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Biophysical Characterization of Lipopolysaccharide and Lipid A Inactivation by Lactoferrin
Biological Chemistry
Abstract
The interaction of bacterial endotoxins (LPS Re and lipid A, the 'endotoxic principle' of LPS) with the endogenous antibiotic lactoferrin (LF) was investigated using various physical techniques and biological assays. By applying Fourier-transform infrared (FTIR) spectroscopy, we find that LF binds to the phosphate group within the lipid A part and induces a rigidification of the acyl chains of LPS. The secondary structure of the protein - as monitored by the amide I band - is, however, not changed. Concomitant with the IR data, scanning calorimetric data indicate a sharpening of the acyl chain phase transition. From titration calorimetric and zeta potential data, saturation of LF binding to LPS was found to lie at a [LF]:[LPS] ratio of 1:3 to 1:5 M from the former and 1:10 M from the latter technique. X-ray scattering data indicate a change of the lipid A aggregate structure from inverted cubic to multilamellar, and with fluorescence (FRET) spectroscopy, LF is shown to intercalate by itself into phospholipid liposomes and may also block the lipopolysaccharide-binding protein (LBP)-induced intercalation of LPS. The LPS-induced cytokine production of human mononuclear cells exhibits a decrease due to LF binding, whereas the coagulation of amebocyte lysate in the Limulus test exhibited concentration-dependent changes. Based on these results, a model for the mechanisms of endotoxin inactivation by LF is proposed.
... Beside its ability as metal binding proteins, lactoferrin binds to various types of mammalian cells, including neutrophils, macrophages (monocytes), natural killer (NK) cells, platelets, enterocytes, hepatocytes, dendritic cells, fibroblasts and osteoblasts [7][8][9], and regulate their migration, growth and differentiation. Lactoferrin is known as highly basic protein, and binds anionic molecules such as DNA, heparin, glycosaminoglycans, and endotoxins [10][11][12]. The multi-functionality of lactoferrin could be explained by diversity of target cells and molecules. ...
... LPS is a major structural component in the outer membrane of the Gram-negative bacteria, and is associated with their pathogenic capability. Lactoferrin binds to the lipid A of LPS with high affinity [10,11]. The lactoferrin-mediated LPS release results in osmotic destruction of the outer membrane, and bacterial death [77,78]. ...
... mCD14 enables LPS to be transferred to the LPS receptor complex, which is composed of Toll-like receptor 4 (TLR4) and MD-2 and induces the release of the pro-inflammatory cytokines [176]. As described above, lactoferrin interacts with lipid A part of LPS with high affinity, and inhibits bindings of LPS/LBP complex to mCD14, and antagonizes the production of pro-inflammatory cytokines [10,11]. Endothelial and epithelial cells do not express mCD14. ...
Lactoferrin is an iron-binding glycoprotein that belongs to the transferrin family. It consists of two globular domains, called N-lobe and C-lobe. Both lobes have the similar polypeptide-folding pattern and one metal-binding site. Its structure is conserved among species. As well as other members of transferrin family, lactoferrin exerts their biological functions by chelating free irons in body fluids in mammals. Lactoferrin acts as bacteriostatic agent, based on their ability to deprive iron essential for bacteria growth. Besides, its iron-chelating activity, lactoferrin positively or negatively regulates host immune response by controlling maturation, migration, cytokine secretion of innate and adaptive immune cells. Lactoferrin is detected in many body fluids, and contributes host defense through the bactericidal and immuno-modulating activities. The difference of the lactoferrin expression among the different species and tissue could be explained by the diversity of lactoferrin gene promoter region.
... Independently from its iron-chelation ability, bLf is able to exert a bactericidal activity through the perturbation of bacterial membranes of both Gram-negative and Gram-positive bacteria via the binding to lipopolysaccharides (LPS) or lipoteichoic acid, respectively . In Gram-negative bacteria, cellular lysis is performed through the direct interaction of bLf with LPS (Appelmelk et al. 1994;Brandenburg et al. 2001) and bLf ability to sequester Ca 2+ thus inducing the LPS release (Rossi et al. 2002). In Gram-positive bacteria, the bactericidal activity of bLf is mediated by electrostatic interactions between the negatively charged of bacterial lipid layer and the positively charged of bLf surface, causing changes in the membrane permeability . ...
... Further studies demonstrated that, beyond its iron-binding capabilities, Lf has bactericidal effects because provokes the lysis of several pathogens through its interaction with the LPS of Gram-negative bacteria (Appelmelk et al. 1994;Brandenburg et al. 2001;Elass et al. 2002;Rosa et al. 2017) and the lipoteichoic acid of Gram-positive bacteria Lu et al. 2021). Moreover, bLf is also able to interact with glycosaminoglycans (GAGs) or heparan sulfate proteoglycans (HSPGs), anionic components of host cells (Hu et al. 2021), and with anionic surface structures of facultative intracellular pathogenic bacteria thus hindering bacterial adhesion and entry (Valenti and Antonini 2005 and references therein). ...
Uropathogenic Escherichia coli (UPEC) strains are the primary cause of urinary tract infections (UTIs). UPEC strains are able to invade, multiply and persisting in host cells. Therefore, UPEC strains are associated to recurrent UTIs requiring long‐term antibiotic therapy. However, this therapy is suboptimal due to the increase of multidrug-resistant UPEC. The use of non-antibiotic treatments for managing UTIs is required. Among these, bovine lactoferrin (bLf), a multifunctional cationic glycoprotein, could be a promising tool because inhibits the entry into the host cells of several intracellular bacteria. Here, we demonstrate that 100 μg/ml bLf hinders the invasion of 2.0 ± 0.5 × 10⁴ CFU/ml E. coli CFT073, prototype of UPEC, infecting 2.0 ± 0.5 × 10⁵ cells/ml urinary bladder T24 epithelial cells. The highest protection (100%) is due to the bLf binding with host surface components even if an additional binding to bacterial surface components cannot be excluded. Of note, in the absence of bLf, UPEC survives and multiplies, while bLf significantly decreases bacterial intracellular survival. After these encouraging results, an observational survey on thirty-three patients affected by recurrent cystitis was performed. The treatment consisted in the oral administration of bLf alone or in combination with antibiotics and/or probiotics. After the observation period, a marked reduction of cystitis episodes was observed (p < 0.001) in all patients compared to the episodes occurred during the 6 months preceding the bLf-treatment. Twenty-nine patients did not report cystitis episodes (87.9%) whereas the remaining four (12.1%) experienced only one episode, indicating that bLf could be a worthwhile and safe treatment in counteracting recurrent cystitis.
... In previous reports, human lactoferrin (LF) was shown to represent a natural defense protein [35]. Recent studies with the recombinant talactoferrin, however, failed to protect septic patients in a clinical study [36]. ...
... A schematic of the processes leading to a neutralization of endotoxin is presented in Figure 12. In this figure it is shown that the bioactive aggregate structure of LPS has a unilamellar/non-lamellar cubic form, whereas in the presence of suitable AMP, but also sequestrants, the LPS aggregate structure is converted into a multilamellar form [3,[14][15][16]24,29,35,[37][38][39]45,47,49,56]. In this aggregate form, the recognition structures of the immune cells, the TLR4-/MD2 system cannot bind to the lipid A backbone. ...
Introduction
Gram-negative bacterial infections represent still a severe problem of human health care, regarding the increase in multi-resistance against classical antibiotics and the lack of newly developed antimicrobials. For the fight against these germs, anti-infective agents must overcome and/or bind to the Gram-negative outer membrane consisting of a lipopolysaccharide (LPS, endotoxin) outer leaflet and an inner leaflet from phospholipids, with additional peripheral or integral membrane proteins (OMP’s).
Areas covered
The current article reviews data of existing therapeutic options and summarizes newer approaches for targeting and neutralizing endotoxins, ranging from in vitro over in vivo animal data to clinical applications by using databases such as Medline.
Expert opinion
Conventional antibiotic treatment of the bacteria leads to their killing, but not necessary LPS neutralization, which may be a severe problem in particular for the systemic pathway. This is the reason why there is an increasing number of therapeutic approaches, which – besides combating whole bacteria – at the same time try to neutralize endotoxin within or outside the bacterial cells mainly responsible for the high inflammation induction in Gram-negative species.
... Lactoferrin shows anti-microbial activity including the inhibition of bacterial adhesion to host cells and colonization of the bronchial epithelium. In addition, it shows an important anti-inflammatory activity in several chronic inflammatory diseases including COPD [197][198][199][200][201][202][203][204][205][206][207][208][209][210][211][212][213][214]. ...
... However, lactoferrin also exerts an ironindependent bactericidal action by interacting directly with LPS from the cellular membrane of Gramnegative bacteria or with LTA from Gram-positive bacteria [209,211]. The anti-microbial activity of lactoferrin is mediated by its N-terminal region, as the pepsindigested cationic peptide exhibits more potency against Gram-negative bacteria [209,212]. ...
Chronic obstructive pulmonary disease (COPD) represents a heightened inflammatory
response in the lung generally resulting from tobacco smoking-induced recruitment and
activation of inflammatory cells and/or activation of lower airway structural cells. Several
mediators can modulate activation and recruitment of these cells, particularly those belonging
to the chemokines (conventional and atypical) family. There is emerging evidence for complex
roles of atypical chemokines and their receptors [such as high mobility group box 1
(HMGB1), antimicrobial peptides, receptor for advanced glycosylation end products (RAGE)
or toll-like receptors (TLRs)] in the pathogenesis of COPD, both in the stable disease and during
exacerbations. Modulators of these pathways represent potential novel therapies for
COPD and many are now in preclinical development. Inhibition of only a single atypical
chemokine or receptor may not block inflammatory processes because there is redundancy in
this network. However, there are many animal studies that encourage studies for modulating
the atypical chemokine network in COPD. Thus, few pharmaceutical companies maintain a
significant interest in developing agents that target these molecules as potential antiinflammatory
drugs. Antibody-based (biological) and small molecule drug (SMD)-based
therapies targeting atypical chemokines and/or their receptors are mostly at the preclinical
stage and their progression to clinical trials is eagerly awaited. These agents will most likely
enhance our knowledge about the role of atypical chemokines in COPD pathophysiology and
thereby improve COPD management.
... To neutralize endotoxins, the binding of peptides to the LPS molecule alone is not sufficient [28]. Studies from other groups have shown that for certain cationic AMPs, a fatty acid substitution of ≥ C8 is necessary for efficient LPS neutralization [29,30]. For this reason, only lipid acylations ≥ C8 were selected for this study. ...
... To neutralize endotoxins, the binding of peptides to the LPS molecule alone is not sufficient [28]. Studies from other groups have shown that for certain cationic AMPs, a fatty acid substitution of ≥ C 8 is necessary for efficient LPS neutralization [29,30]. For this reason, only lipid acylations ≥ C 8 were selected for this study. ...
Acylation of antimicrobial peptides mimics the structure of the natural lipopeptide polymyxin B, and increases antimicrobial and endotoxin-neutralizing activities. In this study, the antimicrobial properties of lactoferrin-based LF11 peptides as well as blood compatibility as a function of acyl chain length were investigated. Beyond the classical hemolysis test, the biocompatibility was determined with human leukocytes and platelets, and the influence of antimicrobial peptides (AMPs) on the plasmatic coagulation and the complement system was investigated. The results of this study show that the acylation of cationic peptides significantly reduces blood tolerance. With increasing acyl chain length, the cytotoxicity of LF11 peptides to human blood cells also increased. This study also shows that acylated cationic antimicrobial peptides are inactivated by the presence of heparin. In addition, it could be shown that the immobilization of LF11 peptides leads to a loss of their antimicrobial properties.
... An iron-independent bactericidal action is exerted by Lf direct interaction with the lipopolysaccharide (LPS) of Gram-negative or with the lipoteichoic acid of Gram-positive bacteria [73,74]. The bactericidal activity of Lf is located in the N-terminal region ( Figure 5), as its derivative cationic peptide, generated by pepsin digestion, called lactoferricin (Lfcin), is several folds ...
... An iron-independent bactericidal action is exerted by Lf direct interaction with the lipopolysaccharide (LPS) of Gram-negative or with the lipoteichoic acid of Gram-positive bacteria [73,74]. The bactericidal activity of Lf is located in the N-terminal region ( Figure 5), as its derivative cationic peptide, generated by pepsin digestion, called lactoferricin (Lfcin), is several folds more active than the intact protein in interacting with LPS and in killing Gram-negative bacteria [75,76]. ...
Human lactoferrin (hLf), an iron-binding multifunctional cationic glycoprotein secreted by exocrine glands and by neutrophils, is a key element of host defenses. HLf and bovine Lf (bLf), possessing high sequence homology and identical functions, inhibit bacterial growth and biofilm dependently from iron binding ability while, independently, bacterial adhesion to and the entry into cells. In infected/inflamed host cells, bLf exerts an anti-inflammatory activity against interleukin-6 (IL-6), thus up-regulating ferroportin (Fpn) and transferrin receptor 1 (TfR1) and down-regulating ferritin (Ftn), pivotal actors of iron and inflammatory homeostasis (IIH). Consequently, bLf inhibits intracellular iron overload, an unsafe condition enhancing in vivo susceptibility to infections, as well as anemia of inflammation (AI), re-establishing IIH. In pregnant women, affected by AI, bLf oral administration decreases IL-6 and increases hematological parameters. This surprising effect is unrelated to iron supplementation by bLf (80 μg instead of 1–2 mg/day), but to its role on IIH. AI is unrelated to the lack of iron, but to iron delocalization: cellular/tissue overload and blood deficiency. BLf cures AI by restoring iron from cells to blood through Fpn up-expression. Indeed, anti-inflammatory activity of oral and intravaginal bLf prevents preterm delivery. Promising bLf treatments can prevent/cure transitory inflammation/anemia/oral pathologies in athletes.
... The interaction of lactoferrin (LF), an important human defense protein, with endotoxins studied by ITC at 37 °C is clearly indicative of an exothermic for LPS and an endothermic process for lipid A, respectively [53], which can be understood by the phase specificity of the binding heat as described above. ...
... This could be proven by the analysis of the LPS head group regions with FTIR, showing strongly changed vibrational bands for the LPS from sensitive strain R595, but nearly no changes in the bands from the head group region of the LPS from the resistant strain Re45.The interaction of various endotoxin-binding proteins with LPS and lipid A was investigated by Brandenburg et al[50][51][52][53]. To this class belong albumin, lactoferrin, hemoglobin, and high-density lipoprotein (HDL). ...
... The secondary structure of the protein, however, was not changed. 88 Bellamy et al. 89 reported that proteolytic digestion of LF with gastric pepsin yields several peptides. A peptide from the N-terminal region, consisting of a loop of 18 amino acid residues formed by a disulphide bond between cysteine residues 20 and 37, is called human lactoferricin (hLFcin) and shows a marked bactericidal activity. ...
