The Pepsin Hydrolysate of Bovine Lactoferrin

Probiotics & Antimicro. Prot. (2010) 2:112–119 DOI 10.1007/s12602-010-9039-2

The Pepsin Hydrolysate of Bovine Lactoferrin Causes a Collapse of the Membrane Potential in Escherichia coli O157:H7 Christopher Murdock • Michael L. Chikindas Karl R. Matthews

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Published online: 16 March 2010 Ó Springer Science + Business Media, LLC 2010

Abstract In the present study, the ability of bovine lactoferrin hydrolysate (LfH) to disrupt the cytoplasmic membrane of Escherichia coli O157:H7 was investigated. Lactoferrin and LfH antimicrobial activities were compared against E. coli O157:H7 and E. coli O157:H7 spheroplasts. The effect of LfH on the cytoplasmic membrane of E. coli O157:H7 cells was determined by evaluating potassium efflux (K?), dissipation of ATP and membrane potential (DW). LfH produced a rapid efflux of potassium ions, a decrease in intracellular levels of ATP coupled with a substantial increase in extracellular ATP levels and a complete dissipation of the DW. The results suggest that LfH causes a collapse of the membrane integrity by pore formation in the inner membrane, leading to the death of the cell. Moreover, the mechanism of action of LfH on E. coli O157:H7 appears to involve an interference with the inner membrane integrity based on experiments using E. coli O157:H7 spheroplasts. Keywords Lactoferrin Antimicrobial peptides Mode of action E. coli O157:H7

Introduction Foodborne illnesses associated with Escherichia coli O157:H7 is a major public health concern in the United States and throughout the world (36). According to the

C. Murdock M. L. Chikindas K. R. Matthews (&) Department of Food Science, Rutgers, The State University of New Jersey, 65 Dudley Road, New Brunswick, NJ 08901-8520, USA e-mail: [email protected]

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Centers for Disease Control, E. coli O157:H7 accounts for approximately 73,000 cases of foodborne illness and nearly 60 deaths per year. Additionally, this same report indicated that over the ten-year study period, these numbers increased. Finally, data also indicate that over 50% of these cases were transmitted by foods [36]. Cases of E. coli O157:H7 have been linked to consumption of contamination ground beef, produce (e.g., lettuce, apples, melons, sprouts) and dairy products (e.g., milk, butter, cheese, ice cream) [36]. E. coli O157:H7 has several characteristics that distinguish it from other strains of E. coli, most importantly are the production of Shiga toxins and a low infectious dose for susceptible populations [42]. Infections caused by E. coli O157:H7 can lead to a debilitating or deadly disease known as hemolytic uremic syndrome (HUS) characterized by hemolytic anemia, thrombocytopenia and renal injury (including failure in severe cases) [41]. The control of microbial pathogens in foods is a significant concern, and numerous methods have been employed to prevent the growth of pathogenic microorganisms in food, including the use of synthetic and natural antimicrobial agents such as lactoferrin (Lf), lysozyme, EDTA, bacteriocins, monolaurin, lactic acid and hydrogen peroxide [12, 15, 16, 34, 35]. The literature is rich with papers describing these antimicrobial compounds, and for some the mechanism of action has been detailed (e.g., nisin) [14, 18, 27]. However, while Lf has received considerable attention over the past several years, the mechanism of action has not been completely elucidated. Lf, a member of the transferrin protein family, is a cationic iron-binding glycoprotein that is found in many exocrine secretions, including milk, tears, saliva and serum and is potentially an extremely powerful and useful antimicrobial agent from milk [4, 5]. Lf is a single-chained molecule

