggesting synergistic effects. These data indicate that LFchimera is a potential candidate to combat the multidrug resistant pathogenic Vibrio species.