Lactoferrin is an iron-binding glycoprotein present in various secretions (e.g. milk, tears, saliva, pancreatic juice, etc.). It is also stored in specific granules of polymorphonuclear granulocytes from which it is released following activation. Lactoferrin exerts a bactericidal activity by damagingthe outer membrane of Gram-negative bacteria, as well as immunoregulatory functions by decreasing the release of interleukin-1 (IL-1), IL-2 and tumor necrosis factor- (TNF-) and enhancing monocyte and natural killer cell cytotoxicity. Lactoferrin binds with high affinity to lipid A, the toxic moiety of the lipopolysaccharide, or endotoxin from Gram-negative bacteria. Lipopolysaccharide interaction with monocytes/macrophages results in the production and release of TNF- , that plays an important role in inducing septic shock. In this respect, it has recently been demonstrated that lactoferrin inhibits the lipopolysaccharide interaction with CD14 on monocytes/macrophages by competition with the lipopolysaccharide binding protein. Therefore, besides its bactericidal activity, lactoferrin may also act by neutralizing the toxic effects of lipopolysaccharide and this protective role against endotoxin lethal shock has been demonstrated in animal models. Moreover, in vitro and in vivo neutralization of endotoxin by a human lactoferrin-derived peptide was also reported and lactoferrin or lactoferrin-derived peptides could represent useful tools for the treatment of endotoxin-induced septic shock. The recent production and characterization of monoclonal antibodies against different epitopes of human lactoferrin, including monoclonal antibodies selectively neutralizinglactoferrin binding to lipid A, may allow a better elucidation of the consequence of lactoferrin-lipopolysaccharideinteraction.
... In this context, it was found that the antiviral activity of lactoferrin makes it a potential immunity enhancer which could be administered along with conventional antivirals [44]. Interestingly, this compound shows anti-SARS-CoV2 activity by itself [45], which seems to be mechanistically independent from its antibacterial and LPS-binding activities [46]. On the other hand, Sohn et al. (2020) [47] discovered that drugs that have been described as inhibitors of the LPS-induced cytokine storm such as the polypeptide Aspidasep (Pep19-2.5) ...
Sepsis is a life-threatening condition caused by the body’s overwhelming response to an infection, such as pneumonia or urinary tract infection. It occurs when the immune system releases cytokines into the bloodstream, triggering widespread inflammation. If not treated, it can lead to organ failure and death. Unfortunately, sepsis has a high mortality rate, with studies reporting rates ranging from 20% to over 50%, depending on the severity and promptness of treatment. According to the World Health Organization (WHO), the annual death toll in the world is about 11 million. One of the main toxins responsible for inflammation induction are lipopolysaccharides (LPS, endotoxin) from Gram-negative bacteria, which rank among the most potent immunostimulants found in nature. Antibiotics are consistently prescribed as a part of anti-sepsis-therapy. However, antibiotic therapy (i) is increasingly ineffective due to resistance development and (ii) most antibiotics are unable to bind and neutralize LPS, a prerequisite to inhibit the interaction of endotoxin with its cellular receptor complex, namely Toll-like receptor 4 (TLR4)/MD-2, responsible for the intracellular cascade leading to pro-inflammatory cytokine secretion. The pandemic virus SARS-CoV-2 has infected hundreds of millions of humans worldwide since its emergence in 2019. The COVID-19 (Coronavirus disease-19) caused by this virus is associated with high lethality, particularly for elderly and immunocompromised people. As of August 2023, nearly 7 million deaths were reported worldwide due to this disease. According to some reported studies, upregulation of TLR4 and the subsequent inflammatory signaling detected in COVID-19 patients “mimics bacterial sepsis”. Furthermore, the immune response to SARS-CoV-2 was described by others as “mirror image of sepsis”. Similarly, the cytokine profile in sera from severe COVID-19 patients was very similar to those suffering from the acute respiratory distress syndrome (ARDS) and sepsis. Finally, the severe COVID-19 infection is frequently accompanied by bacterial co-infections, as well as by the presence of significant LPS concentrations. In the present review, we will analyze similarities and differences between COVID-19 and sepsis at the pathophysiological, epidemiological, and molecular levels.
... It also reduced the levels of TNF-α and IL-10 and protein expression of NF-κBp65, COX-2 and iNOS. The interaction of lactoferrin with lipopolysaccharide (LPS) is linked to its role in downregulation of pro-inflammatory cytokines expression through its Lfc domain [94][95][96]. Of quite interest, it has been shown that Lfc on its own can also neutralize the action of LPS [97,98]. ...
Lactoferrin, a pleiotropic and multivalent natural protein, derived from bovine and camel milk has become the center of attention in current scientific arena due to its divers immune-modulation and inflammation related properties. Lactoferrin has immense role in host immune defenses as several important immune cells have surface receptors specific for lactoferrin. It has been studied at a great depth for its contribution to immune system as covered in this chapter at the interface between innate and adaptive defenses. The medicinal and biological benefits of lactoferrin are due to its diverse chemical structure. Furthermore, the anti and pro inflammation characteristics of the molecule makes it of utmost interest in medical and therapeutic field. Lactoferrin has the potential to serve as a clinical marker in a number to inflammation related maladies and can be used as a treatment option in oxidative stress mediated inflammation related disorders and for harmful immune allergies. Future research on lactoferrin can not only present it as a prognostic or diagnostic biomarker but also as a remedial solution to cure inflammation related disorders.
... Lactoferrin's antibacterial activity on Gram-positive bacteria is attributed to its binding to iron, leading to the inhibition of bacterial growth via restriction of the avail- ability of iron as a nutrient for bacteria, and to its effects on lipoteichoic and teichoic acids, which leads to depolarization and disruption of bacterial membranes than on the cytoplasmic contents (Brandenburg et al., 2001;Orsi, 2004;Liu et al., 2011). ...
Background: Lactoferrin is a natural biological active cationic protein that can be used as a yogurt additive to inhibit the growth of foodborne pathogens. Objectives: The present study evaluated the antimicrobial effects of lactoferrin against Bacillus cereus, Enterococcus faecalis, and Candida albicans inoculated in laboratory prepared yogurt at refrigerator temperature. Methods: The pre-warmed skimmed milk was inoculated by commercial starter and then divided into 3 parts, each inoculated by different inoculum and a certain concentration of lactoferrin (0.5% and 1.5%) and incubated at 42oC for 4 h till curd formation. Tenfold serial dilutions were performed for each group and refrigerated at4±1oC for up to 14 days to be examined every day. Results: The obtained results showed that the treated yogurt samples with lactoferrin had significant reductions in B. cereus, Ent. faecalis, and C. albicans counts than untreated samples (control positive). Generally, yogurt samples treated with 1.5% lactoferrin showed the highest reduction percentages on B. cereus and C. albicans than 0.5% lactoferrin. In addition, B. cereus showed more susceptibility to lactoferrin than Ent. faecalis; the maximum reduction of the inoculated B. cereus was observed on the sixth day of the incubation to be 99.99%. Maximum reduction of the tested C. albicans was observed after the seventh day. Conclusion: The application of lactoferrin showed a potentially significant antimicrobial effect against B. cereus, Ent. faecalis, and C. albicans in refrigerating conditions, so lactoferrin is recommended to be used in yogurt production for safe product manufacturing.
... Alteration to the outer membrane permeability results in the release of LPS and consequent damage to the bacteria [59]. Lactoferrin, by affecting LPS or other surface proteins, triggers additional bacterial effect and potentiates activity of natural antibacterial such as lysozyme [60]. ...
Pregnancy is a period which requires special care and attention. Maintaining health during pregnancy helps to avoid birth related complications and is the best way of promoting a healthy birth. Besides a daily intake of folic acid, iron, iodine, vitamin D3 and A, calcium and polyunsaturated fatty-acids, as recommended by health agencies, supplementation of lactoferrin - a protein of multidirectional biological activity and proven safety of use - seems to be beneficial. A wide range of lactoferrin biological roles (including regulation of iron balance, modulation of immune responses, antimicrobial, antiviral, antioxidant, and anti-inflammatory activity) may contribute to better pregnancy and birth related outcomes.
... It would even seem that LF could stimulate the proliferation of probiotic bacteria [63]. The exploration of the LF peptide mechanisms of action for the antibacterial effects mainly revealed (i) the sequestration of bacteria and their deprivation in iron (essential for their survival) by LF given its ironbinding ability [64]; (ii) the potential of LF in interacting with LPS of gram-negative bacteria, thus considerably hampering their growth [8,65,66]; (iii) the LF competency of binding with receptors on microorganisms to obstruct the transport of nutrients, thereby restraining bacterial synthesis and metabolism [67,68]; (iv) the inhibition of microbial enzyme activity by LF [67]; (v) the LF-mediated prevention of biofilm formation by bacteria [69]; (vi) the capacity of LF to release antibacterial peptide lactoferricin and other bioactive peptides following hydrolysis by pepsin [70][71][72]; and (vii) the direct antimicrobial activities exerted by LF [73]. It should be noted that the impact of LF is not limited to bacteria, but can also extend to antivirals [74][75][76], antiprotozoal [77], and antifungal [78] activities. ...
Milk-derived bioactive proteins have increasingly gained attention and consideration throughout the world due to their high-quality amino acids and multiple health-promoting attributes. Apparently, being at the forefront of functional foods, these bioactive proteins are also suggested as potential alternatives for the management of various complex diseases. In this review, we will focus on lactoferrin (LF) and osteopontin (OPN), two multifunctional dairy proteins, as well as to their naturally occurring bioactive LF–OPN complex. While describing their wide variety of physiological, biochemical, and nutritional functionalities, we will emphasize their specific roles in the perinatal period. Afterwards, we will evaluate their ability to control oxidative stress, inflammation, gut mucosal barrier, and intestinal microbiota in link with cardiometabolic disorders (CMD) (obesity, insulin resistance, dyslipidemia, and hypertension) and associated complications (diabetes and atherosclerosis). This review will not only attempt to highlight the mechanisms of action, but it will critically discuss the potential therapeutic applications of the underlined bioactive proteins in CMD.
... Several mechanisms of the antimicrobial activity of lactoferrin have been described. Mechanisms that have long been known include its iron-binding ability that results in sequestrating and depriving bacteria of iron required for their growth, as well as its ability to interact with lipopolysaccharide of Gram-negative bacteria thus impeding their growth [96,97]. More recent studies have shown that lactoferrin can eliminate biofilms formed by potential pathogens, preventing interactions between microbes and the gut epithelium [98,99]. ...
Early-life gut microbiota plays a role in determining the health and risk of developing diseases in later life. Various perinatal factors have been shown to contribute to the development and establishment of infant gut microbiota. One of the important factors influencing the infant gut microbial colonization and composition is the mode of infant feeding. While infant formula milk has been designed to resemble human milk as much as possible, the gut microbiome of infants who receive formula milk differs from that of infants who are fed human milk. A diverse microbial population in human milk and the microbes seed the infant gut microbiome. Human milk contains nutritional components that promote infant growth and bioactive components, such as human milk oligosaccharides, lactoferrin, and immunoglobulins, which contribute to immunological development. In an attempt to encourage the formation of a healthy gut microbiome comparable to that of a breastfed infant, manufacturers often supplement infant formula with prebiotics or probiotics, which are known to have a bifidogenic effect and can modulate the immune system. This review aims to elucidate the roles of human milk and formula milk on infants’ gut and health.
... Aerosolized bovine Lf was reducing pulmonary bacterial load, pulmonary iron overload, and infiltrating leukocytes in a cystic fibrosis mouse model of Pseudomonas aeruginosa chronic lung infection (Cutone et al. 2019). On the other hand, Lf exerted its anti-inflammation role by direct binding with the LPS of Gram-negative bacteria (Brandenburg, et al. 2001). Lf may prevent injuryinduced oxidative stress and subsequent 'cytokine storm', hence, it has the potential to reduce SARS-CoV-2-induced cytokine storm (Zimecki, et al. 2021), and was proposed for a clinical trial to evaluate and verify its effect in COVID-19 pandemic (Campione et al. 2020). ...
Lactoferrin (Lf), a multiple functional natural immune protein, is widely distributed in mammalian milk and glandular secretions (bile, saliva, tears and nasal mucosal secretions, etc.). In the previous study, we found that Lf plays an anti-inflammatory and anti-tumorigenesis role in AOM/DSS (azoxymethane/dextran sulfate sodium) induced mouse colitis-associated colon cancer model. Although we found that Lf has anti-inflammatory effects in chronic inflammation, its specific role and mechanisms in acute inflammation have not been clarified. Here, we reported that the expression levels of Lf were significantly increased when the organism was infected by Gram-negative bacteria. We then explored the role and potential mechanism of Lf in lipopolysaccharide (LPS)-induced acute inflammation. In the LPS-induced acute abdominal inflammation model, Lf deficiency aggravated inflammatory response and promoted macrophage chemotaxis to the inflammation site. Lf inhibited macrophage chemotaxis by suppressing the expression of macrophage-associated chemokines Ccl2 and Ccl5. Highly activated NF-κB signaling in Lf−/− mice was responsible for the high expression of Ccl2 and Ccl5. Our results suggested that the anti-inflammatory effect of Lf offers a new potential treatment for acute inflammatory diseases.
... Lactoferrin has a bacteriostatic effect through iron-binding, leading to iron deprivation of the microorganisms. In addition, LF affects bacteria directly by destabilizing the outer membrane of Gram-negative bacteria through binding of bacterial LPS [25][26][27][28]. Moreover, LF can cause selective permeation of ions through the inner membrane [29], and it interferes with bacterial adherence on host cells through disruption of the bacterial Type III secretion system (TTSS) [24,30,31]. ...
Chlamydia suis (C. suis) resides in the intestines of pigs and tetracycline-resistant strains are emerging worldwide. Intestinal infections are often subclinical. However, the gut is regarded as a C. suis reservoir and clinical infections have been associated with enteritis, conjunctivitis, pneumonia and reproductive failure. C. suis was found in boar semen and venereal transmission occurred. We studied the anti-Chlamydia suis activity of ovotransferrin (ovoTF) and bovine lactoferrin (bLF). Pre-incubation of C. suis with bLF or ovoTF had no significant effect on overall chlamydia replication (mean fluorescence area) in McCoy cells. The addition of ovoTF to the culture medium had no effect on bacterial replication, but the addition of 0.5 or 5 mg/mL of bLF significantly reduced the inclusion size by 17% and 15% respectively. Egg components are used for cryopreservation of boar semen. When inoculating an ovoTF-containing and Chlamydia suis-spiked semen sample in McCoy cells, a significant reduction in inclusion number (by 7%) and overall replication (by 11%) was observed. Thus, we showed that transferrins possess anti-chlamydial activity. Moreover, ovoTF addition to semen extenders might reduce C. suis venereal transmission. Further research is needed to unravel the mechanisms behind the observations and to enhance the effect of transferrins on C. suis.
