Proton NMR Spectra of Peracetylated Derivatives of α,α-Trehalose and Sucrose in the Presence of a Europium Shift Reagent

The possibility of using recombinant human lactoferrin from rice (rhLF) makes it necessary to study its differences from the protein of milk. In this work, the binding of different iron-saturated forms of rhLF to Caco-2 cells was studied. Iron-saturated rhLF bound in higher proportion than the apo-form, but, the data obtained for specific binding were not compatible with receptor-mediated binding. Competition assays showed the same binding capacity for human milk lactoferrin as for rhLF to Caco-2 cells. Another basic protein of milk, lactoperoxidase, was found to compete with rhLF for binding to Caco-2 cell membranes, suggesting an electrostatic interaction. The transport of iron (⁵⁹Fe) bound to rhLF and to citrate and the transport of rhLF (¹²⁵I-labeled) were studied on Caco-2 monolayers. Transport of iron was found to be significantly greater when bound to citrate than to rhLF. The amount of intact lactoferrin that traversed the Caco-2 monolayers was very low, suggesting degradation of it across these cells.     

Utilization of lactoferrin-bound and transferrin-bound iron by Campylobacter jejuni

Campylobacter jejuni NCTC 11168 was capable of growth to levels comparable with FeSO₄ in defined iron-limited medium (minimal essential medium alpha [MEMα]) containing ferrilactoferrin, ferritransferrin, or ferri-ovotransferrin. Iron was internalized in a contact-dependent manner, with 94% of cell-associated radioactivity from either ⁵⁵Fe-loaded transferrin or lactoferrin associated with the soluble cell fraction. Partitioning the iron source away from bacteria significantly decreased cellular growth. Excess cold transferrin or lactoferrin in cultures containing ⁵⁵Fe-loaded transferrin or lactoferrin resulted in reduced levels of ⁵⁵Fe uptake. Growth of C. jejuni in the presence of ferri- and an excess of apoprotein reduced overall levels of growth. Following incubation of cells in the presence of ferrilactoferrin, lactoferrin became associated with the cell surface; binding levels were higher after growth under iron limitation. A strain carrying a mutation in the cj0178 gene from the iron uptake system Cj0173c-Cj0178 demonstrated significantly reduced growth promotion in the presence of ferrilactoferrin in MEMα compared to wild type but was not affected in the presence of heme. Moreover, this mutant acquired less ⁵⁵Fe than wild type when incubated with ⁵⁵Fe-loaded protein and bound less lactoferrin. Complementation restored the wild-type phenotype when cells were grown with ferrilactoferrin. A mutant in the ABC transporter system permease gene (cj0174c) showed a small but significant growth reduction. The cj0176c-cj0177 intergenic region contains two separate Fur-regulated iron-repressible promoters. This is the first demonstration that C. jejuni is capable of acquiring iron from members of the transferrin protein family, and our data indicate a role for Cj0178 in this process.     

The binding of lactoferrin to glycosaminoglycans on enterocyte-like HT29-18-C1 cells is mediated through basic residues located in the N-terminus

Although lactoferrins (Lfs) isolated from milk of various mammals exhibit a close structural relationship, they show species-specific binding to cells. To define the specificity of recognition of human (hLf), bovine (bLf) and murine (mLf) lactoferrin by human intestinal cells, we analysed the binding of the three proteins to a subclone derived from human carcinoma cell line HT29. We observed that hLf and bLf interact with two types of binding sites (K_d: 63±22 nM; 0.7±0.2 μM) while mLf was recognized only by the lowest affinity binding sites with a lower number of binding sites. Using N-terminal deleted human Lf variants, we found that the sequence G¹RRRR⁵ is mainly responsible for the interactions with HT29 cells. Lactoferrin-binding sites on the surface of HT29 cells were further identified as heparan sulphate and chondroitin sulphate glycosaminoglycans. We conclude that the presence of the sequence A¹PRK⁴ in bLf and K¹ATT⁴ in mLf provides an insight into why the interaction of bLf with cell membrane-associated glycosaminoglycans is similar to that of hLf and why binding of these lactoferrin species differs from that of murine Lf.     