Probiotics & Antimicro. Prot. (2010) 2:112–119

with a molecular weight of approximately 78 kDa, composed of two lobes with four domains that facilitate the reversible binding of two iron (Fe?) ions (or other divalent cations) between the inner faces of the inter domain clefts [18]. Lf exerts antimicrobial activity against Gram-negative and Gram-positive bacteria [1, 2, 8, 9, 13, 15, 29, 30, 34, 39]. However, when Lf binds divalent cations (e.g., Fe?, Ca?, Mg?), it loses much if not all of its antimicrobial activity, therefore, its activity would most likely be reduced in foods [5, 31, 38]. Studies investigating the antimicrobial activity of Lf in food systems do not demonstrate the same activity. Payne et al. [34] and Murdock and Matthews [28] demonstrated that Lf alone or in combination with EDTA or lysozyme in UHT milk had little practical effect against E. coli O157:H7, Pseudomonas fluorescens, S. Typhimurium or L. monocytogenes. Treatment of Lf with pepsin yields antibacterial peptides composed of two chains with varying lengths, typically 25 residues, corresponding to a region of the surface helix near the N-terminus [24, 25], that are termed lactoferricin (Lfcin) when purified. Additionally, the peptides lose the ability to bind divalent cations, which have been demonstrated to deplete the antimicrobial activity of the parent molecule, Lf [5, 30, 38]. Lfcin were reported to exhibit antibacterial activity against E. coli, P. fluorescens, L. monocytogenes and Bacillus cereus in peptone yeast extract glucose (PYG) medium at concentrations 4–80 times lower than the native molecule [13]. Similarly, Shin et al. [39] demonstrated the enhanced activity of lactoferrin hydrolysate (LfH) and Lfcin against E. coli O157:H7 in peptone broth. Others have reported a reduction of approximately 0.7 and 2 log CFU E. coli O157:H7 in 1% peptone solution containing 50 or 100 lg/mL Lfcin B, respectively [43]. Additionally, LfH was found to inhibit the growth of E. coli in carrot juice, ground beef and UHT milk [9, 28]. The mechanism of action of Lfcin peptide, which has the greatest antimicrobial activity, has yet to be defined. Lfcin was originally thought to inhibit cell growth by limiting the amount of iron in the environment; however, further studies demonstrated that it might act as a more general chelator, releasing lipopolysaccharide (LPS) from the outer membrane of Gram-negative bacteria [15]. These peptides can also interact with the binding sites on the LPS, causing distortion in the outer membrane integrity and ultimately forming pores or ‘‘blebs’’ [10]. Other research indicated that Lf and LfH might act by damaging the outer cell wall and destabilizing the cytoplasmic membrane [37, 45]. The objective of this study was to determine the activity of bovine LfH against the cytoplasmic membrane of E. coli O157:H7 as the potential mechanism of cell death.

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Materials and Methods Bacteria E. coli O157:H7 ATCC 43895 was streaked for purity on tryptic soy agar (TSA, Becton–Dickinson, USA), cultured in peptone yeast extract glucose (PYG) broth (1% peptone, 0.025% yeast extract and 1% glucose) and stocks were maintained in a 50:50 glycerol/ peptone broth at -20 °C. Working cultures were obtained by transferring a loop of frozen stock culture to TSA plate, incubating overnight at 35 ± 2 °C and transferring an isolated colony to PYG broth of sufficient volume for the experiment. Cultures were incubated in PYG broth at 35 ± 2 °C under agitation for 8–12 h for log-phase cells and 16–24 h for stationary-phase cells. Preparation of Lactoferrin and Lactoferrin Hydrolysate Bovine Lf obtained from Immu Cell Corporation (Portland, ME, USA) was stored at -20 °C until needed for individual studies. Lactoferrin was re-suspended at the appropriate concentrations needed for each individual study in sterile water (Fisher Scientific Water LC–MS Ultra Pure ChromosolvÒ), PYG broth or the appropriate buffer as indicated, and the solution was filter sterilized by passing through 0.45- and 0.22-lm pore size cellulose acetate filters (Pall Corporation AcrodiscÒ, Nalgene) in sequence. All experiments were performed using freshly prepared lactoferrin. The bovine Lf was determined to be [95% pure (data not shown) [28]. LfH was prepared using the methods described by Bellamy et al. [4, 5], and the resulting lyophilized powder was stored in sealed containers at -20 °C until re-suspended for use in individual experiments. Briefly, to prepare LfH, Lf was dissolved in sterile water (Fisher Scientific) at a concentration of 5% (5 g/100 mL). The pH was adjusted to 3.0 ± 0.1 using sterile 1 N HCl. Porcine pepsin (Sigma) was added to the solution to a final concentration of 3.0% weight to weight of substrate to pepsin. The mixture was incubated for 4 h at 37 °C under agitation. Following incubation, the reaction was terminated (i.e., inactivate the pepsin) by placing the solution in a preheated 80 °C water bath for 15 min. The mixture was subsequently cooled in an ice bath to room temperature. After cooling, the pH of the solution was adjusted to 7.0 ± 0.1 using sterile 1 N NaOH. The solids were removed by centrifugation (15,0009g, 30 min). To ensure removal of the heat-inactivated pepsin, the supernatant was passed through a 30,000 MW cutoff filter. The resulting supernatant was lyophilized and stored at -20 °C until re-suspended for each individual study. Based on SDS– PAGE, the preparation was [95% pure and contained peptides of approximately 8 and 3.5 kDa (data not shown) [28].