... It has been established that the lipid A component of the LPS is a known drug target for antimicrobial therapeutics (Morrison et al., 1976;Cohen et al., 1992;Cutone et al., 2014). One of the mechanisms by which LF acts as an antimicrobial agent is through binding to pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS), thereby disrupting the bacterial membrane integrity and activating the chemical signaling pathway (Ellison et al., 1988;Appelmelk et al., 1994;Brandenburg et al., 2001). This leads to the secretion of pro-inflammatory responses that downregulates the release of cytokine production (Farnaud and Evans, 2004;Legrand and Mazurier, 2010). ...
The problem of antibiotic resistance has prompted researchers around the globe to search for new antimicrobial agents. Antimicrobial proteins and peptides are naturally secreted by almost all the living organisms to fight infections and can be safer alternatives to chemical antibiotics. Lactoferrin (LF) is a known antimicrobial protein present in all body secretions. In this study, LF was digested by trypsin, and the resulting hydrolysates were studied with respect to their antimicrobial properties. Among the hydrolysates, a 21-kDa basic fragment of LF (termed lactosmart) showed promise as a new potent antimicrobial agent. The antimicrobial studies were performed on various microorganisms including Shigella flexneri, Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli as well as fungal pathogens such as Candida albicans, Candida tropicalis, and Candida glabrata. In addition, the lipopolysaccharide (LPS)-binding properties of lactosmart were studied using surface plasmon resonance technique in vitro, along with docking of LPS and molecular dynamics (MD) simulation studies. The results showed that lactosmart had better inhibitory effects against pathogenic microorganisms compared to LF. The results of docking and MD simulation studies further validated the tighter binding of LPS to lactosmart compared to LF. The two LPS-binding sites have been characterized structurally in detail. Through these studies, it has been demonstrated that in native LF, only one LPS-binding site remains exposed due to its location being on the surface of the molecule. However, due to the generation of the lactosmart molecule, the second LPS-binding site gets exposed too. Since LPS is an essential and conserved part of the bacterial cell wall, the pro-inflammatory response in the human body caused by LPS can be targeted using the newly identified lactosmart. These findings highlight the immense potential of lactosmart in comparison to native LF in antimicrobial defense. We propose that lactosmart can be further developed as an antibacterial, antifungal, and antibiofilm agent.
... LF achieves this by disrupting binding of LPS to the Toll-like receptor 4/myeloid differentiation protein 2 (TLR4-MD-2) PRR complex on cell surfaces and thereby cease the intracellular signalling cascade causing NF-κB activation (da Silva Correia, Soldau, Christen, Tobias, & Ulevitch, 2001). This action by LF could be achieved by electrostatic binding to the toxic lipid A moiety of LPS, to sCD14, to LPS-binding protein, and/or to the LPS/sCD14 complex (Appelmelk et al., 1994;Baveye et al., 2000;Brandenburg, Jürgens, Müller, Fukuoka, & Koch, 2001;Drago-Serrano, de La Garza-Amaya, Luna, & Campos-Rodríguez, 2012;Elass-Rochard et al., 1998). Increased LF inhibition of LPS-mediated activation of the NF-κB pathway in the present study (Fig. 4A) occurred when the LF: LPS molecular concentration became greater, suggesting increased binding events. ...
Lactoferrin (LF) is a multifunctional glycoprotein which, when thermally processed, undergoes significant physicochemical changes. The link between such changes and the bioactivity of LF is not well characterised and requires much research. In this work, bovine LF solutions (1%, w/v, protein, pH 7) were thermally processed using high temperature short time conditions (72, 80, 85 or 95°C with 15 s holding times). Following this, it was shown that LF and heat induced LF aggregates were largely resistant to simulated infant gastric, but not intestinal, digestion. Also, the efficacy of LF bactericidal activity, and inhibition of lipopolysaccharide-induced NF-κB activation were negatively impacted by thermal processing. This study confirmed that the efficacy of LF bio-functionalities was affected by the extent of heat-induced changes in protein structure whereby processing conditions of least severity (i.e. pasteurisation) had the least impact on bioactivity.
... In Gram-positive bacteria, the bactericidal activity of Lf is mediated by electrostatic interactions between the negatively charged lipid layer and the positively charged Lf surface, causing changes in the membrane permeability [20]. In Gram-negative bacteria, cellular lysis is performed through (i) direct interaction of Lf with lipopolysaccharide (LPS) [185][186][187] and (ii) Lf ability to sequester Ca(II) thus inducing the release of LPS [188]. Moreover, independent of iron-chelation, Lf is also able to inhibit: (i) bacterial adhesion and invasion through its competitive binding with surface components of host cells and/or bacteria, thus decreasing bacterial-host cell interaction and bacterial internalization [34,[189][190][191][192], (ii) bacterial intracellular survival through still unknown mechanisms [34,180] and (iii) biofilm formation [193]. ...
The connection between inflammation and cancer is well-established and supported by genetic, pharmacological and epidemiological data. The inflammatory bowel diseases (IBDs), including Crohn’s disease and ulcerative colitis, have been described as important promoters for colorectal cancer development. Risk factors include environmental and food-borne mutagens, dysbalance of intestinal microbiome composition and chronic intestinal inflammation, with loss of intestinal epithelial barrier and enhanced cell proliferation rate. Therapies aimed at shutting down mucosal inflammatory response represent the foundation for IBDs treatment. However, when applied for long periods, they can alter the immune system and promote microbiome dysbiosis and carcinogenesis. Therefore, it is imperative to find new safe substances acting as both potent anti-inflammatory and anti-pathogen agents. Lactoferrin (Lf), an iron-binding glycoprotein essential in innate immunity, is generally recognized as safe and used as food supplement due to its multifunctionality. Lf possesses a wide range of immunomodulatory and anti-inflammatory properties against different aseptic and septic inflammatory pathologies, including IBDs. Moreover, Lf exerts anti-adhesive, anti-invasive and anti-survival activities against several microbial pathogens that colonize intestinal mucosa of IBDs patients. This review focuses on those activities of Lf potentially useful for the prevention/treatment of intestinal inflammatory pathologies associated with colorectal cancer development.
... Due to its ability to bind iron, lactoferrin does not allow it to be used by microorganisms, thus inhibiting their development and exerting a bacteriostatic effect [12,13]. It also acts as a bactericidal agent, regardless of iron binding from the environment [14,15]. The lysozyme's lytic character is closely correlated with the growth phase of the colony. ...
Simple Summary
Mastitis is one of the most common diseases of high-yielding dairy cows, and over 90% of cases are caused by Streptococcus spp., Enterobacteriaceae, or Staphylococcus spp. Whey proteins are very important in relation to cows’ bacteriostatic and bactericidal properties. It is therefore important to determine the relationship between the content of individual proteins and the bacterial strain. This study aimed to determine the influence of Staphylococcus spp., Streptococcus spp., and Enterobacteriaceae on the level of bioactive whey proteins and oxidative stress markers. From the herd, 60 multiparous cows with diagnosed mastitis were selected. Samples were taken for analyses from each cow individually from each quarter and pooled, which gave 60 samples. This study has shown that the levels of whey proteins and oxidative stress markers changed depending on the bacterial strain inducing inflammation, and selected whey proteins can be a marker for the diagnosis of individual mastitis-inducing strains.
Abstract
Mastitis is one of the most common diseases of high-yielding dairy cows, and over 90% of cases are caused by Streptococcus spp., Enterobacteriaceae, or Staphylococcus spp. Certain groups of proteins are very significant in terms of the cow’s antioxidant, bacteriostatic, and germicidal properties: lysozyme (Lz), lactoferrin (Lf), and β-lactoglobulin (BLG). This study aimed to determine the influence of Staphylococcus spp., Streptococcus spp., and Enterobacteriaceae on the secretion of bioactive whey proteins and oxidative stress markers. From the herd, 60 multiparous cows with diagnosed mastitis were selected. Samples were taken individually from each quarter and pooled, which gave 60 samples. Enterobacteriaceae did not affect the BLG synthesis, whereas lysozyme and lactoferrin responded to a high concentration of these bacterial strains. In the case of Staphylococcus spp. infection, the BLG level increased. These strains did not affect the levels of di-malonic aldehyde (MDA), lactoferrin, and lysozyme. In contrast, they were significantly influenced by Streptococcus spp. In summary, the levels of whey proteins and oxidative stress markers changed depending on the bacterial strain inducing inflammation. Lysozyme and lactoferrin may be markers of udder inflammation caused by Enterobacteriaceae and Streptococcus spp., whereas β-lactoglobulin may prove useful in diagnosing Staphylococcus spp. induced mastitis.
... In contact with Gram (−) bacteria, LF combines with its surface proteins, causing the release of lipopolysaccharide, which results in an increase in membrane permeability, intracellular concentration of antibacterial factors, and intracellular osmotic pressure. [12]. Gram (+) bacteria break down by combining the positively charged proteins with the bacterial membrane. ...
The impact of cow mammary gland diseases on the quality of colostrum is not conclusively defined; research results are conflicting. However, it is widely believed that mastitis lowers the level of immunoglobulins and the quality of the colostrum produced. Therefore, the aim of this study was to determine the influence of somatic cell counts (SCC) on the colostrum immunostimulating and chemical components. The experiment was conducted on an experimental organic dairy farm in which a herd of approximately 250 cows was kept in a freestall housing system, with the average performance exceeding 6,000 kg of milk per lactation. Colostrum and milk samples were taken individually from each cow seven times during the experiment: from the first to second day after calving–twice per day, and from the third to fifth day after calving–once per day. Therefore, after preliminary analyses, the cows were divided into two groups based on the cytological quality of their colostrum at the first collection: 1. SCC ≤400,000 cells/ml (good quality colostrum; GCC– 18 cows), 2. SCC ≥ 400,000 cells/ml (low quality colostrum; LCC– 22 cows). The study found almost double the concentration of immunoglobulins and essential fatty acids in first milking colostrum in the GCC group than in colostrum from the LCC group. In addition, an increase in the concentration of lysozyme in first milking colostrum was associated with a decrease in the concentration of immunoglobulins. In addition, the increase in the level of lysozyme was associated with a decrease in the concentration of immunoglobulins. In conclusion, the SCC of first milking colostrum can be used as an indicator of colostrum quality.
... Similarly to intact LF, also LF-derived cryptides show a variety of immunomodulatory functions ( Table 1). Intact LF, LFcinB, LFcinH and LFampin, can exert anti-inflammatory effects by scavenging bacterial endotoxins, such as LPS and LTA [24,[84][85][86][87][88][89][90][91]. The anti-inflammatory effects can be mediated also by other mechanisms not based on the scavenging ability, for example, both LFcinB and LFcinH inhibit the classical complement pathway [92]. ...
Cationic antimicrobial peptides (CAMPs), also known as host defence peptides (HDPs), are essential evolutionarily conserved components of innate immunity, constitutively or inducibly expressed in response to invasion by pathogens. In addition to a direct antimicrobial action, they are able to synergistically operate with other defence molecules to combat infection by neutralization of endotoxins, chemokine-like activities, induction of angiogenesis and wound repair. The importance of CAMPs has been highlighted in animal models and supported by observations in patient studies. CAMPs are attractive alternative candidates to antibiotic treatment, because they offer several advantages over the currently used drugs, moreover, knowledge on these peptides, especially regarding the intertwinement between their structure, function and mechanism of action, could be applied in the rational design of antimicrobial/anti-inflammatory/wound healing enhancing drugs. CAMPs combat pathogens by targeting bacterial membranes and essential membrane-related functions, and, in some cases, also target intracellular components. Despite differences in their size and sequence, many of them share a net positive charge and fold into amphipathic structures after contact with bacterial surfaces or endotoxins like lipopolysaccharides and lipoteichoic acid. Due to their peculiar mechanism, acquisition of resistance towards these peptides would be difficult for the bacteria. Very interestingly it has been demonstrated that several proteins, including proteins apparently not involved in immunity, can behave as sources of CAMPs hidden in their primary structures and released by the action of host and/or bacterial proteases. The existence of these "cryptic CAMPs" suggests that the panel of antimicrobial peptides present in higher eukaryotes and the variety of functions they perform could be much wider and more complex than previously suspected. This review focuses on source, structure and mechanism of action of cryptic CAMPs, with special attention to their immunomodulatory functions.
... It is important to underline that Lfs are also able to exert bactericidal activity especially against Gram-negative pathogenic bacteria. This function is iron independent and due to the direct interaction of Lf molecules with the LPS (Appelmelk et al. 1994;Brandenburg et al. 2001). The latter activity is exerted by a cationic sequence of 25 amino-acids located in the N-terminal region. ...
Human and bovine lactoferrin (hLf and bLf) are multifunctional iron-binding glycoprotein constitutively synthesized and secreted by glandular epithelial cells and by neutrophils following induction. HLf and bLf possess very high similarity of sequence. Therefore, most of the in vitro and in vivo studies are carried out with commercial bLf (cbLf), available in large quantities and recognized by Food and Drug Administration (FDA, USA) as a safe substance. Physico-chemical heterogeneity of different cbLf preparations influences their effectiveness. CbLf iron-saturation affects thermal stability and resistance to proteolysis. Moreover, other metal ions such as Al(III), Cu(II), Mg(II), Mn(II), Zn(II) are chelated by cbLf, even if at lower affinity than Fe(III). Ca(II) is also sequestered by the carboxylate groups of sialic acid present on glycan chains of cbLf thus provoking the release of LPS, contributing to bactericidal activity. Similarly to more than 50% of eukaryotic proteins, cbLf possesses five N-glycosylation sites, also contributing to the resistance to proteolysis and, putatively, to the protection of intestinal mucosa from pathogens. CbLfs possess several functions as anti-microbial, anti-biofilm, anti-adhesive, anti-invasive and anti-inflammatory activities. They are also relevant modulators of iron and inflammatory homeostasis. However, the efficacy of cbLfs in exerting several functions can be erratic mainly depending from integrity, degree of iron and other metal ions saturation, N-glycosylation sites and chains, desialylated forms, Ca(II) sequestration, presence of contaminants and finally the ability to enter inside nucleus.
... Only later was it demonstrated that LF can kill bacteria through an iron-independent mechanism by direct interaction of LF with the bacterial cell surface [63]. Large positively charged areas on the surface of LF facilitate direct interaction with negatively charged Lipid A, a component of the lipopolysaccharides of gram-negative bacteria [64]. This interaction can damage the bacterial membrane, altering the outer membrane permeability, resulting in the release of lipopolysaccharide [65]. ...
The provision of essential and non-essential amino acids for breast-fed infants is the major function of milk proteins. In addition, breast-fed infants might benefit from bioactivities of milk proteins, which are exhibited in the intestine during the digestive phase and by absorption of intact proteins or derived peptides. For lactoferrin, osteopontin and milk fat globule membrane proteins/lipids, which have not until recently been included in substantial amounts in infant formulas, in vitro experiments and animal models provide a convincing base of evidence for bioactivities, which contribute to the protection of the infant from pathogens, improve nutrient absorption, support the development of the immune system and provide components for optimal neurodevelopment. Technologies have become available to obtain these compounds from cow´s milk and the bovine compounds also exhibit bioactivities in humans. Randomized clinical trials with experimental infant formulas incorporating lactoferrin, osteopontin, or milk fat globule membranes have already provided some evidence for clinical benefits. This review aims to compare findings from laboratory and animal experiments with outcomes of clinical studies. There is good justification from basic science and there are promising results from clinical studies for beneficial effects of lactoferrin, osteopontin and the milk fat globule membrane complex of proteins and lipids. Further studies should ideally be adequately powered to investigate effects on clinically relevant endpoints in healthy term infants.