Seminal plasma of brown trout,Salmo trutta fario (L.) contains a factor able to retain iron at acid pH,typical feature of lactoferrin

Blood and seminal plasma of brown trout Salmo trutta fario were analyzed for their iron binding potential adopting two different methods. Seminal plasma showed an iron binding capacity that was retained even if samples were exposed at acid pH, similarly to mammalian lactoferrin that binds ferric iron also at acid pH. This suggests that the iron binding capacity is determined by a factor having a lactoferrin-like activity. Moreover, trout seminal plasma proteins were also analyzed in their pattern by sodium dodecyl sulphate polyacrylamide gel elecrophoresis (SDS-PAGE) and electroblotted onto nitrocellulose membrane. When seminal plasma was subjected to immunoblotting using goat anti-bovine lactoferrin antibodies as a probe, only a single band having an apparent molecular weight of around 80 kDa was specifically detected, showing that this protein has homology with bovine lactoferrin.     

How the surface functionalized nanoparticles affect conformation and activity of proteins: Exploring through protein-nanoparticle interactions

To understand the effect of counter ions (Na⁺) on the secondary conformation and functionality of the lysozyme, we have studied the interaction of lysozyme with counterion associated iron oxide nanoparticles (IONPs). The investigation was carried out at pH 7.4 and 9.0, with three different types of NPs, namely, bare IONPs, low molecular weight chitosan modified IONPs (LMWC-IONPs) and the counterion (Na⁺) associated sodium tripolyphosphate IONPs (STP-LMWC-IONPs) and confirmed by using various spectroscopy techniques. The difference in UV–vis absorbance (ΔA) between native and STP-LMWC-IONPs interacted hen egg white lysozyme (HEWL) was greater than that between native and NPs interacted HEWL at pH 9.0 compared with pH 7.4. Furthermore, STP-LMWC-IONPs exhibited quenching effect on lysozyme fluorescence spectrum at pH 9.0 due to binding of Na⁺ counterions to the protein, confirming denaturation of the latter. After HEWL interaction with STP-LMWC-IONPs (pH 9.0), CD spectra revealed a conformational change in the secondary structure of HEWL. Also, counterion induced lysozyme inactivation, due to interaction with nanoparticles at pH 9.0, was confirmed by enzymatic activity assay involving lysis of Micrococcus lysodeikticus. In conclusion, pH 9.0 was observed to be a more favorable condition, compared to pH 7.4, for the strongest electrostatic interaction between lysozyme and NPs. We postulate that the counterions in nanoparticle surface-coating can ameliorate protein misfolding or unfolding and also prevent their aggregation and, therefore, can be considered as a powerful and potential therapeutic strategy to treat incurable neurodegenerative disorders.     

Human lactoferrin receptor activity in non-encapsulated Haemophilus influenzae

Since the ability of bacteria to compete with lactoferrin for iron contributes to the pathogenesis of mucosal infections, the presence of lactoferrin receptor activity in non-encapsulated Haemophilus influenzae was investigated. The growth of 18 H. influenzae isolates from the sputum samples of chronic bronchitis patients and of six of seven H. influenzae throat isolates from healthy adults was stimulated by iron saturated human lactoferrin. Apo-lactoferrin did not stimulate the growth of H. influenzae. Human lactoferrin binding to iron limited bacteria was detected for 16 H. influenzae strains from chronic bronchitis patients and for five of seven isolates from healthy adults. We conclude that the majority of H. influenzae isolates tested bind human lactoferrin and that the iron from lactoferrin is used for growth.     