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Preparation of Spheroplasts

Potassium Efflux Assay

E. coli O157:H7 spheroplasts were generated using modifications of methods described previously [6]. From a frozen culture, a plate of TSA was streak inoculated and incubated for 18–24 h at 35 ± 2 °C. Nutrient broth was inoculated with a colony picked from the TSA plate and incubated for 18–24 h at 35 ± 2 °C under agitation. Subsequently, 100 ll of the culture was transferred to 10 mL of Penn Assay Medium (Difco) and incubated for 18–24 h at 35 ± 2 °C under agitation. To initiate transformation of the wild-type cells to spheroplasts, Penicillin G (1,000 IU/mL), lysozyme (200 mg/mL) and EDTA (0.05% wt/v) were added to the medium and the culture incubated for an additional 18–24 h at 35 ± 2 °C under agitation. The sample was then separated into two parts, and 1 mL was transferred to fresh Penn Assay Medium and incubated for 18–24 h at 35 ± 2 °C under agitation. The remaining portion of the sample was treated a second time with filter-sterilized Penicillin G (1,000 IU/mL), lysozyme (200 mg/mL) and EDTA (0.05% wt/v) and incubated for an additional 18–24 h at 35 ± 2 °C under agitation. The sample was then transferred to fresh Penn Assay Medium and incubated for an additional 18–24 h at 35 ± 2 °C under agitation. Photo micrographs were obtained throughout the process to document spheroplast formation (data not shown).

The effect of LfH on membrane permeability was determined through measuring K? leakage from LfH-treated E. coli O157:H7 with a potassium ion electrode (Jenco Electronics, Ltd, San Diego, CA) and a Jenco Microprocessor pH/Mv/Ion/Temp Meter (Jenco Electronics, LTD). To evaluate the effect of LfH against E. coli O157:H7, the ion potential response generated using the potassium ion electrode was converted to a percentage based on a baseline (untreated cells) and a maximum efflux (permeabilized cells) [32]. In brief, E. coli O157:H7 cells were grown to an OD600 of approximately 1.0 in 50 mL of sterile PYG broth and collected by centrifugation (15,0009g, 5 min) at 5 °C. The cells were washed twice with cold 10 mM Tris acetate, 100 mM NaCl, pH 7.4 ± 0.1 buffer and re-suspended in the same buffer to an OD600 of approximately 30 and retained on ice until used for each experiment. All cultures were used within 30 min. The concentrated cells were diluted 1:10 in fresh 10 mM Tris acetate, 100 mM NaCl, pH 7.4 ± 0.1 buffer prior to use in each experiment. The potassium efflux was measured over time for untreated cells, permeabilized and treated (exposed to 4,000 and 8,000 lg/mL LfH) cells. In each experiment, the efflux was monitored and recorded approximately every 30 s, excluding the total efflux from permeabilized cells, which was recorded as an initial reading and monitored for any increase over the same time period.