... 26,27 Other studies revealed that LF is able to act bacteriostatically by competing for iron (e.g., with Salmonella typhimurium), 28 by disrupting the bacterial cell membrane through its serine protease activity, 29 or via binding to the lipid A portion of lipopolysaccharides (LPSs). 30 Moreover, LF impedes host cell invasion of enteropathogens, including Y. enterocolitica, through degradation of surface-expressed or secreted virulence factors. 31 On the basis of these findings, we aim to elucidate putative barrier protective properties of LF. ...
The iron-binding glycoprotein lactoferrin (LF) is naturally present in human breast milk. Several studies suggest that LF contributes to infant health and development owing to a variety of protective effects, including antimicrobial and anti-inflammatory features. Therefore, we aimed to elucidate its protective properties on intestinal epithelial barrier dysfunction induced by infection or inflammation using the human epithelial cell culture models HT-29/B6 and T84. During barrier perturbation induced by the proinflammatory cytokine tumor necrosis factor α (TNF-α), bovine LF restored tight junction (TJ) morphometry and inhibited TNF-α-induced epithelial apoptosis. This resulted in an attenuation of the TNF-α-induced decrease in transepithelial resistance (TER) and increases in permeability of fluorescein and FITC-dextran (4 kDa) and was as effective as the apoptosis inhibitor Q-VD-Oph. The enteropathogenic bacterium Yersinia enterocolitica is a frequent cause of diarrhea in early childhood. This involves focal changes in TJ protein expression and localization. LF diminished the Y. enterocolitica-induced drop in TER in the present in vitro model, which was paralleled by an inhibition of the Yersinia-induced reduction of claudin-8 expression via c-Jun kinase signaling. In conclusion, LF exerts protective effects against inflammation- or infection-induced barrier dysfunction in human intestinal cell lines, supporting its relevance for healthy infant development.
... Her bir lob α-heliks ve β-plaka yapı formunda iki alt domain içermektedir. [34][35][36][37][38][39]. Ayrıca Gram-pozitif bakterilere karşı antibakteriyel aktivitesini hücre duvarında bulunan taykoik asitlerle reaksiyona girmek suretiyle de gösterebilmektedir (Şekil 2) [40]. ...
ÖZET
Transferrin protein ailesi içinde yer alan laktoferrin, basta süt olmak üzere birçok sıvıda bulunan demir bağlayan bir glikoproteindir. Laktoferrin antibakteriyal, antifungal, antiviral, antiprotozoal, antioksidant, antikanserojen, kemik sağlığını iyilestirici, bağırsakta demir absorpsiyonunu düzenleyici, antienflamatuar, immünomodulatör gibi birçok biyolojik aktiviteye sahip olduğundan çok fonksiyonlu protein olarak değerlendirilmektedir. Bu derlemede inek sütü laktoferrinin kimyasal ve yapısal özellikleri ile biyolojik aktiviteleri hakkında bilgiler verilmektedir. ABSTRACT
Lactoferrin is an iron-binding glycoprotein of the transferrin family and it is mainly found in milk and in most biological fluids. Lactoferrin is a multifunctional protein since it has many biological activities including antibacterial, antifungal, antiviral, antiprotozoal, antioxidant and anticarcinogenic activity, improvement of bone health, regulator of iron absorption in intestine, anti-inflammatory activity, and immunomodulatory. In this review article, we focus on the chemical and structural properties, and biological activities of bovine milk lactoferrin.
... residues near the N terminus of the lactoferrin from many mammalian species(Tomita et. al., 1994;Vorland et. al., 1998;Elass -Rochard et. al., 1998;Nibbering et. al., 2001). This positive cluster binds to the lipid A part of lipopolysaccharide molecules present on the outer membrane of clinically relevant bacterial species(Appelmelk et. al., 1994;Brandenburg et. al., 2001). In particular the binding takes place to the phosphate group within the lipid A part, inducing a rigidification of the acyl chains of lipopolysaccharide(Brandenburg et. al., 2001). Rossi et. al. reported that lactoferrin can bind Ca 2+ releasing significant amounts of lipopolysaccharide from Gram negative bacteria without the need of d ...
Milk is a highest quality source of well balanced nutrients and also displays a range of biological activities that affects digestion, metabolic responses to absorbed nutrients, growth & development of specific organs, and resistance to disease. Bioactive proteins such as lactoferrin (Lf) have been isolated over decades ago and showed their importance in stimulating immune system in the infants through breast milk in addition to immunoglobulin present in the milk. In addition to immune system stimulation, Lf also has antibacterial activity and antioxidant activity in infant and adult of human as well as animal health. In this review paper, antibacterial properties of lactoferrin have been discussed along with its future perspectives.
... For example, bovine lactoferrin inhibits Yersinia spp. login in epithelial cells (11). Lactoferrin also causes apoptosis in cells (12). ...
... Synthesized lipid A from Peptide Institute, Inc. (Osaka, Japan) was used for confirmation of the endotoxin inactivation mechanism. This is because lipid A is an active site of endotoxins [14][15][16][17][18][19][20][21]. Chemical structure of endotoxin and lipid A from E. coli is presented in Figures 2 and 3, respectively. ...
... Aside from iron chelation, lactoferrin also exhibits bacteriocidal effects through membrane destabilization (Jenssen and Hancock, 2009). Lactoferrin binds directly to Lipid A of LPS in the outer membrane of Gramnegative bacteria (Appelmelk et al., 1994) causing release of LPS from the bacterial cell membrane with resultant membrane destabilization (Ellison et al., 1988) and LPS inactivation (Fig. 2) via chemical modification (Brandenburg et al., 2001). Lactoferrin also mediates the inflammatory response to LPS by translocating to host cell nucleus (Fig. 2) and inhibiting production of pro-inflammatory cytokines IL-1b, IL-6 and TNF-a via blockade of the NF-kB pathway (Haversen et al., 2002). ...
BACKGROUND At the interface of the external environment and the mucosal surface of the female reproductive tract (FRT) lies a first-line
defense against pathogen invasion that includes antimicrobial peptides (AMP). Comprised of a unique class of multifunctional,
amphipathic molecules, AMP employ a wide range of functions to limit microbial invasion and replication within host cells
as well as independently modulate the immune system, dampen inflammation and maintain tissue homeostasis. The role of AMP
in barrier defense at the level of the skin and gut has received much attention as of late. Given the far reaching implications
for women's health, maternal and fetal morbidity and mortality, and sexually transmissible and polymicrobial diseases, we
herein review the distribution and function of key AMP throughout the female reproductive mucosa and assess their role as
an essential immunological barrier to microbial invasion throughout the reproductive cycle of a woman's lifetime.
... 54 Also, the addition of human LF or LFderived peptides leads to a reduction in the secretion of TNF-a, whereas the effect on the phase transition is different depending on the hydrophobicity of the peptides and the LPS used for the induction of fluidisation or rigidification of the acyl chains. 46,55 In summary, we could demonstrate that the peptide Hbg-35 is able to enhance the immune stimulatory effect of LPS, similar to the entire Hbg chain and the complete Hb protein. Our results strongly support that LPS disaggregation induced by Hbg-35 is the main factor in enhancing its biological activity, reinforcing the importance of LPS supramolecular structure in the cell activation process. ...
Endotoxins (LPS) are highly potent immune stimulatory molecules and are mainly known for triggering Gram-negative sepsis. However, besides their toxic effects, this stimulatory function may be advantageous, for example when used as an adjuvant during vaccination. Thus, there is always a narrow range between the useful wake-up of the immune system and its overwhelming reaction, which can lead to diseases like sepsis. This raises the question of which conformational properties are responsible for making the LPS aggregates more or less potent. As described previously, the size, type and form of LPS aggregates play a major role in their immune stimulatory activity. In this study we investigate the role of these parameters. On the one hand, we use a peptide (Pep19-2.5; Aspidasept) that causes a change of the LPS aggregate structure into a less toxic state; on the other hand, we use a potent immune stimulating peptide (Hbγ-35), leading to higher toxicity. We have found opposing effects on LPS aggregate conformations allowing a better understanding of the processes of immune stimulation.
Breast milk is a miraculous nutrient specially produced for each baby. Breast milk, the essential source of infant nutrition, provides different benefits with every component it contains. Skin-to-skin contact with the mother during breastfeeding reduces the baby’s stress, eases the baby’s adaptation to the world, and contributes to neurobehavioral development [1]. Breast milk has many more benefits further than growth and development. The formation of the gastrointestinal microbiota, reduction in the risk of necrotizing enterocolitis (NEC), protection against various diseases such as oral candidiasis, otitis media, and respiratory, gastrointestinal, and urinary tract infections, and a reduction in the mortality risk in the first months of life for any reason are some of the short-term benefits provided by breast milk [1–5].KeywordsBreastfeedingMaternal Bacterial InfectionsBreast MilkNewborn
Peptide antibiotics have gathered attention given the urgent need to discover antimicrobials with new mechanisms of action. Their extended role as immunomodulators makes them interesting candidates for the development of compounds with dual mode of action. The objective of this study was to test the anti-inflammatory capacity of a recently reported chimeric peptidomimetic antibiotic (CPA) composed of polymyxin B nonapeptide (PMBN) and a macrocyclic β-hairpin motif (MHM). We investigated the potential of CPA to inhibit lipopolysaccharide (LPS)-induced activation of RAW264.7 macrophages. In addition, we elucidated which structural motif was responsible for this activity by testing CPA, its building blocks, and their parent compounds separately. CPA showed excellent LPS neutralizing activity for both smooth and rough LPSs. At nanomolar concentrations, CPA completely inhibited LPS-induced nitric oxide, TNF-α, and IL-10 secretion. Murepavadin, MHM, and PMBN were incapable of neutralizing LPS in this assay, while PMB was less active compared to CPA. Isothermal titration calorimetry showed strong binding between the CPA and LPS with similar binding characteristics also found for the other compounds, indicating that binding does not necessarily correlate with neutralization of LPS. Finally, we showed that CPA-killed bacteria caused significantly less macrophage activation than bacteria killed with gentamicin, heat, or any of the other compounds. This indicates that the combined killing activity and LPS neutralization of CPA can prevent unwanted inflammation, which could be a major advantage over conventional antibiotics. Our data suggests that immunomodulatory activity can further strengthen the therapeutic potential of peptide antibiotics and should be included in the characterization of novel compounds.
Purpose
The goal of this study was to review the role of human milk in shaping the infant intestinal microbiota and the potential of human milk bioactive molecules to reverse trends of increasing intestinal dysbiosis and dysbiosis-associated diseases.
Methods
This narrative review was based on recent and historic literature.
Findings
Human milk immunoglobulins, oligosaccharides, lactoferrin, lysozyme, milk fat globule membranes, and bile salt–stimulating lipase are complex multifunctional bioactive molecules that, among other important functions, shape the composition of the infant intestinal microbiota.
Implications
The co-evolution of human milk components and human milk–consuming commensal anaerobes many thousands of years ago resulted in a stable low-diversity infant microbiota. Over the past century, the introduction of antibiotics and modern hygiene practices plus changes in the care of newborns have led to significant alterations in the intestinal microbiota, with associated increases in risk of dysbiosis-associated disease. A better understanding of mechanisms by which human milk shapes the intestinal microbiota of the infant during a vulnerable period of development of the immune system is needed to alter the current trajectory and decrease intestinal dysbiosis and associated diseases.
Background:
Lactoferrin (Lf) is a natural iron-binding protein involved in coordinating specific and non-specific immune responses in humans. It exhibits broad spectrum antimicrobial properties against bacteria, viruses, and fungi. Owing to its high affinity for ferric (Fe+++) ions, Lf is responsible for controlling the oxidative stress in the body, and thus protects cells from oxygen injury. In addition, Lf is a natural immunomodulatory molecule that regulates the activity of the immune system.
Highlight:
Lactoferrin present in saliva plays an important role in maintaining oral hygiene. It exhibits protective function on mucosal surfaces, which constitute a barrier between the host and the external environment. Thus, Lf may be considered as the first line of defense protein that is associated with oral mucosal immunity.
Conclusion:
Many studies indicate that lactoferrin supplementation is safe and beneficial for human health. The aim of this review is to discuss the effects of Lf on oral microflora, highlighting the potential significance of this protein in dental therapy and prevention of oral diseases.
With a rapidly growing number of bacterial strains displaying resistance against conventional antibiotics, the development of novel types of antimicrobial agents represents an important health challenge. Antimicrobial peptides (AMPs) has attracted interest in this context, as these can be designed to display potent broad-spectrum antimicrobial as well as anti-inflammatory effects, but simultaneously low toxicity against human cells. Much of the work on AMPs has been focused on membrane interactions of monomeric AMPs, and how these can be controlled by peptide design to obtain selective disruption of bacterial membranes. However, a growing body of research has demonstrated that AMPs offer opportunities as antimicrobials beyond this through their self-assembly. An overview is therefore provided of the current understanding of the interplay between AMP aggregation and antimicrobial effects, including the role of oligomerization and self-assembly on membrane interactions and antimicrobial effects, AMP interactions with amyloid-forming peptides/proteins, AMP self-assemblies as antimicrobial biomaterials, and AMP-induced flocculation of bacteria and bacterial lipopolysaccharides as a novel pathway for confinement of infection and inflammation.
Enterohaemorhagic Escherichia coli (EHEC) are a group of bacteria responsible for numerous
foodborne outbreaks in humans which can lead to bloody diarrhea, haemorrhagic uremic
syndrome and in some cases in children even in death. E. coli O157:H7 is the most well known
serotype of this group. Ruminants are the most important reservoir of these microorganisms
and many outbreaks have been traced to ruminant’s origin. Ruminants typically continue to
shed bacteria in their feces for weeks to months. Carcasses of non-colonized animals have
regularly been found to be contaminated with E. coli O157:H7 in the abattoir, suggesting that
cross-contamination during meat processing can be a major source of the contamination of beef
products and subsequently of human’s infection. Therefore reducing E. coli O157:H7 shedding
by ruminants would be a way to control the infection of humans. Up till now, no efficient
treatment is available against E. coli O157:H7 infection and some traditional treatments,
including antibiotic therapy may even increase the chance of developing severe disease in
humans. Therefore, there is need for alternative ways to reduce the risk of E. coli O157:H7
infection of humans for instance by diminishing or preventing E. coli O157:H7 excretion at the
farm level.