Nuclear magnetic resonance and ultraviolet difference spectral studies of the binding properties of turkey egg white lysozyme. Consequences of the replacement of Asp 101 by glycine

The nuclear magnetic resonance spectrum of the ¹⁹F nuclei in N-trifluoroacetylated chitotriose was studied in the presence of turkey lysozyme. In contrast to results previously obtained with hen lysozyme, the ¹⁹F nmr spectrum of the complex did not show any striking pH dependence. It was, in fact, very similar at all pH's to the spectrum of the trisaccharide complexed with hen lysozyme at low pH, where Asp 101 is protonated. The replacement of Asp 101 in turkey lysozyme by a glycine is thought to account for this difference and the results allow unequivocal assignment of a value of 4.2 to the pK_a of Asp 101 in hen lysozyme. The dissociation constant of the chitotriose-turkey lysozyme complex was measured at various pH's using uv difference methods and compared with that previously reported for the hen lysozyme-chitotriose complex. Again, the results could be attributed to the loss in binding energy due to the absence of Asp 101. In contrast to chitotriose, the binding of chitobiose and methyl-2-acetamido-2-deoxy-β-d-glucopyranoside as studied by both uv difference and nmr methods is the same within experimental error for turkey and hen lysozyme. The results obtained for binding of chitobiose suggest that Asp 101 does not contribute as much to the binding energy of the disaccharide as was previously thought. Finally, the specific activities of both of these lysozymes against Micrococcus lysodeikticus were found to be identical.     

Are lactoferrin receptors in Gram-negative bacteria viable vaccine targets?

A number of important Gram-negative pathogens that reside exclusively in the upper respiratory or genitourinary tract of their mammalian host rely on surface receptors that specifically bind host transferrin and lactoferrin as a source of iron for growth. The transferrin receptors have been targeted for vaccine development due to their critical role in acquiring iron during invasive infection and for survival on the mucosal surface. In this study, we focus on the lactoferrin receptors, determining their prevalence in pathogenic bacteria and comparing their prevalence in commensal Neisseria to other surface antigens targeted for vaccines; addressing the issue of a reservoir for vaccine escape and impact of vaccination on the microbiome. Since the selective release of the surface lipoprotein lactoferrin binding protein B by the NalP protease in Neisseria meningitidis argues against its utility as a vaccine target, we evaluated the release of outer membrane vesicles, and transferrin and lactoferrin binding in N. meningitidis and Moraxella catarrhalis. The results indicate that the presence of NalP reduces the binding of transferrin and lactoferrin by cells and native outer membrane vesicles, suggesting that NalP may impact all lipoprotein targets, thus this should not exclude lactoferrin binding protein B as a target.     

Specific adsorption of phosphate ions on proteins evidenced by capillary electrophoresis and reversed-phase high-performance liquid chromatography

Specific adsorption of phosphate ions at pH=7.0 was studied on different proteins, either counter-ions of phosphate (lysozyme, lactoferrin) or co-ion of phosphate (α-lactalbumin). The theoretical electrophoretic mobility of globular proteins lysozyme and α-lactalbumin (apo and holo (+1 calcium per molecule) forms) was compared with those measured by capillary electrophoresis in phosphate at pH 7.0, versus the ionic strength (I) in the range 0–0.775 mol L⁻¹. The specific adsorption of phosphate ions was evidenced by difference. From the experimental charge number (Z^eff) of protein in phosphate medium, a phosphate content per protein molecule was determined at pH=7.0.

• •For lactoferrin (pI=8–9), the electrophoretic mobility (μ) was constant and negative, highlighting a charge reversal due to phosphate adsorption.
• •For α-lactalbumin (holo form) experimental μ was roughly constant and more negative than predicted. Z^eff increased continuously from −4 to −11 in the ionic strength range from 0.005 to 0.775 mol l⁻¹, respectively. Accordingly, one to six phosphates were bound per molecule, respectively.
• •For lysozyme, experimental electrophoretic mobility was positive but lower than predicted. Z^eff was only discrete values +5 for I in the range 0.001–0.020 mol l⁻¹ and about +3 in the range 0.050–0.500 mol l⁻¹, whereas the theoretical Z value was +7 at pH=7.0. Lysozyme bounds one phosphate at low ionic strength and about two — three at higher ionic strength.

Reversed-phase HPLC confirms that adsorption of phosphate is different for the three proteins.     