Lf and LfH Sensitivity Assay The ATP Assay The antimicrobial activity of Lf and LfH was determined using a modification of the methods described by Bellamy et al. [4, 5] and Shin et al. [39]. Minimum inhibitory concentrations (MIC) of Lf and LfH were determined using a temperature-controlled Dynex 96-well plate reader (MRX; Dynex Tech Inc., Chantilly, VA, USA) with software (Revelation) measuring optical density (OD) at 630 nm. PYG was added to all wells, and an equivalent volume of Lf or LfH re-suspended in PYG added to the first well and serially diluted (1:2 dilutions). The concentrations of Lf or LfH tested were 8,000, 4,000, 2,000, 1,000, 500, 250, 125 and 62.5 lg/mL. Logarithmic growth phase cell cultures of E. coli O157:H7 and the E. coli O157:H7 spheroplasts were added to corresponding wells to achieve ca. 104 CFU/well. The microtiter plate was incubated at 35 ± 2 °C for 18–24 h, and the OD630 of each well read every 15 min following two repeats of shaking for 5 s. The MIC was considered the lowest concentration of the Lf or LfH that showed no increase in OD. The experiment was conducted twice, in triplicate. Growth curves for wild-type and spheroplast cells were conducted using MIC levels of Lf and LfH.

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The effect of LfH on the cellular ATP content of E. coli O157:H7 was determined using the method described previously [7]. Briefly, E. coli O157:H7 cells were grown to mid-log phase in PYG broth and collected by centrifugation (15,0009g, 5 min) at 5 °C. The pellet was re-suspended and washed twice in equal volumes of 50 mM HEPES (Fisher Scientific, Pittsburgh, PA.). Cells were re-suspended in one half the original HEPES buffer volume and retained on ice for all studies. For the purpose of energization, the cells were re-suspended in one half of their original volume in 50 lM HEPES buffer containing 0.2% glucose and allowed to remain at room temperature for 20 min prior to use for time-dependent assays. All cultures were used within 30 min. Negative controls were prepared with 5,000 and 10,000 lg/mL LfH in HEPES buffer (e.g., antimicrobial buffer mix). Antimicrobial buffer mix was diluted 1:10 in fresh buffer and combined with an equal portion of ATP Assay Mix (enzyme/substrate preparation containing luciferase, luciferin, MgSO4, DTT, EDTA, BSA and tricine buffer salt). Measurements were taken immediately.


To determine extracellular levels of ATP, 100 lL of the cell suspension was diluted 1:10 in fresh buffer and combined with an equal portion of ATP Assay Mix. ATP measurements were taken using the commercially available ATP Bioluminescent Assay Kit (Sigma, St. Louis, MO.). This procedure uses the enzyme luciferase to oxidize luciferin to adenyl-luciferin in the presence of limiting ATP as the amount of ATP in the sample is proportional to the amount of light emitted and detected by a spectrophotometer (Luminoskan TL Plus luminometer, Labsystems Oy, Helsinki, Finland). Measurements were taken immediately. All samples were tested in duplicate, and all assays were repeated in triplicate. To determine total levels of ATP (intracellular and extracellular ATP), 100 lL of the cell suspension was diluted 1:10 in fresh buffer and centrifuged (10,0009g, 30 s). The resulting pellet was mixed with 10 lL dimethylsulfoxide (DMSO), held at room temperature for 5 min to permeabilize the cells, and then 990 lL of fresh buffer was added to the pellet. Equal portions of the permeabilized cell suspension and ATP Assay Mix were combined. Measurements were taken immediately. All samples were tested in duplicate, and all assays were repeated in triplicate. Time-dependent changes in intracellular and extracellular ATP levels were determined following exposure of the cell suspension to 5,000 and 10,000 lg/mL LfH. Intracellular ATP was determined by calculating the difference in the total and extracellular ATP concentrations. The cell dry weight (CDW) was determined according to Lee et al. [26], and Montville and Chikindas (personal communication) and used for normalization of all ATP values. DW Dissipation Assay The effect of LfH on the transmembrane electrical potential of the cells (DW) was determined using the method described by Herranz et al. [23]. E. coli O157:H7 cells were grown to mid-log phase in 50 mL PYG broth and centrifuged (15,0009g, 5 min) at 5 °C. The pellet was re-suspended and washed twice in equal volumes of 50 mM Potassium HEPES (K-HEPES) buffer (Fisher Scientific) pH 7.0 ± 0.1 with a final re-suspension in 1/100 of the original K-HEPES buffer volume in buffer containing 100 mM KPO4 (Sigma), 20% glucose (Sigma), 5 mM HEPES and 1 mM K-EDTA (Sigma) and retained on ice prior to use. All cultures were used within 30 min. The fluorescent probe, 3,3’-dipropyltyiadicarbocyanine iodide (Di-S-C3-(5)), was used to monitor changes in DW. The fluorescence measurements (PerkinElmer fluorescence spectrometer LS 50B, Fremont, CA) were initiated followed by the addition of 10 lL of Di-S-C3-(5) and 10 lL of the cell concentrate in buffer with quick mixing with the pipette. After the stabilization of the fluorescence signal, 5-lL nigericin (MP Biomedicals, Solon, OH) stock 5 mM, final concentration