The first part of this thesis reviews current literature. Chapter 1 of this thesis gives an
overview of the literatures on the pathogenesis of E. coli O157:H7 in human and ruminants. Also
the reported intervention strategies in ruminants were discussed. Chapter 2 reviews present
knowledge on lactoferrin as the main antibacterial immunomodulatory protein of milk. The
focus of this chapter is on the antimicrobial mechanism of lactoferrin and its peptides, in
particular the antimicrobial mechanism against gram-negative pathogens. In addition the role of
lactoferrin as immunomodulatory protein is briefly reviewed.
A second part of the thesis describes the aims. Following questions were addressed in this
thesis:
√ What is the pattern of antimicrobial susceptibility among E. coli isolates from dairy cows in Iran?
√ Can lactoferrin, a natural antimicrobial protein of milk, be used to prevent or reduce
colonisation of the ovine intestinal tract with E. coli O157:H7 and so reduce or prevent fecal
shedding?
√ Is systemic vaccination of sheep with type III secretion system proteins and intimin g a possible
strategy to reduce fecal shedding of E. coli O157:H7 as in cattle?
A third part, comprising Chapters 3 to 7, presents the experimental work of this thesis.
In Chapter 3 the antimicrobial susceptibility of E. coli isolates from milk and healthy cattle
feces in Iran has been investigated. The results identified ceftiofur ( 6%) and colistin as the most
effective antibiotics. However the incidence of colistin resistance (14%) among the isolates was
remarkably higher than in previous reports on antibiotic susceptibility of commensal bacteria.
The highest percentage of acquired resistance was detected for tetracycline (46%), followed by
ampicillin (43%). %). For the other tested antimicrobials (amoxicillin-clavulanic acid,
chloramphenicol, enrofloxacin, florfenicol, spectinomycin, sulfafurazole and trimethoprim)
resistance varied between 30 and 17%. Seventy-five percent of the isolates showed multiple
antimicrobial resistance. This means resistance to three or more antimicrobial agents. This
overall high antimicrobial resistance should alert veterinarians and authorities to take measures
for decreasing antimicrobial usage and should stimulate research towards alternative strategies
for treating cattle against bacterial infections.
Since traditional treatment including antibiotic therapy is not a suitable option in the control
and/or treatment of E. coli O157:H7, the use of lactoferrin, a natural antimicrobial protein of
milk has been investigated in the present thesis for reducing E. coli O157:H7 shedding in
ruminants feces. First the effect of lactoferrin on this bacterium was tested. In Chapter 4
lactoferrin from bovine and human milk were compared for their direct effect on E. coli
O157:H7 growth. Bovine lactoferrin (bLF) had a significantly stronger inhibitory effect than
human lactoferrin (hLF). The non iron-saturated form of bovine lactoferrin (apo-bLF) reduced E.
coli O157:H7 growth significantly stronger than the iron-saturated form. In addition, all
lactoferrins, apart of their origin or iron saturation level, reduced E. coli O157:H7 attachment to
Caco-2 cells at a dose that had no effect on bacterial growth. Therefore other factors rather than simply scavenging iron from the environment and/or reducing bacterial growth plays a role
in the anti-adhesive effect of lactoferrin. In Chapter 4 we showed that this effect is at least in
part due to a catalytic effect of lactoferrin on TTSS proteins of E. coli O157:H7; EspA and EspB
which are essential for E. coli O157:H7 colonisation were cleaved. This proteolytic activity of
lactoferrin has already been shown on the TTSS apparatus of EPEC, a similar pathogen. In
conclusion, the results reported in this chapter, support the idea that lactoferrin may reduce the
colonisation of E. coli O157:H7 in vivo.
To obtain more insights in the effect of lactoferrin on the interaction of E. coli O157:H7 with
the sheep intestinal mucosa, a sheep intestinal explant model was developed. In Chapter 5 we
examined using this sheep specific in vitro model 1) the tropism of E. coli O157:H7 for ileal or
large intestinal mucosa, 2) the intestinal mucosal cytokine response induced by the mucosal E.
coli O157:H7 colonisation and 3) the effect of lactoferrin on colonisation and cytokine response.
The data presented in this chapter show that E. coli O157:H7 preferentially binds to ileal follicleassociated
epithelium, but can also bind to the normal absorptive ileal mucosa and colonic
mucosa, although to a lesser extent. We also demonstrated that this bacterial attachment could
enhance IL-8 and TNF-α mRNA expression with the increase in expression being correlated with
the number of bacteria attaching to the sheep intestinal explant. Furthermore results presented
in this chapter clearly showed that lactoferrin inhibits E. coli O157:H7 attachment to the
explants with lower IL-8 and TNF-α mRNA expression as a consequence.
In Chapter 6, we examined if lactoferrin, administered via the oral route, could reduce E. coli
O157:H7 excretion of sheep. It is not evident that the administration via this route would be
effective since the protein perhaps could become degraded in the rumen. Therefore, lactoferrin
was solublized in a bicarbonate buffer which can close the esophageal groove allowing
lactoferrin to pass rumen, reticulum and omasum so reaching the abomasum. Oral lactoferrin
was given every 12 hours for 30 days in two different doses (1.5 or 0.15 g) and the inoculation
with E. coli O157:H7 occurred 24 hours after the first lactoferrin administration. Both dosages
reduced the number of E. coli O157:H7 in sheep feces as well as the duration of fecal excretion.
Furthermore, sheep, which received the high dose of lactoferrin (1.5 g, every 12 hours) showed
a significantly higher serum antibody response against EspA and EspB in comparison with the control group. Since the peak of this antibody response was seen when the excretion of E. coli
O157:H7 had completely ceased, it could be that this immune response plays a role in reducing
the bacterial excretion in sheep orally treated with lactoferrin. Results of this study show that
lactoferrin can become a new tool in controlling E. coli O157:H7 but also other EHEC infections
by decreasing their excretion by reservoir animals.
In the last chapter of the experimental part of the present thesis, the potential of systemic
vaccination against E. coli O157:H7 in sheep was examined. Vaccination studies have previously
been performed in cattle. Some of these studies in which cattle were systemically vaccinated
with an antigen mixture containing several type III secretion system (TTSS) proteins could
significantly reduce shedding. In this chapter we wanted to know if similar result could be
obtained in sheep. This could allow us to study in a next step the mechanism of this protection
and to further optimize vaccination in sheep. Sheep were intramuscularly vaccinated with a
mixture of the TTSS proteins EspA and EspB and the surface antigen intimin g in incomplete
Freund’s adjuvant or received only the adjuvant. The data obtained in Chapter 7 showed that
the vaccination significantly reduced the fecal excretion of the bacteria. These results are
consistent with the previous studies in cattle. Although the vaccination strongly increased the
level of serum IgG against intimin, EspA and EspB, the mechanism for the observed E. coli
O157:H7 reduction in the feces still needs to be determined. Results suggest that besides oral
lactoferrin treatment also vaccination could become an important tool to reduce E. coli
O157:H7 excretion by sheep.
The fourth part of the thesis consists of the general discussion and future perspectives.
Although important steps were set towards new strategies in the control of E. coli O157:H7
infections, many questions still remain to be resolved such as: 1) Is there a tropism for the rectal
mucosa in sheep and can lactoferrin via the oral route reach the rectal mucosa and clear the
colonizing bacteria?; 2) What is the mechanism for the enhanced antibody response following
lactoferrin treatment?; 3) Is this antibody response also present in the mucosa?; 4) Can we
develop an administration form that is feasible in practice?; 5) What is the mechanism of the
reduction in excretion following vaccination?; 6) Can we still improve this clearance using more or different antigens and different adjuvants?; 7) What is the duration of this immune
mechanism in the mucosa?
Future research has to direct these questions. For some of these questions the first steps to
resolve them have already been taken.
The objective of the present study is the investigation of possibilities for boosting peptide anti-inflammatory effects by tryptophan end-tagging, including identification of underlying mechanisms for this. In doing so, effects of tryptophan end-tagging of KYE21 (KYEITTIHNLFRKLTHRLFRR), a peptide derived from heparin co-factor II, on membrane and lipopolysaccharide (LPS) interactions were investigated by ellipsometry, NMR, fluorescence spectroscopy, and circular dichroism measurements. Through its N-terminal W stretch, WWWKYE21 displays higher membrane binding, liposome rupture, and bacterial killing than unmodified KYE21. Analogously, W-tagging promotes binding to E. coli LPS and to its endotoxic lipid A moiety. Furthermore, WWWKYE21 causes more stable peptide/LPS complexes than KYE21, as evidenced by detailed NMR studies, adopting a pronounced helical conformation, with a large hydrophobic surface at the N-terminus due to the presence of W-residues, and a flexible C-terminus due to presence of several positively charged arginine residues. Mirroring its increased affinity for LPS and lipid A, WWWKYE21 displays strongly increased anti-inflammatory effect due to a combination of direct lipid A binding, peptide-induced charge reversal of cell membranes for LPS scavenging, and peptide-induced fragmentation of LPS aggregates for improved phagocytosis. Importantly, potent anti-inflammatory effects were observed at low cell toxicity, demonstrated for both monocytes and erythrocytes.
The bactericidal mechanism of human lactoferrin (LF) was analyzed by employing bioelectrochemistry technique. The result showed that LF bound to lipopolysaccharides (LPS) and caused release of LPS, resulting in formation of ion channels in the mimic biomembrane. Furthermore, a survey of microenvironment factors for bactericidal activity of LF was conducted. In weakly alkaline condition of pH 8, LF exhibited maximally bactericidal activity. The bactericidal activity of LF was closely related to its concentration. In addition, the bactericidal activity would decrease with a higher concentration of metal ions. Divalent cations showed greater influence on the bactericidal activity than that of monovalent cations.
Lactoferrin (Lf), a natural defence iron-binding protein, is present in exocrine secretions that are commonly exposed to normal flora: milk, tears, nasal exudate, saliva, bronchial mucus, gastrointestinal fluids, cervicovaginal mucus and seminal fluid. Additionally, Lf is produced in polymorphonuclear leukocytes and is deposited by these circulating cells in septic sites. A principal function of Lf is that of scavenging non-protein-bound iron in body fluids and inflamed areas so as to suppress free radical-mediated damage and decrease accessibility of the metal to invading bacterial, fungal and neoplastic cells. Adequate sources of bovine and recombinant human Lf are now available for development of commercial applications. Among the latter are use of Lf in food preservation, fish farming, infant milk formula and oral hygiene. other readily accessible body compartments for Lf administration include skin, throat and small intestine. Further research is needed for possible medicinal use in colon and systemic tissues. Although Lf is a natural product and should be highly biocompatible, possible hazards have been documented.
Statement of significance:
Tryptophan (Trp) residues show a strong preference for the interfacial region of biological membranes, and this property endows Trp-containing peptides with the unique ability to interact with the surface of bacterial cell membranes. In this manuscript, we report the membrane interaction of Trp-containing peptide to address whether bacterial LPS is responsible for the different susceptibilities of Gram-negative bacteria to Trp-containing peptides. Based on the data collected, we propose a molecular mechanism for the peptide-LPS interactions that allows the peptides to traverse or prevents them from transversing the LPS layer and the target inner membrane. The data should help in the development of a potent broad-spectrum antibiotic for future therapeutic purposes.
Bacterial infections, with the most severe form being sepsis, can often not be treated adequately leading to high morbidity and lethality of infected patients in critical care units. In particular, the increase in resistant bacterial strains and the lack of new antibiotics are main reasons for the worsening of the current situation, As a new approach, the use of antimicrobial peptides (AMPs) seems to be promising, combining the ability of broad-spectrum bactericidal activity and low potential of induction of resistance. Peptides based on natural defense proteins or polypeptides such as lactoferrin, Limulus anti-lipopolysaccharide factor (LALF), cathelicidins, and granulysins are candidates due to their high affinity to bacteria and to their pathogenicity factors, in first line lipopolysaccharide (LPS, endotoxin) of Gram-negative origin.
Objective:
The aim of this study was to evaluate the effect of supplementation with bovine lactoferrin (bLf) from iron-fortified formulas on diarrhea and respiratory tract infections (RTIs) in weaned infants.
Methods:
In this prospective, multicenter, controlled intervention study, 260 infants ages 4 to 6 mo who previously were exclusively breastfed but weaned were randomized into two groups: a lactoferrin-fortified formula milk group (fortified group, FG, containing lactoferrin 38 mg/100 g milk) and a no lactoferrin-fortified milk (control group, CG); breastfed infants were enrolled and served as a reference group (breastfed group, BG). The intervention duration was 3 mo. The morbidity of diarrhea and RTIs were collected during supplementation.
Results:
The results of the study demonstrated evidence of a lower incidence rate of respiratory-related illnesses and fewer symptoms of running nose, cough, and wheezing for infants in the FG and BG groups compared with those in the CG (P < 0.05). Despite the undistinguished incidence rate of vomiting, nausea, and colic, the occurrences of diarrhea-related illnesses were significantly lower for children in the FG and BG than for those in CG (P < 0.05).
Conclusion:
The beneficial effects on infectious morbidity over 3 mo highlighted the potential of bLF supplementation for previously weaned infants; these findings may be applicable to other infants living in similar socioeconomic districts.
The lack of newly developed antibiotics, together with the increase in multi-resistance of relevant pathogenic bacteria in the last decades, represents an alarming signal for human health care worldwide. The number of severely infected persons increases not only in developing but also in highly industrialized countries. This relates in first line to the most severe form of a bacterial infection, sepsis and the septic shock syndrome, with high mortality on critical care units. No particular anti-sepsis drug is available, and the therapy with conventional antibiotics more and more fails to provide a survival benefit. Due to the fact that the pharmaceutical industry has withdrawn to a high degree from the development of anti-infectious agents, a huge challenge for health care is approaching in the 21(st) century. In this article, these problems are outlined and possible alternatives are presented which may be helpful to solve the problem.
Treating infections caused by multidrug-resistant Gram-negative pathogens is challenging, and there is concern regarding the toxicity of the most effective antimicrobials for Gram-negative pathogens. We hypothesized that conjugating a fatty acid moiety onto a peptide dimer could maximize the interaction with lipopolysaccharide (LPS) and facilitate the permeabilization of the LPS barrier, thereby improving potency against Gram-negative pathogens. We systematically designed a series of N-lipidated peptide dimers that are active against Gram-negative bacteria, including carbapenem-resistant Enterobacteriaceae (CRE). The optimized lipid length was 6-10 carbons. At these lipid lengths, the N-lipidated peptide dimers exhibited strong LPS permeabilization. Compound 23 exhibited synergy with select antibiotics in most of the combinations tested. 23 and 32 also displayed rapid bactericidal activity. Importantly, 23 and 32 were non-hemolytic at 10 mg/mL, with no cellular or in vivo toxicity. These characteristics suggest that these compounds can overcome the limitations of current Gram-negative-targeted antimicrobials such as polymyxin B.