Camel lactoferrin, a transferrin-cum-lactoferrin: crystal structure of camel apolactoferrin at 2.6 A resolution and structural basis of its dual role

Camel lactoferrin is the first protein from the transferrin superfamily that has been found to display the characteristic functions of iron binding and release of lactoferrin as well as transferrin simultaneously. It was remarkable to observe a wide pH demarcation in the release of iron from two lobes. It loses 50 % iron at pH 6.5 and the remaining 50 % iron is released only at pH values between 4.0 and 2.0. Furthermore, proteolytically generated N and C-lobes of camel lactoferrin showed that the C-lobe lost iron at pH 6.5, while the N-lobe lost it only at pH less than 4.0. In order to establish the structural basis of this striking observation, the purified camel apolactoferrin was crystallized. The crystals belong to monoclinic space group C2 with unit cell dimensions a=175.8 A, b=80.9 A, c=56.4 A, beta=92.4 degrees and Z=4. The structure has been determined by the molecular replacement method and refined to an R-factor of 0.198 (R-free=0.268) using all the data in the resolution range of 20.0-2.6 A. The overall structure of camel apolactoferrin folds into two lobes which contain four distinct domains. Both lobes adopt open conformations indicating wide distances between the iron binding residues in the native iron-free form of lactoferrin. The dispositions of various residues of the iron binding pocket of the N-lobe of camel apolactoferrin are similar to those of the N-lobe in human apolactoferrin, while the corresponding residues in the C-lobe show a striking similarity with those in the C-lobes of duck and hen apo-ovotransferrins. These observations indicate that the N-lobe of camel apolactoferrin is structurally very similar to the N-lobe of human apolactoferrin and the structure of the C-lobe of camel apolactoferrin matches closely with those of the hen and duck apo-ovotransferrins. These observations suggest that the iron binding and releasing behaviour of the N-lobe of camel lactoferrin is similar to that of the N-lobe of human lactoferrin, whereas that of the C-lobe resembles those of the C-lobes of duck and hen apo-ovotransferrins. Hence, it correlates with the observation of the N-lobe of camel lactoferrin losing iron at a low pH (4.0-2.0) as in other lactoferrins. On the other hand, the C-lobe of camel lactoferrin loses iron at higher pH (7.0-6.0) like transferrins suggesting its functional similarity to that of transferrins. Thus, camel lactoferrin can be termed as half lactoferrin and half transferrin.     

Binding of Extracellular Matrix Proteins by Enterococci

Forty-four enterococcal strains isolated from human clinical specimens were investigated for binding of ¹²⁵I-labeled fibronectin, vitronectin, thrombospondin, lactoferrin, and collagen type I and IV, and for cell surface hydrophobicity. Most strains expressed low binding of iodine-labeled human fibronectin, collagen I and IV, and higher binding of human vitronectin, human lactoferrin, and human thrombospondin. Bacteria grown in Todd-Hewitt broth exhibited increased binding to vitronectin and thrombospondin. In particle agglutination assays (PAA), Enterococcus faecalis strains reacted strongly with coated latex beads in contrast to E. faecium strains, which generally did not react. The ability of enterococci to bind ECM proteins was affected by heating and proteolytic digestion, suggesting that some protein-binding components become surface exposed after treatment with proteases. The binding of ¹²⁵I-labeled proteins to E. faecalis strain E70 was inhibited when cells were preincubated with unlabeled proteins. Preincubating cells with sulfated polymers such as dextran sulfate (M _r 5000 and 8000), pentosan sulfate and heparin decreased binding of vitronectin, lactoferrin, and thrombospondin. The binding of lactoferrin and thrombospondin was also decreased when bacteria were preincubated with galactose, fucose, and mannosamine, but not with mannose. All of 30 E. faecalis strains expressed pronounced surface hydrophobicity, but 10 of 14 E. faecium strains showed hydrophilic cell surface. Received: 22 April 1996 / Accepted: 29 June 1996     

ANIONIC SITES OF HUMAN ERYTHROCYTE MEMBRANES : I. Effects of Trypsin, Phospholipase C, and pH on the Topography of Bound Positively Charged Colloidal Particles