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12.5 lM in 95% ethanol with 1 mM K-EDTA was added with quick mixing using the pipette. Subsequently, either buffer with 1 mM K-EDTA or LfH (5,000 and 10,000 lg/mL) in buffer with 1 mM K-EDTA was added to the cuvette. In all assays, 10-lL valinomycin (MP Biomedicals) stock 2 mM, final concentration 10 lM in 95% ethanol with 1 mM K-EDTA was added to collapse any remaining residual DW. A PerkinElmer fluorescence spectrometer LS 50B with excitation and emission wavelengths of 643 and 666 nm, respectively, with a 10-nm slit width and a 700-s assay duration with reading every 0.1 s was used for all assays. Membrane Permeability Assay The membrane permeability of E. coli O157:H7 was determined using the LIVE/DEADÒ BacLightTM Bacterial Viability Kit (Invitrogen Molecular Probes, Eugene, Oregon). E. coli O157:H7 cells were grown to mid-log phase in 50 mL PYG broth and collected by centrifugation (15,0009g, 5 min) at 5 °C. The pellet was re-suspended and washed twice in equal volumes of sterile water and then re-suspended in one half the original volume of sterile water and retained on ice prior to all studies. All cultures were used within 2 h. To determine the effect of LfH on E. coli O157:H7, the cells were mixed with MIC and 29 MIC levels of LfH and incubated at room temperature for 1 h. Following incubation, equal portions of the 29 stock solution of SYTOÒ -9/propidium iodide and the treated culture were mixed in a microfuge tube and incubated at room temperature in the dark for 15 min. Cells were visualized using an Olympus BH2-RFCA fluorescence microscope fitted with a Pixera camera. Five random fields were counted from each of three slides. All assays were conducted twice. The assay is based on membrane integrity, and therefore would demonstrate damage to the membrane caused by LfH. The assay utilizes a mixture of two stains, SYTOÒ-9 a membrane-permeable green-fluorescent nucleic acid stain, and propidium iodide a membrane-impermeable red-fluorescent nucleic acid stain. Under fluorescence microscopy, cells with intact membranes appear green and cells with damaged membranes appear red. Controls included cells that were not treated with LfH and cells treated with 70% isopropyl alcohol.