Antimicrobial peptides (AMPs) have attracted considerable recent interest as potential therapeutics, motivated by increasing resistance development against conventional antibiotics. This brief overview summarizes some key aspects related to the interaction of AMPs with bacterial and cell membranes, as well as with membrane components, which is at the core of the mode-of-action of these compounds. Throughout, studies on peptide interactions with model lipid membranes and membrane components are correlated to biological results on antimicrobial and anti-inflammatory effects of AMPs, and translated into therapeutic considerations.
Due to rapidly increasing resistance development against conventional antibiotics, as well as problems associated with diseases either triggered or deteriorated by infection, antimicrobial and anti-inflammatory peptides have attracted considerable interest during the last few years. While there is an emerging understanding of the direct antimicrobial function of such peptides through bacterial membrane destabilization, the mechanisms of their anti-inflammatory function are less clear. We here summarize some recent results obtained from our own research on anti-inflammatory peptides, with focus on peptide-(lipo)polysaccharide interactions.
Copyright © 2014 Elsevier Inc. All rights reserved.
Investigations are reported into the interaction of lipid A, the ‘endotoxic principle’ of bacterial lipopolysaccharide (LPS), with phospholipid membranes in the absence and presence of an acute-phase lipid transport
protein, lipopolysaccharide-binding protein (LBP) applying Fourier-transform infrared (FTIR) and fluorescence resonance energy transfer (FRET) spectroscopy. In the absence of LBP, intermixing of phospholipids with lipid A takes place on the time-scale of hours, while in the presence of LBP this process takes place
in the order of minutes. A comparison of chemically different lipid A shows that a prerequisite for the intercalation of lipid A into the phospholipid membrane is a sufficiently high negative charge density of lipid A. Variations in the lipid A acyl chain fluidity may modulate the intercalation, whereas the type of lipid A aggregate structure has no influence on the intercalation.The intercalation is a necessary, but not sufficient prerequisite for
cell activation. Only lipid A with a conical molecular shape and a tilt angle of more than 40° of the backbone with respect to the direction of the acyl chains induces cytokine induction in human mononuclear cells, while lipid A with a cylindrical shape and a small tilt angle does not exhibit this biological activity but may act antagonistically. This antagonistic effect may be explained by blocking of the binding-sites of the putative
signal-transducing protein, possibly an ion channel, by the antagonist.
The iron-binding protein lactoferrin (Lf) is a constituent of neutrophil secondary granules and is discharged into the surrounding medium when neutrophils are activated. Lf released from neutrophils phagocytosing opsonized particles inhibits proliferation of mixed lymphocyte cultures (MLC) and has also been shown to inhibit granulopoiesis, suppress antibody production, and regulate natural killer cell activity. All of these processes are controlled by cytokines, suggesting that Lf may modulate immune responses by inhibiting cytokine activity. When MLC were cultured in round-bottomed wells to crowd the cells together, Lf, 50% saturated with iron, inhibited both proliferation and interleukin-2 (IL-2) release into the supernatants. Inhibition was concentration-dependent and lost at concentrations of Lf greater than 10(-12) mol/L. Lf at 10(-10) mol/L inhibited release of tumor necrosis factor-alpha (TNF) and interleukin-1 beta (IL-1) into MLC supernatants, as well as inhibiting IL-2 release. TNF in the supernatant was significantly reduced at 5 and 24 hours, becoming less and losing significance by 72 hours. IL-1 in the supernatant was not significantly reduced at 5 and 24 hours, becoming significant at 48 and 72 hours. IL-2 was significantly reduced at 48 and 72 hours and followed the same time course as proliferation. Inhibition was blocked by specific antiserum to Lf, but not by a preimmune serum. Lf, 10(-10) mol/L, also inhibited the production of TNF (49.15% +/- 7.98%; n = 10, P = .032) and IL-1 (42.67% +/- 6.72%; n = 6, P = .032) from endotoxin-stimulated mononuclear cells. As with MLC, inhibition was dose-dependent and abrogated by specific antiserum. Lf did not block the biological action of TNF, IL-1, or IL-2 in specific assays using cytokine-sensitive cell lines. These data suggest that Lf, released from activated neutrophils, acts as a negative feedback mechanism to prevent recruitment and activation of leukocytes in sites of inflammation.
Lactoferrin is an iron-binding protein that has been detected in secretions that bathe human mucosal tissues. Previous studies have shown that, when this protein is in the iron-free state, it is capable of a direct bactericidal effect on Streptococcus mutans and Vibrio cholerae. The present study demonstrates variable susceptibilities for a variety of different microorganisms. The list of susceptible organisms includes gram-positive and gram-negative microbes, rods and cocci, facultative anaerobes, and aerotolerant anaerobes. Similar morphological and physiological types are represented among the lalctoferrin-resistant bacteria. S. mutans was more resistant to lactoferrin when grown on a sucrose-contaning medium than when it was grown on brain heart infusion broth without added scurose. When a lactoferrin-sensitive, avirulent strain of Streptococcus pneumoniae was passed through mice, the resultant virulent culture became lactoferrin resistant. Since organisms of the same species and even of the same strain (S. pneumoniae) can differ in susceptibility to lactoferrin, it appears that accessibility to the lactoferrin target site may account for differences in susceptibility. It appears that there may be a relation between virulence and resistance to lactoferrin.
The turnover of phospholipid fatty acid moieties of bone marrow-derived macrophages was analyzed by separate determination of degrading and acylating activities. Acylating activities were followed in intact cells by incubation with excess arachidonic acid and degradation of phospholipids was followed in cells prelabeled with fatty acids. Significant phospholipase A2 activity was detectable only if the reutilization of liberated fatty acid was inhibited , e.g. by p-chloromercuribenzoate. It was of interest that the divalent cation ionophore A 23187 and various antiphlogistic drugs like indomethacin, diclofenac, and acetylsalicylic acid were found to inhibit the acylation reaction. These compounds led to increased levels of free arachidonic acid in stimulated, as well as in unstimulated cells. Increased activities of phospholipase A2 were achieved by treatment with the bivalent cation ionophore A 23187 and with zymosan. The effect of zymosan obtained from various sources was found to be exclusively due to contamination of tee zymosan particles with phospholipase A2 activity. Even when the cellular phospholipase activity was increased by the addition of exogenous phospholipase activity contained in the zymosan particles, degradation of cellular phospholipids was not measurable unless the reacylation was inhibited. These results suggest that in the cells studied, the level of free arachidonic acid is mainly controlled by the activity of the lysophosphatide acyltransferase.
CD14 is a 55-kD protein found as a glycosylphosphatidylinositol (GPI)-anchored protein on the surface of monocytes, macrophages, and polymorphonuclear leukocytes, and as a soluble protein in the blood. Both forms of CD14 participate in the serum-dependent responses of cells to bacterial lipopolysaccharide (LPS). While CD14 has been described as a receptor for complexes of LPS with LPS-binding protein (LBP), there has been no direct evidence showing whether a ternary complex of LPS, LBP, and CD14 is formed, or whether CD14 binds LPS directly. Using nondenaturing polyacrylamide gel electrophoresis (native PAGE), we show that recombinant soluble CD14 (rsCD14) binds LPS in the absence of LBP or other proteins. Binding of LPS to CD14 is stable and of low stoichiometry (one or two molecules of LPS per rsCD14). Recombinant LBP (rLBP) does not form detectable ternary complexes with rsCD14 and LPS, but it does accelerate the binding of LPS to rsCD14. rLBP facilitates the interaction of LPS with rsCD14 at substoichiometric concentrations, suggesting that LBP functions catalytically, as a lipid transfer protein. Complexes of LPS and rsCD14 formed in the absence of LBP or other serum proteins strongly stimulate integrin function on PMN and expression of E-selectin on endothelial cells, demonstrating that LBP is not necessary for CD14-dependent stimulation of cells. These results suggest that CD14 acts as a soluble and cell surface receptor for LPS, and that LBP may function primarily to accelerate the binding of LPS to CD14.
Human lactoferrin (hLf), a glycoprotein released from neutrophil granules during inflammation, and the lipopolysaccharide (LPS)-binding protein (LBP), an acute-phase serum protein, are known to bind to the lipid A of LPS. The LPS-binding sites are located in the N-terminal regions of both proteins, at amino acid residues 28 to 34 of hLf and 91 to 108 of LBP. Both of these proteins modulate endotoxin activities, but they possess biologically antagonistic properties. In this study, we have investigated the competition between hLf and recombinant human LBP (rhLBP) for the binding of Escherichia coli 055:B5 LPS to the differentiated monocytic THP-1 cell line. Our studies revealed that hLf prevented the rhLBP-mediated binding of LPS to the CD14 receptor on cells. Maximal inhibition of LPS-cell interactions by hLf was raised when both hLf and rhLBP were simultaneously added to LPS or when hLf and LPS were mixed with cells 30 min prior to the incubation with rhLBP. However, when hLf was added 30 min after the interaction of rhLBP with LPS, the binding of the rhLPS-LBP complex to CD14 could not be reversed. These observations indicate that hLf competes with rhLBP for the LPS binding and therefore interferes with the interaction of LPS with CD14. Furthermore, experiments involving competitive binding of the rhLBP-LPS complex to cells with two recombinant mutated hLfs show that in addition to residues 28 to 34, another basic cluster which contains residues 1 to 5 of hLf competes for the binding to LPS. Basic sequences homologous to residues 28 to 34 of hLf were evidenced on LPS-binding proteins such as LBP, bactericidal/permeability-increasing protein, and Limulus anti-LPS factor.
LPS-binding protein (LBP) recognizes bacterial LPS and transfers it to CD14, thereby enhancing host cell stimulation, eventually resulting in pathogenic states such as septic shock. Recently, LBP also was shown to detoxify LPS by transferring LPS into HDL particles in vitro. Thus, the predominant in vivo function of LBP has remained unclear. To investigate the biological activity of acute phase concentrations of recombinant murine LBP, high concentrations of LBP were investigated in vitro and in vivo. Although addition of low concentrations of LBP to a murine macrophage cell line enhanced LPS-induced TNF-alpha synthesis, acute phase concentrations of LBP blocked this effect in comparison to low-dose LBP. When injected into mice intraperitoneally, LBP inhibited LPS-mediated cytokine release and prevented hepatic failure resulting in a significantly decreased mortality rate in LPS-challenged and D-galactosamine-sensitized mice, as well as in a murine model of bacteremia. These results complement a recent study revealing LBP-deficient mice to be dramatically more susceptible to an intraperitoneal Salmonella infection as compared with normal mice. We conclude that acute phase LBP has a protective effect against LPS and bacterial infection and may represent a physiologic defense mechanism against infection. Despite the limitations of any murine sepsis model, the results shown may imply that LBP could have beneficial effects during gram-negative peritonitis in humans.
Innate immune recognition is mediated by a system of germline-encoded receptors that recognize conserved molecular patterns that are associated with microbial pathogens. These receptors are coupled to signal transduction pathways that control expression of a variety of inducible immune-response genes. Toll receptors and the associated signaling pathways of nuclear factor kappaB may represent the most ancient host defense system found in mammals, insects and plants.
TLR4 is a member of the recently identified Toll-like receptor family of proteins and has been putatively identified as Lps, the gene necessary for potent responses to lipopolysaccharide in mammals. In order to determine whether TLR4 is involved
in lipopolysaccharide-induced activation of the nuclear factor-κB (NF-κB) pathway, HEK 293 cells were transiently transfected
with human TLR4 cDNA and an NF-κB-dependent luciferase reporter plasmid followed by stimulation with lipopolysaccharide/CD14
complexes. The results demonstrate that lipopolysaccharide stimulates NF-κB-mediated gene expression in cells transfected
with the TLR4 gene in a dose- and time-dependent fashion. Furthermore, E5531, a lipopolysaccharide antagonist, blocked TLR4-mediated
transgene activation in a dose-dependent manner (IC50∼30 nm). These data demonstrate that TLR4 is involved in lipopolysaccharide signaling and serves as a cell-surface co-receptor for
CD14, leading to lipopolysaccharide-mediated NF-κB activation and subsequent cellular events.
Toll-like receptor 4 (TLR4) is a mammalian homologue of Drosophila Toll, a leucine-rich repeat molecule that can trigger innate responses against pathogens. The TLR4 gene has recently been shown to be mutated in C3H/HeJ and C57BL/10ScCr mice, both of which are low responders to lipopolysaccharide (LPS). TLR4 may be a long-sought receptor for LPS. However, transfection of TLR4 does not confer LPS responsiveness on a recipient cell line, suggesting a requirement for an additional molecule. Here, we report that a novel molecule, MD-2, is requisite for LPS signaling of TLR4. MD-2 is physically associated with TLR4 on the cell surface and confers responsiveness to LPS. MD-2 is thus a link between TLR4 and LPS signaling. Identification of this new receptor complex has potential implications for understanding host defense, as well as pathophysiologic, mechanisms.
The iron-binding protein lactoferrin (Lf) is a constituent of neutrophil secondary granules and is discharged into the surrounding medium when neutrophils are activated. Lf released from neutrophils phagocytosing opsonized particles inhibits proliferation of mixed lymphocyte cultures (MLC) and has also been shown to inhibit granulopoiesis, suppress antibody production, and regulate natural killer cell activity. All of these processes are controlled by cytokines, suggesting that Lf may modulate immune responses by inhibiting cytokine activity. When MLC were cultured in round-bottomed wells to crowd the cells together, Lf, 50% saturated with iron, inhibited both proliferation and interleukin-2 (IL-2) release into the supernatants. Inhibition was concentration-dependent and lost at concentrations of Lf greater than 10(-12) mol/L. Lf at 10(-10) mol/L inhibited release of tumor necrosis factor-alpha (TNF) and interleukin- 1 beta (IL-1) into MLC supernatants, as well as inhibiting IL-2 release. TNF in the supernatant was significantly reduced at 5 and 24 hours, becoming less and losing significance by 72 hours. IL-1 in the supernatant was not significantly reduced at 5 and 24 hours, becoming significant at 48 and 72 hours. IL-2 was significantly reduced at 48 and 72 hours and followed the same time course as proliferation. Inhibition was blocked by specific antiserum to Lf, but not by a preimmune serum. Lf, 10(-10) mol/L, also inhibited the production of TNF (49.15% +/- 7.98%; n = 10, P = .032) and IL-1 (42.67% +/- 6.72%; n = 6, P = .032) from endotoxin-stimulated mononuclear cells. As with MLC, inhibition was dose-dependent and abrogated by specific antiserum. Lf did not block the biological action of TNF, IL-1, or IL-2 in specific assays using cytokine-sensitive cell lines. These data suggest that Lf, released from activated neutrophils, acts as a negative feedback mechanism to prevent recruitment and activation of leukocytes in sites of inflammation.