The effects of pH, trypsin, and phospholipase C on the topographic distribution of acidic anionic residues on human erythrocytes was investigated using colloidal iron hydroxide labeling of mounted, fixed ghost membranes. After glutaraldehyde fixation at pH 6.5–7.5, the positively charged colloidal particles were bound to the membranes in small randomly distributed clusters. The clusters of anionic sites were reversibly aggregated by incubation at pH 5.5 before fixation at the same pH. These results correlate with the distribution of intramembranous particles found by Pinto da Silva (J. Cell Biol. 53:777), with the exception that the distribution of anionic sites on a majority of the fixed ghosts at pH 4.5 was aggregated instead of dispersed. The randomly distributed clusters could be nonreversibly aggregated by trypsin or phospholipase C treatment of intact ghosts before glutaraldehyde fixation. Previous glutaraldehyde fixation prevented trypsin and pH induced aggregation of the colloidal iron sites. Evidence that N-acetylneuraminic acid groups are the principal acidic residues binding colloidal iron was the elimination of greater than 85% of the colloidal iron labeling to neuraminidase-treated cell membranes. Colloidal iron binding N-acetylneuraminic acid residues may reside on membrane molecules such as glycophorin, a sialoglycoprotein which contains the majority of the N-acetylneuraminic acid found on the human erythrocyte membrane.     

Pyrophosphate-Mediated Iron Acquisition from Transferrin in Neisseria meningitidis Does Not Require TonB Activity

The ability to acquire iron from various sources has been demonstrated to be a major determinant in the pathogenesis of Neisseria meningitidis. Outside the cells, iron is bound to transferrin in serum, or to lactoferrin in mucosal secretions. Meningococci can extract iron from iron-loaded human transferrin by the TbpA/TbpB outer membrane complex. Moreover, N. meningitidis expresses the LbpA/LbpB outer membrane complex, which can extract iron from iron-loaded human lactoferrin. Iron transport through the outer membrane requires energy provided by the ExbB-ExbD-TonB complex. After transportation through the outer membrane, iron is bound by periplasmic protein FbpA and is addressed to the FbpBC inner membrane transporter. Iron-complexing compounds like citrate and pyrophosphate have been shown to support meningococcal growth ex vivo. The use of iron pyrophosphate as an iron source by N. meningitidis was previously described, but has not been investigated. Pyrophosphate was shown to participate in iron transfer from transferrin to ferritin. In this report, we investigated the use of ferric pyrophosphate as an iron source by N. meningitidis both ex vivo and in a mouse model. We showed that pyrophosphate was able to sustain N. meningitidis growth when desferal was used as an iron chelator. Addition of a pyrophosphate analogue to bacterial suspension at millimolar concentrations supported N. meningitidis survival in the mouse model. Finally, we show that pyrophosphate enabled TonB-independent ex vivo use of iron-loaded human or bovine transferrin as an iron source by N. meningitidis. Our data suggest that, in addition to acquiring iron through sophisticated systems, N. meningitidis is able to use simple strategies to acquire iron from a wide range of sources so as to sustain bacterial survival.     

Quantitative studies of the non-productive binding of lysozyme to partially N-acetylated chitosans: Binding of large ligands to a one-dimensional binary lattice studied by a modified McGhee and von Hippel model

This paper considers the non-productive (inhibitory) binding of chitosans to lysozyme from chicken egg white. Chitosans are linear, binary heteropolysaccharides consisting of 2-acetamido-2-deoxy-β-d-glucose (GlcNAc; A-unit) and 2-amino-2-deoxy-β-d-glucose (GlcN, D-unit). The active site cleft of lysozyme can bind six consecutive sugar residues in subsites named A–F, and specific binding of chitosan sequences to lysozyme occurs with A-units in subsite C. Chitosans with different fractions of A-units (F_A) induced nearly identical changes in the ¹H NMR spectrum of lysozyme upon binding, and the concentration of bound lysozyme could be determined. The data were analysed using a modified version of the McGhee and von Hippel model for binding of large ligands to one-dimensional homogeneous lattices. The average value of the dissociation constant for different sequences that may bind to lysozyme (K^ave_D) was estimated, as well as the number of chitosan units covered by lysozyme upon binding. K^ave_D decreased with increasing F_A-values at pH* 3 and 4.5, while the opposite was true at pH* 5.5. Contributions from different hexamer sequences to K^ave_D of the chitosans were considered, and the data revealed interesting features with respect to binding of lysozyme to partially N-acetylated chitosans. The relevance of the present data with respect to understanding lysozyme degradation kinetics of chitosans is discussed.     