Results and Discussion Generation of E. coli O157:H7 Spheroplasts and Comparison of the Antimicrobial Activity of Lf and LfH on E. coli O157:H7 Cells and Spheroplasts The outer membrane consists of proteins, phospholipids, lipopolysaccharides (LPS) as well as other structures

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Induced Potassium Efflux from LfH-Treated E. coli O157:H7 Cells The release of significant amounts of potassium by a bacterial cell indicates that the membrane integrity is disrupted; therefore, the membrane potential may be dissipated as a consequence of the significant loss in cellular ion balance [3, 32]. Extracellular potassium (K?) concentrations of E. coli O157:H7 cells were determined prior to and after exposure to MIC levels of LfH. Untreated control samples were monitored over time to determine the K? efflux and relative amount of efflux due to normal cell culture growth and death. Results showed there was a 3–5% efflux of K? in the culture, most likely associated with normal cell death. The addition of MIC and 29 MIC concentrations of LfH resulted in a rapid K? efflux of 18–22% (within 1 min) followed by a slow steady increase to 40% within 10 min (Fig. 3). This represented ca. eightfold increase in K? efflux over the

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(e.g., porins) that transverse the outer membrane. The primary site of action for Lf has not yet been definitively determined. Research suggests that the antibacterial activity might be due to a destabilization or disruption of the outer membrane due to release of LPS. Additionally, the binding of Lf to porin may be involved by interference with the transfer of materials in and out of the cell [10, 17, 30, 37, 44, 45]. Yamauchi et al. [45] demonstrated a 58 and 45% release of LPS in the presence of Lf and Lfcin, respectively. The associated viability studies demonstrated a 1–2 log reduction for the whole molecule versus a 4–6 log reduction for the peptide (Lfcin). Other researchers have reported similar results [19, 44]. Additionally, researchers have demonstrated that Lf and Lfcin both bound to porins and caused a reduction in channel conductance (e.g., porin channel interference) and only slight reductions in cell viability (\2 log) [17, 30, 37]. The release of LPS and porin binding appear to have a bactericidal effect; however, the complete mechanism of action of Lf still has not been elucidated. To reduce or eliminate the potential for interaction of LfH with the outer membrane, via LPS or porin channels, E. coli O157:H7 spheroplasts were used [20]. Spheroplasts are characterized by partial or complete loss of the outer membrane and lack of cell rigid structure [33, 40]. Changes in cell morphology during development of E. coli O157:H7 spheroplasts were documented (data not shown). The inhibition of growth or killing of spheroplasts should be limited compared to wildtype cells if the outer membrane LPS or porin channels are required as part of the mechanism by which Lf/LfH exert antimicrobial activity. Results demonstrate similar growth patterns for intact control cells and spheroplast cells untreated or treated at MIC levels of Lf (4 mg/mL) and LfH (2 mg/mL; Figs. 1, 2).


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Fig. 2 Lactoferrin hydrolysate (2 mg/mL) inhibits growth of wildtype E. coli O157:H7 and spheroplast cells. Black square O157:H7 wild type, black triangle O157:H7 spheroplast, black circle negative control, square O157:H7 WT with LfH, triangle O157:H7 spheroplast with LfH

untreated control samples. Shin et al. [39] reported that Lfcin interacted with the cytoplasmic membrane resulting in formation of ‘‘debris in the cytoplasm’’. Haukland et al. [21] reported that lactoferricin B was able to cross the cytoplasmic membrane and become localized in the cytoplasm. It is presumed that cationic peptides, including Lfcin and LfH, that potentially form pores, act by thinning the membrane or by destabilizing the membrane permeability causing a loss of intracellular constituents [21, 22]. Loss of Intracellular and Extracellular ATP from LfH-Treated E. coli O157:H7 Cells Due to the size of the ATP molecule, it cannot freely pass across intact cell membranes. However, sufficient damage to the cell membrane by stress or exposure to select antimicrobial compounds can result in the release of ATP. The change in extracellular and intracellular levels of ATP for control and treated cells is shown in Fig. 4. Extracellular


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ion gradient to produce energy (e.g., ATP). When antimicrobials interrupt this balance, it can lead to a loss of membrane potential and ultimately cell death.