Neutrophils can inactivate lipopolysaccharide (LPS), thereby blocking the ability of LPS to prime fresh neutrophils for enhanced fMLP-triggered release of superoxide. Here we show that inactivation of LPS by neutrophils was primarily due to lactoferrin. A time course for inactivating LPS showed that neutrophils (5 million/ml) took 30 min to inactivate 10 ng/ml LPS. Mononuclear cells could not inactivate LPS under the same conditions. Experiments with radioactive LPS showed that inactivated LPS remained in the medium and was not taken up or destroyed by the neutrophils during inactivation. Inactivated IPS still gelled Limulus lysate and primed monocytes. Cell-free medium from neutrophil suspensions also inactivated LPS. A single LPS-inactivating factor was purified from medium by heparin-agarose chromatography. SDS-PAGE showed a single band at 80 kDa, which was identified as lactoferrin by immunoblotting. Antilactoferrin immunoglobulin G removed the LPS-inactivating activity from purified lactoferrin and cell-free medium. Surprisingly, even purified neutrophil lactoferrin required 30 min to inactivate LPS, indicating inherently slow binding of lactoferrin to LPS J. Leukoc. Biol. 57: 865–874; 1995.
Lactoferrin is an 80-kDa, iron-binding glycoprotein present in milk and, to a lesser extent, in exocrine fluids such as bib and tears. It consists of a single-chain polypeptide with two gobular lobes and is relatively resistant to proteolysis. The complete cDNAs for lactoferrin from human milk, neutrophils, and bovine milk have been reported, and recombinant proteins have been produced. Owing to its iron-binding properties, lactoferrin has been proposed to play a role in iron uptake by the intestinal mucosa and to act as a bacteriostatic agent by withholding iron from iron-requiring bacteria. Its presence in neutrophils and its release during inflammation suggest that lactoferrin is also involved in phagocytic killing and immune responses. Additionally, lactoferrin may function in ways not related to iron-binding, e.g. as a growth factor and as a bactericidal agent. This review attempts to evaluate these proposed functions and their biological significance in more detail.
The parameters defining the optical design for X-ray scattering and diffraction experiments with synchrotron radiation are derived and the various approaches are reviewed. A few selected examples illustrate the advantages of synchrotron radiation methods for various applications on poorly ordered systems.
The general theory of Fourier self-deconvolution, i.e., spectral deconvolution using Fourier transforms and the intrinsic line-shape, is developed. The method provides a way of computationally resolving overlapped lines that can not be instrumentally resolved due to their intrinsic
linewidth. Examples of the application of the technique to synthetic and experimental infrared spectra are presented, and potential applications are discussed. It is shown that lines in spectra having moderate signal/noise ratios (∼1000) can readily be reduced in width by a factor of 3.
The method is applicable to a variety of spectroscopic techniques.
Attenuated total reflection Fourier-transform infrared spectroscopy of thin hydrated films of soluble and membrane protein included in a phospholipid bilayer is shown to provide useful information as to the secondary structure of the protein. The analysis of the amide I band of deuterated samples by Fourier self-deconvolution followed by a curve fitting was performed by a new procedure in which all the input parameters are generated by the computer rather than by the investigator. The results of this analysis provide a correct estimation of the -helix and β-sheet structure content with a standard deviation of 8.6% when X-ray structures are taken as a reference. We also show that the orientation of the different secondary structures resolved by the Fourier self-deconvolution/curve-fitting procedure and of the phospholipid acyl chains can be simultaneously evaluated for membrane proteins reconstituted in a lipid bilayer. Of special interest for reconstitution of membrane proteins, the lipid/protein ratio can be accurately and quickly determined from the infrared spectrum.
Lipopolysaccharides (LPS, endotoxin) represent a major virulence factor of Gram-negative bacteria, which can cause septic shock in mammals, including man. The lipid anchor of LPS to the bacterial outer membrane, lipid A, exhibits a peculiar chemical structure, harbours the ‘endotoxic principle’ of LPS and is also responsible for the expression of pathophysiological effects. Chemically modified lipid A can be endotoxically inactive, but may express strong antagonistic activity against endotoxically active LPS. By applying orientation measurements with attenuated total reflectance (ATR) infrared spectroscopy on hydrated lipid A samples, we show here that these different biological activities are directly correlated to the intrinsic conformation of lipid A. Bisphosphoryl-hexaacyl lipid A molecules with an asymmetric (4/2) distribution of the acyl chains linked to the diglucosamine backbone have a large tilt angle (> 45 °) of the diglucosamine backbone with respect to the membrane surface, a conical molecular shape (larger cross-section of the hydrophobic than the hydrophilic moiety), and are endotoxically highly active. Monophosphoryl hexaacyl lipid A has a smaller tilt angle, and the conical shape is less expressed in favour of a more cylindrical shape. This correlates with decreasing endotoxic activity. Penta- and tetraacyl lipid A or hexaacyl lipid A with a symmetric acyl chain distribution (3/3) have a small tilt angle (< 25 °) and a cylindrical shape and are endotoxically inactive, but may be antagonistic.
A position sensitive proportional counter is described which shows some novel features in the method of position encoding compared with previous systems. The encoding is done by an inductance???capacity (L???C) line connected to cathode strips, rather than by resistance???capacity encoding (high resistance) using the anode wire. The spatial resolution for a counter length of 8 cm is 0.2 mm (which corresponds to 0.01?? for the geometry used here). Counting can be at high rates for indefinite periods.
The hardware and software of the data appraisal, evaluation and display systems used for synchrotron radiation experiments at the EMBL Outstation on the storage ring DORIS are described.The system is based on CAMAC hardware and modular software and has been extensively used for a variety of applications in EXAFS and small angle scattering and diffraction.
The unique germfree, colostrum-deprived, immunologically "virgin" piglet model was used to evaluate the ability of lactoferrin (LF) to protect against lethal shock induced by intravenously administered endotoxin. Piglets were fed LF or bovine serum albumin (BSA) prior to challenge with intravenous Escherichia coli lipopolysaccharide (LPS), and temperature, clinical symptoms, and mortality were tracked for 48 h following LPS administration. Prefeeding with LF resulted in a significant decrease in piglet mortality compared to feeding with BSA (16.7 versus 73.7% mortality, P < 0.001). Protection against the LPS challenge by LF was also correlated with both resistance to induction of hypothermia by endotoxin and an overall increase in wellness, as quantified by a toxicity score developed for these studies. In vitro studies using a flow cytometric assay system demonstrated that LPS binding to porcine monocytes was inhibited by LF in a dose-dependent fashion, suggesting that the mechanism of LF action in vivo may be inhibition of LPS binding to monocytes/macrophages and, in turn, prevention of induction of monocyte/macrophage-derived inflammatory-toxic cytokines.
The cationic polypeptide polymyxin B, has been demonstrated to form a stable molecular complex with the lipid A region of bacterial lipopolysaccharides. The interaction between polymyxin B and LPS appears to be stoichiometric, with several lines of evidence suggesting that one molecule of polymyxin B binds one monomer unit of LPS. The formation of LPS-polymyxin B complexes results in the generation of higher mol. wt aggregates with decreased isopynic density.
Leukocytes respond to lipopolysaccharide (LPS) at nanogram per milliliter concentrations with secretion of cytokines such as tumor necrosis factor-alpha (TNF-alpha). Excess secretion of TNF-alpha causes endotoxic shock, an often fatal complication of infection. LPS in the bloodstream rapidly binds to the serum protein, lipopolysaccharide binding protein (LBP), and cellular responses to physiological levels of LPS are dependent on LBP. CD14, a differentiation antigen of monocytes, was found to bind complexes of LPS and LBP, and blockade of CD14 with monoclonal antibodies prevented synthesis of TNF-alpha by whole blood incubated with LPS. Thus, LPS may induce responses by interacting with a soluble binding protein in serum that then binds the cell surface protein CD14.
For the past ten years, several groups were engaged in synthetic studies of lipid A, namely the lipid portion of bacterial lipopolysaccharides (LPS) that has been assumed to be the bioactive center of LPS, but has not been unanimously approved. Among them, Shiba, Kusumoto, and colleagues, Osaka, Japan have synthesized most energetically and successfully a variety of counterparts of lipid As, biosynthetic lipid A precursors, and their analogs. The endotoxic and related bioactivities of these synthetic compounds were studied by Japanese and German groups, including ours. In 1985, one of the compounds, having an acylation and phosphorylation pattern in beta(1-6)-D-glucosamine disaccharide which was proposed for Escherichia coli F515 lipid A was found to be exhibit full endotoxic and related bioactivities identical to those of the bacterial product. The study was extended by synthesis and examination of bioactivities of variously acylated D-glucosamine di- and monosaccharide phosphates, which correspond to structural components of lipid As, and their analogs or derivatives. Thus, structural requirements have been fairly well elucidated. In this article, first we will review the progress of synthetic and biological studies, with particular emphasis on chemical structure--bioactivities relationships of lipid As, and then we will discuss possible usefulness of some less or nontoxic lipid A-related synthetic compounds in clinical and preventive medicine.
By utilizing Limulus Amebocyte Lysate (LAL) and a chromogenic peptide substrate it is now possible to determine endotoxin concentrations quantitatively down to 10 EU/L (1 pg/mL) in a two stage assay. The optimal reaction conditions found for the two stages of the method, the endotoxin reaction with LAL and the measurement of the activation with the chromogenic substrate, are briefly described. The properties of the final kit reagents have also been investigated and the results are included. Finally, the usefulness of the present method is demonstrated by results obtained from testing therapeutical products as well as clinical plasmas. Some factors which may be critical in the performance of the assay are discussed.
Application of vibrational spectroscopy to the problem of structure determination of molecules of biological interest goes back to the early uses of raman and infrared spectroscopy in the study of organic molecules. For reviews of earlier work the reader is referred to compilations by Kohlrausch (1943) and by Jones and Sandorfy (1956), whereas more recently a comprehensive discussion has been presented by Bellamy (1975). These compilations accentuate the correlation of vibrational spectra with molecular structure from an essentially empirical point of view and culminate in the establishment of empirical correlation charts. For typical examples the reader is referred to Weast (1974) and Bellamy (1975). There have been many treatments of the theoretical basis of molecular vibrational spectroscopy. Among them the classical work by Herzberg (1945) and by Wilson et al. (1955) should be mentioned. Applications of infrared spectroscopy (IR) to structure problems of biological interest have been summarized by Susi (1969), Fraser and MacRae (1973), and Wallach and Winzler (1974). It was remarked quite eraly that relevant structural information about biological systems often requires study in aqueous solution, which forms the natural environment for most biologically important systems. Besides critical control of experimental conditions and samples the conventional methods of raman spectroscopy may be applied to aqueous solutions in a quite straightforward manner, cf. the contribution by Lord and Mendelson, Chapter 8.
The activation of monocytes, macrophages, and neutrophils by lipopolysaccharides (LPS) involves a serum protein, LPS binding protein (LBP), and a membrane protein, CD14. Evidence to date suggests a pathway in which serum LPS first binds to LBP. The LPS-LBP complexes then interact with CD14, leading to cellular activation. This scheme is supported by experiments in which either LBP or CD14 are blocked by antibodies as well as experiments in which LBP is added to serum free media and CD14 is expressed on cells which normally lack CD14. Discovery of this pathway suggests novel approaches to anti-LPS therapy.
Lactoferrin (LF), a cationic 80-kDa protein present in polymorphonuclear leukocytes and in mucosal secretions, is known to have antibacterial effects on gram-negative bacteria, with a concomitant release of lipopolysaccharides (LPS, endotoxin). In addition, LF is known to decrease LPS-induced cytokine release by monocytes and LPS priming of polymorphonuclear leukocytes. Its mechanism of action is incompletely understood. We have now demonstrated by in vitro-binding studies that LF binds directly to isolated lipid A and intact LPS of clinically relevant serotypes of the species which most frequently cause bacteremia (Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa), as well as to lipid A and LPS of mucosal pathogens (among others, Neisseria meningitides and Haemophilus influenzae). Binding to LPS was inhibitable by lipid A and polymyxin B but not by KDO (3-deoxy-D-manno-octulosonate), a glycoside residue present in the inner core of LPS. Binding of LF to lipid A was saturable, and an affinity constant of 2 x 10(9) M-1 was calculated for the LF-lipid A interaction. Our data may explain, in part, the mechanism whereby LF exerts its antibacterial and anti-endotoxic effects. Further studies on the ability of LF to block the detrimental effects of LPS, both in vitro and in vivo, are warranted.
The effects of bovine lactoferrin on the serum cytokine levels, induced by lipopolysaccharide (LPS) in mice, are described. Bovine lactoferrin (BLF) introduced intravenously, 24 hours before i.v. injection of 50 micrograms of LPS, significantly lowered the serum concentration of TNF-alpha. Doses of BLF lower than 100 micrograms as well as pretreatment of mice with BLF on days 6-2 or 12-2 hours before LPS challenge, were not effective. Moreover, BLF induces by itself a relatively high level of IL-6, peaking at 1 hour following injection. Pretreatment of LPS-injected mice with BLF causes, in addition, a small but statistically significant drop in IL-6 level. Human albumin, used as a control protein, did not cause any changes in the cytokine levels. The data reported herein provide a satisfactory explanation with regard to preventive activity of LF in infection.
The three-dimensional supramolecular structures and the states of order of the acyl chains of lipid A from different Gram-negative species were investigated at 40°C, high water content (80–90%), and different [lipid A]/[Mg²⁺] molar ratios using synchrotron radiation X-ray diffraction and Fourier-transform infrared spectroscopy. Measurements were made on free lipid A from Salmonella minnesota R595, mono- and bi-phosphoryl, as well as those from the non-enterobacterial strains Rhodobacter capsulatus 37 b4, Rhodopseudomonas viridis F, and Rhodocyclus gelatinosus 29/1. Parallel to differences in their chemical primary structure, the structural polymorphisms and states of order at 37°C of the non-enterobacterial lipid A were found to be different from those of enterobacterial lipid A. A clear correlation between the supramolecular structure and previously determined biological activities was found. Lipid A with a strong preference for lamellar structures (Rb. capsulatus and Rp. viridis) are endotoxically inactive and lack cytokine-inducing capacity; the compounds assuming a mixed lamellar/nonlamellar structure (monophosphoryl lipid A from S. minnesota) are of lower toxicity in vivo, but may induce cytokines in vitro; those lipid A with a strong tendency to form non-lamellar inverted structures (lipid A from S. minnesota and Rc. gelatinosus) exhibit full endotoxicity in vitro and in vivo. In contrast, anti-complementary activity is most pronounced for compounds with lamellar and least expressed for those with inverted structures. The states of order at 37°C vary non-systematically, exhibiting the highest values for lipid A of S. minnesota and the lowest for that of Rc. gelatinosus.
Lactoferrin is an 80-kDa, iron-binding glycoprotein present in milk and, to a lesser extent, in exocrine fluids such as bile and tears. It consists of a single-chain polypeptide with two gobular lobes and is relatively resistant to proteolysis. The complete cDNAs for lactoferrin from human milk, neutrophils, and bovine milk have been reported, and recombinant proteins have been produced. Owing to its iron-binding properties, lactoferrin has been proposed to play a role in iron uptake by the intestinal mucosa and to act as a bacteriostatic agent by withholding iron from iron-requiring bacteria. Its presence in neutrophils and its release during inflammation suggest that lactoferrin is also involved in phagocytic killing and immune responses. Additionally, lactoferrin may function in ways not related to iron-binding, e.g. as a growth factor and as a bactericidal agent. This review attempts to evaluate these proposed functions and their biological significance in more detail.