Human lactoferrin binds and removes the hemoglobin receptor protein of the periodontopathogen Porphyromonas gingivalis

Porphyromonas gingivalis possesses a hemoglobin receptor (HbR) protein on the cell surface as one of the major components of the hemoglobin utilization system in this periodontopathogenic bacterium. HbR is intragenically encoded by the genes of an arginine-specific cysteine proteinase (rgpA), lysine-specific cysteine proteinase (kgp), and a hemagglutinin (hagA). Here, we have demonstrated that human lactoferrin as well as hemoglobin have the abilities to bind purified HbR and the cell surface of P. gingivalis through HbR. The interaction of lactoferrin with HbR led to the release of HbR from the cell surface of P. gingivalis. This lactoferrin-mediated HbR release was inhibited by the cysteine proteinase inhibitors effective to the cysteine proteinases of P. gingivalis. P. gingivalis could not utilize lactoferrin for its growth as an iron source and, in contrast, lactoferrin inhibited the growth of the bacterium in a rich medium containing hemoglobin as the sole iron source. Lactoferricin B, a 25-amino acid-long peptide located at the N-lobe of bovine lactoferrin, caused the same effects on P. gingivalis cells as human lactoferrin, indicating that the effects of lactoferrin might be attributable to the lactoferricin region. These results suggest that lactoferrin has a bacteriostatic action on P. gingivalis by binding HbR, removing it from the cell surface, and consequently disrupting the iron uptake system from hemoglobin.     

CaMKII Binding to GluN2B Is Differentially Affected by Macromolecular Crowding Reagents

Binding of the Ca²⁺/calmodulin(CaM)-dependent protein kinase II (CaMKII) to the NMDA-type glutamate receptor (NMDAR) subunit GluN2B controls long-term potentiation (LTP), a form of synaptic plasticity thought to underlie learning and memory. Regulation of this interaction is well-studied biochemically, but not under conditions that mimic the macromolecular crowding found within cells. Notably, previous molecular crowding experiments with lysozyme indicated an effect on the CaMKII holoenzyme conformation. Here, we found that the effect of molecular crowding on Ca²⁺/CaM-induced CaMKII binding to immobilized GluN2B in vitro depended on the specific crowding reagent. While binding was reduced by lysozyme, it was enhanced by BSA. The ATP content in the BSA preparation caused CaMKII autophosphorylation at T286 during the binding reaction; however, enhanced binding was also observed when autophosphorylation was blocked. Importantly, the positive regulation by nucleotide and BSA (as well as other macromolecular crowding reagents) did not alleviate the requirement for CaMKII stimulation to induce GluN2B binding. The differential effect of lysozyme (14 kDa) and BSA (66 kDa) was not due to size difference, as both dextran-10 and dextran-70 enhanced binding. By contrast, crowding with immunoglobulin G (IgG) reduced binding. Notably, lysozyme and IgG but not BSA directly bound to Ca²⁺/CaM in an overlay assay, suggesting a competition of lysozyme and IgG with the Ca²⁺/CaM-stimulus that induces CaMKII/GluN2B binding. However, lysozyme negatively regulated binding even when it was instead induced by CaMKII T286 phosphorylation. Alternative modes of competition would be with CaMKII or GluN2B, and the negative effects of lysozyme and IgG indeed also correlated with specific or non-specific binding to the immobilized GluN2B. Thus, the effect of any specific crowding reagent can differ, depending on its additional direct effects on CaMKII/GluN2B binding. However, the results of this study also indicate that, in principle, macromolecular crowding enhances CaMKII binding to GluN2B.     