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Fig. 3 Lactoferrin hydrolysate induces time-dependent efflux of K? from E. coli O157:H7 cells. Black circle untreated cellular K? efflux, black square 5,000 lg/mL exposure, black triangle 10,000 lg/mL exposure

levels of ATP were significantly lower than intracellular ATP levels in control samples both initially and after 30 min of incubation. Initially, there was approximately 1 mM extracellular ATP/g of cells dry weight (CDW) and 27 mM intracellular ATP/g CDW that was typical for E. coli (range 15–36 nMol/g). This demonstrated little change over time in untreated control samples, most likely due to normal cell leakage and death [11]. Final levels in untreated control samples were approximately 2 mM extracellular ATP/g CDW and 25 mM intracellular ATP/g CDW. However, the addition of MIC and 29 MIC levels of LfH caused a slow progressive change in ATP concentration. Intracellular levels of ATP decreased nearly 50% from approximately 27 mM/g CDW to 14 mM/g CDW, while extracellular levels increased approximately fivefold from 2 to 9 mM/g CDW. The significant decrease in the intracellular level of ATP coupled with the simultaneous increase in extracellular ATP suggests cell membrane damage was causing a cellular ATP efflux (Fig. 4). The bioenergetics of a cell is essentially how a cell utilizes chemical and electrical systems to maintain membrane integrity and produce energy. Within a bacterial cell, the cytoplasmic membrane utilizes the energy (e.g., ATP) to maintain the balance of the ion gradient and utilizes the

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The DW component of the proton motive force was qualitatively examined by utilizing the fluorescent probe Di-SC3-(5). The addition of MIC and 29 MIC levels of LfH caused a rapid dissipation of DW in E coli O157:H7 cells. A similar dissipation was observed in the control assay when valinomycin was added. Valinomycin is a potassium ionophore that can dissipate residual membrane potential (Fig. 5). Cationic peptides have a high affinity for negatively charged surface structures such as lipopolysaccharides on Gram-negative bacteria. Once closely associated with the outer membrane, cationic peptides, such as Lfcin or LfH, may gain access to the inner or cytoplasmic membrane via permeabilization (removal of LPS) or self-promoted uptake. In each case, the actual action of the antimicrobial is associated with damage to the cytoplasmic membrane and not the minor damage to the outer membrane. After the LfH gains access to the cytoplasmic membrane, it creates pores that cause a large potassium efflux and a significant loss of ATP. This causes a disruption in the ion/energy balance maintained by the cytoplasmic membrane (membrane potential). If the damage is severe, for example, a 40% efflux of potassium and fivefold increase in extracellular ATP that occurred in the present study, the cell can no longer maintain the membrane potential. Change in Membrane Permeability of E. coli O157:H7 Following Treatment with LfH The disruption of membrane integrity of E. coli O157:H7 cells following treatment with MIC and 29 MIC levels of LfH was demonstrated using a differential staining method. The method is typically used to differentiate live from dead bacteria based on the membrane permeability to two nucleic acid stains. Results of the assay suggested that LfH causes disruption of membrane integrity and eventually cell death (Table 1). Approximately, 50% of the cells had membrane damage when exposed to MIC levels of LfH.

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Fig. 4 Lactoferrin hydrolysate induces time-dependent dissipation of ATP in E. coli O157:H7 cells. Untreated cellular ATP levels (black circle intracellular, circle extracellular), treated (dashed line) cellular ATP levels (black square intracellular, square extracellular)

The results of the present study suggest that the pepsin hydrolysate of lactoferrin (LfH) exerts its antimicrobial action on the inner membrane of E. coli O157:H7 by forming pores in the membrane. The pores lead to a loss of energy and ion balance, resulting in a collapse of DW and a

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Fig. 5 Determination of membrane potential (DW) in E. coli O157:H7 cells alone and in the presence of LfH. Control, upper line; treated sample, lower line


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loss of viability of the foodborne pathogen, E. coli O157:H7. Future research should focus on determining the potential for a docking molecule or outer membrane binding sites. Acknowledgments The authors thank Dr. John Rejman and the Immu Cell Corporation for providing Lf. The work described in this paper was funded in part by the New Jersey Agricultural Experiment Station.

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