We have demonstrated that egg-white lysozyme (EW-LZM) bound to lipopolysaccharide (LPS), reduced the lethal toxicity and the biological activity of LPS. In this study, the interaction of LPS with murine lysozyme (M-LZM) and the modulation of biological activities were investigated. M-LZM was prepared from the culture supernatant of the murine macrophage cell line RAW264.7 by ion-exchange and gel filtration chromatographies and dialysis. Two types of M-LZM, murine M lysozyme (MM-LZM) and murine P lysozyme (MP-LZM), were purified from the supernatant. The enzymatic activities of both MM-LZM and MP-LZM were inhibited by LPS and their effects were affected by the temperature and the ionic strength. TNF-alpha production from RAW264.7 by LPS was inhibited by mixing with MM-LZM and MP-LZM. MP-LZM inhibited TNF-alpha production stronger than MM-LZM. Considering these facts, we suggested that M-LZM, like EW-LZM, make a complex with LPS to reduce the toxicity of LPS together with inhibiting the enzymatic activity.
The discovery of endotoxin dates from the late nineteenth century when Richard Pfeiffer, then working in Berlin, characterized endotoxins as heat-stable and cell-associated molecules (Westphal et al. 1977), thus distinguishing them from the heat-labile and proteinous exotoxins which are actively secreted by bacteria (Bhakdi et al. 1994). They were first found to be produced by Vibrio cholerae bacteria and later by Salmonella and Serratia. Endotoxins, due to their various potent biological activities soon attracted worldwide scientific interest. Initial chemical analyses of purified endotoxin indicated that it consists essentially of polysaccharide and lipid, and it was therefore termed lipopolysaccharide (LPS). Today the terms endotoxin (Wolff 1904) and lipopolysaccharide (Shear and Turner 1943) are used synonymously for the same molecule.
Human milk is in several ways anti-inflammatory. This study investigates whether or not human milk lactoferrin (LF) in comparison with bovine LF can affect the IL-6 release from human cells. Human, as well as bovine, LF and a bactericidal pepsin-derived fragment of bovine LF (lactoferricin B) were found to suppress the IL-6 response in a monocytic cell line (THP-1) when stimulated by lipopolysaccharide (LPS). The suppression of bovine LF was similar to or higher than that of human LF. Lactoferricin B was the strongest inhibitor of the LPS-induced IL-6 response. A time-dependence regarding the inhibitory capacity of LF was found. For human LF, the strongest inhibition was observed when added 15-30 min after the addition of LPS. Addition of LF before the LPS induced an approximately 45% reduction of the IL-6 response. The results suggest an anti-inflammatory activity of both human and bovine LF, and of the LF fragment lactoferricin B through their suppressive effects on the cytokine release.
Lipopolysaccharides (LPS, endotoxin) stimulate mononuclear cells to release cytokines which initiate endotoxic effects. Interaction of LPS at low concentrations with target cells is CD14-dependent whereas at high LPS concentrations it is CD14-independent. Here, we demonstrate by resonance energy transfer (RET) technique that nonspecific, CD14-independent intercalation of LPS into membrane systems can be mediated by lipopolysaccharide-binding protein (LBP). It is proposed that in this pathway, LBP breaks down LPS aggregates, transports the smaller units to and inserts them into the phospholipid cell matrix. We furthermore show that LBP also mediates the intercalation of other negatively charged amphiphilic molecules. We propose a model explaining CD14-independent cell activation at high endotoxin concentrations.
We have investigated the mechanisms of interaction of the recombinant N-terminal portion of bactericidal/permeability-increasing protein, rBPI21, with lipopolysaccharide (LPS) isolated from enterobacterial deep rough mutant strains. Experimentally, the ability of rBPI21 to form monolayers at the air/water interface and its action on lipid monolayers were analyzed. We have further studied the interaction of rBPI21 with aggregates from phospholipids and Re mutant LPS by infrared and resonance energy transfer spectroscopy and laser Doppler velocimetry. From monolayer experiments, the molecular area of a single rBPI21 molecule was estimated to be about 12 nm2. At lateral pressures of </=25 mN/m, rBPI21 incorporated into monolayers from negatively charged LPS and phosphatidylglycerol (PG) but not into those from neutral phosphatidylcholine. rBPI21 incorporated not only into monolayers but also into liposomes made from or containing negatively charged phospholipids, reducing the absolute value of the zeta-potential of LPS and PG aggregates. Furthermore, due to intercalation, rBPI21 caused the rigidification of the acyl chains of lipids in the gel as well as in the fluid phase and significantly immobilized their phosphate groups. High concentrations of Mg2+ ions were found to have a protective effect against the action of rBPI21. On the basis of these results, the biophysical characteristics of rBPI21 are discussed and a model is proposed as to how the rBPI21-induced influence on lipid monolayers and bilayers could explain rBPI21-mediated effects on the bacterial membrane.
Synthetic lipid A analogues and partial structures were analyzed and compared with natural hexaacyl lipid A from E. coli applying Fourier transform infrared spectroscopy. The investigations comprised (i) the measurement of the beta <=> alpha phase transition of the acyl chains via monitoring of the symmetric stretching vibration of the methylene groups, (ii) an estimation of the supramolecular aggregate structures evaluating vibrations from the interface like ester carbonyl and applying theoretical calculations (iii) a determination of the inter- and intramolecular conformations monitoring functional groups from the interface and the diglucosamine backbone (ester carbonyl, phosphate). The phase transition temperature Tc was found to be nearly a linear function of the number of acyl chains for most bisphosphoryl compounds indicating comparable packing density, whereas the deviating behaviour of some samples indicated a higher packing density. From the determination of the supramolecular aggregate structures (cubic, HII) of natural hexaacyl lipid A by X-ray small-angle diffraction, the existence of the same aggregate structures also for the synthetic hexaacyl lipid A was deduced from the nearly identical thermotropism of the ester carbonyl band. From this, a good approximation of the supramolecular structures of all synthetic samples was possible on the basis of the theory of Israelachvili. The analysis of the main phosphate band, together with that of the Tc data and former colorimetric results, allowed the establishment of a model of the intermolecular conformations of neighbouring lipid A/LPS molecules. The biological relevance of the findings is discussed in terms of the strongly varying biological activity (between high and no activity) of the samples.
The aggregate structures of lipid A, the 'endotoxic principle' of bacterial lipopolysaccharide (LPS), from rough mutant Salmonella enterica sv. Minnesota R595 was analyzed at different water content, cation (Mg2+) concentration, and temperature applying synchrotron radiation X-ray diffraction and, in selected cases, freeze-fracture electron microscopy. The X-ray diffraction spectra prove the existence of different lamellar, mixed lamellar/cubic, various cubic, and inverted hexagonal (HII) structures depending on ambient conditions. The three mainly bicontinuous cubic phases Q224, Q229, and Q230 can be observed between 30 and 50 degrees C in narrow water and cation concentration ranges. Above 50 degrees C, Q212 an intermediate phase between bicontinuous and micellar is adopted. In freeze-fracture electron microscopic experiments, cubic structures of these symmetries are not readily detected, which can be understood in the light of changes in hydration during freezing and the metastability of these phases. However, 'lipidic particles' closely related to cubic phases are observed. Above 65-70 degrees C, the existence of the HII phase with hexagonal periodicities dH between 4.0 and 6.0 nm for different hydration states is shown using both techniques. Possible biological implications for the preference of lipid A for nonlamellar structures are discussed.
The binding of lysozyme to bacterial deep rough mutant lipopolysaccharide (LPS) Re and to its lipid moiety lipid A, the 'endotoxic principle' of LPS, was investigated using biophysical techniques. The beta<-->alpha gel to liquid crystalline phase transition, the nature of the functional groups of the endotoxins, the secondary structure of lysozyme, and competition with polymyxin B were studied by Fourier-transform infrared spectroscopy (FTIR); the supramolecular aggregate structure of the endotoxins was determined with synchrotron radiation X-ray diffraction and the binding stoichiometry with microcalorimetry. The results were compared with those found with zwitterionic and negatively charged phospholipids. It can clearly be shown that lysozyme binds electrostatically to charged groups of the endotoxin molecules with the consequence of acyl-chain rigidification and an initiation of a transition from inverted cubic to multilamellar structures. The binding stoichiometry of endotoxin and lysozyme is a 3:1 molar ratio for both LPS Re and lipid A, indicating a dominant binding of lysozyme to the lipid A-phosphates. This could be confirmed by the analysis of a phosphate vibration and by the use of a dephospho LPS. Parallel to lysozyme binding to endotoxin, a conformational change of the secondary structure in the protein from mainly alpha helix to more unordered structures takes place, while the residual beta-sheet substructure does not exhibit a clear concentration dependence. Binding is found to be specific for the endotoxins since, for the zwitterionic phosphatidylcholine, no binding is observed and, for the negatively charged phosphatidylglycerol, only very weak binding is found. The results are discussed in the context of the ability of lysozyme to reduce endotoxicity.
Endotoxin (lipopolysaccharide [LPS]) is the major pathogenic factor of gram-negative septic shock, and endotoxin-induced death is associated with the host overproduction of tumor necrosis factor alpha (TNF-alpha). In the search for new antiendotoxin molecules, we studied the endotoxin-neutralizing capacity of a human lactoferrin-derived 33-mer synthetic peptide (GRRRRSVQWCAVSQPEATKCFQWQRNMRKVRGP; designated LF-33) representing the minimal sequence for lactoferrin binding to glycosaminoglycans. LF-33 inhibited the coagulation of the Limulus amebocyte lysate and the secretion of TNF-alpha by RAW 264.7 cells induced by lipid A and four different endotoxins with a potency comparable to that of polymyxin B. The first six residues at the N terminus of LF-33 were critical for its antiendotoxin activity. The endotoxin-neutralizing capacity of LF-33 and polymyxin B was attenuated by human serum. Coinjection of Escherichia coli LPS (125 ng) with LF-33 (2.5 microg) dramatically reduced the lethality of LPS in the galactosamine-sensitized mouse model. Significant protection of the mice against the lethal LPS challenge was also observed when LF-33 (100 microg) was given intravenously after intraperitoneal injection of LPS. Protection was correlated with a reduction in TNF-alpha levels in the mouse serum. These results demonstrate the endotoxin-neutralizing capability of LF-33 in vitro and in vivo and its potential use for the treatment of endotoxin-induced septic shock.
Until about 10 years ago the exact mechanisms controlling cellular responses to the endotoxin - or lipopolysaccharide (LPS) - of Gram-negative bacteria were unknown. Now a considerable body of evidence supports a model where LPS or LPS-containing particles (including intact bacteria) form complexes with a serum protein known as LPS-binding protein; the LPS in this complex is subsequently transferred to another protein which binds LPS, CD14. The latter is found on the plasma membrane of most cell types of the myeloid lineage as well as in the serum in its soluble form; LPS binding to these two forms of CD14 results in the activation of cell types of myeloid and nonmyeloid lineages, respectively.
Lactoferrin is an iron-binding glycoprotein found in exocrine secretions of mammals and released from neutrophilic granules during inflammation. This review describes the biological roles of lactoferrin in host defence. Secreted lactoferrin exerts antimicrobial action either by chelation of iron or by destabilization of bacterial membranes. Furthermore, lactoferrin modulates the inflammatory process, mainly by preventing the release of cytokines from monocytes and by regulating the proliferation and differentiation of immune cells. Some of these activities are related to the ability of lactoferrin to bind lipopolysaccharides (LPS) with high affinity. Indeed, recent in vitro studies indicate that lactoferrin is able to compete with the LPS-binding protein for LPS binding and therefore to prevent the transfer of LPS to CD14 present at the surface of monocytes. Moreover, the prophylactic properties of lactoferrin against septicemia in vivo have been demonstrated. Taken as a whole, these observations strongly suggest that lactoferrin is one of the key molecules which modulate the inflammatory response.
Lactoferrin is an iron-binding glycoprotein found in milk, exocrine secretions of mammals, and in secondary granules from polymorphonuclear neutrophils. This review describes the wide spectrum of functions ascribed to lactoferrin, with special emphasis on the antimicrobial properties of this protein, and its derived peptides.
The acyl chain packing of various endotoxins and phospholipids was monitored via the main wide-angle reflection between 0.410 and 0.460 nm by wide-angle X-ray scattering (WAXS) and via the absorption band of the symmetric stretching vibration of the methylene groups v(s)(CH(2)) around 2849 to 2853 cm(-1) by Fourier-transform infrared spectroscopy. The lipids investigated included various rough mutant (R) and smooth form (S) lipopolysaccharides (LPS) differing in the length of the sugar portion, lipid A, the "endotoxic principle" of LPS, and various saturated and unsaturated phospholipids with different head groups under a near physiological (>/=85%) water content. The packing density of the saturated endotoxin acyl chains is lower than those of saturated phospholipids but similar to those of monounsaturated phospholipids, each in the gel phase. The hydrophobic moiety of endotoxins thus exhibits significant conformational disorder already in the gel phase. The acyl chain packing of the endotoxins decreases with increasing length of the sugar chain lengths, which seems to be relevant to the observed differences in biological activity. For Re-LPS with different counterions (salt forms), in the presence of externally added cations or at reduced water content (50%), no change of the acyl chain packing density is deduced in the X-ray data, although the FT-IR data indicate its increase. The position of the v(s)(CH(2)) vibration is, thus, only a relative measure of lipid order, in particular when lipids with different head groups and in the presence of external agents are compared.
Lipopolysaccharide (LPS) represents a major virulence factor of Gram-negative bacteria ('endotoxin') that can cause septic shock in mammals including man. The lipid anchor of LPS to the outer membrane, lipid A, has a peculiar chemical structure, harbours the 'endotoxic principle' of LPS and is responsible for the expression of pathophysiological effects. Chemically modified lipid A can be endotoxically inactive, but may express strong antagonistic activity against LPS, a property that can be utilized in antisepsis treatment. We show here that these different biological activities are directly correlated with the molecular shape of lipid A. Only (hexaacyl) lipid A with a conical/concave shape, the cross-section of the hydrophobic region being larger than that of the hydrophilic region, exhibited strong interleukin-6 (IL-6)-inducing capacity. Most strikingly, a correlation between a cylindrical molecular shape of lipid A and antagonistic activity was established: IL-6 induction by enterobacterial LPS was inhibited by cylindrically shaped lipid A except for compounds with reduced headgroup charge. The antagonistic action is interpreted by assuming that lipid A molecules intercalate into the cytoplasmic membrane of mononuclear cells, and subsequently blocking of the putative signaling protein by the lipid A with cylindrical shape.