Adherence Inhibition of Cronobacter sakazakii to Intestinal Epithelial Cells by Lactoferrin

Cronobacter sakazakii is now recognized as an opportunistic pathogen and has been implicated in rare but severe cases of necrotizing enterocolitis, meningitis, and sepsis in neonates. The first step in bacterial pathogenesis requires that the organism adheres to host cells surfaces; therefore, agents that inhibit adherence might be useful for preventing infections. Lactoferrin, an iron binding protein found in milk, has been shown to inhibit bacterial adherence by direct interaction and disruption of bacterial surfaces. Therefore, the goal of this research was to assess the ability of two different types of bovine lactoferrin, alone and in combination with a 1:1 blend of galactooligosaccharides and polydextrose, to inhibit adherence of C. sakazakii to a HEp-2 human cell line. Results showed that the adherence of C. sakazakii was significantly reduced at a minimum lactoferrin concentration of 10 mg/ml. However, in combination with the oligosaccharide blend, no synergistic effect was observed in adherence inhibition. These results suggest that lactoferrin might interact with C. sakazakii and directly inhibit adhesion to tissue culture cells.     

The specificity of protection against cationic antimicrobial peptides by lactoferrin binding protein B

A variety of Gram-negative pathogens possess host-specific lactoferrin (Lf) receptors that mediate the acquisition of iron from host Lf. The integral membrane protein component of the receptor, lactoferrin binding protein A specifically binds host Lf and is required for acquisition of iron from Lf. In contrast, the role of the bi-lobed surface lipoprotein, lactoferrin binding protein B (LbpB), in Lf binding and iron acquisition is uncertain. A common feature of LbpBs from most species is the presence of clusters of negatively charged amino acids in the protein’s C-terminal lobe. Recently it has been shown that the negatively charged regions from the Neisseria meningitidis LbpB are responsible for protecting against an 11 amino acid cationic antimicrobial peptide (CAP), lactoferricin (Lfcin), derived from human Lf. In this study we investigated whether the LbpB confers resistance to other CAPs since N. meningitidis is likely to encounter other CAPs from the host. LbpB provided protection against the cathelicidin derived peptide, cathelicidin related antimicrobial peptide (mCRAMP), but did not confer protection against Tritrp 1 or LL37 under our experimental conditions. When tested against a range of rationally designed synthetic peptides, LbpB was shown to protect against IDR-1002 and IDR-0018 but not against HH-2 or HHC10.     

Production of human lysozyme in biofilm reactor and optimization of growth parameters of Kluyveromyces lactis K7

Lysozyme (1,4-β-N-acetylmuramidase) is a lytic enzyme, which degrades the bacterial cell wall. Lysozyme has been of interest in medicine, cosmetics, and food industries because of its anti-bactericidal effect. Kluyveromyces lactis K7 is a genetically modified organism that expresses human lysozyme. There is a need to improve the human lysozyme production by K. lactis K7 to make the human lysozyme more affordable. Biofilm reactor provides high biomass by including a solid support, which microorganisms grow around and within. Therefore, the aim of this study was to produce the human lysozyme in biofilm reactor and optimize the growth conditions of K. lactis K7 for the human lysozyme production in biofilm reactor with plastic composite support (PCS). The PCS, which includes polypropylene, soybean hull, soybean flour, bovine albumin, and salts, was selected based on biofilm formation on PCS (CFU/g), human lysozyme production (U/ml), and absorption of lysozyme inside the support. To find the optimum combination of growth parameters, a three-factor Box–Behnken design of response surface method was used. The results suggested that the optimum conditions for biomass and lysozyme productions were different (27 °C, pH 6, 1.33 vvm for biomass production; 25 °C, pH 4, no aeration for lysozyme production). Then, different pH and aeration shift strategies were tested to increase the biomass at the first step and then secrete the lysozyme after the shift. As a result, the lysozyme production amount (141 U/ml) at 25 °C without pH and aeration control was significantly higher than the lysozyme amount at evaluated pH and aeration shift conditions (p?<?0.05).