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. Author manuscript; available in PMC: 2013 Sep 1.
Published in final edited form as: J Appl Microbiol. 2012 Jul 24;113(3):714–722. doi: 10.1111/j.1365-2672.2012.05376.x

Mode of action and safety of lactosporin, a novel antimicrobial protein produced by Bacillus coagulans ATCC 7050

Shadi Riazi 1, Sara E Dover 2, Michael L Chikindas 1,*
PMCID: PMC3419795  NIHMSID: NIHMS389657  PMID: 22737982

Abstract

Aims

To determine the mechanism of action of antimicrobial protein, lactosporin, against Gardnerella vaginalis and to evaluate its safety in-vitro.

Methods and Results

Bacillus coagulans ATCC 7050 was grown at 37 °C for 18 hours. The cell free supernatant was concentrated 10-fold and screened for antimicrobial activity against indicator strain Micrococcus luteus. The mode of action of lactosporin was determined by measuring the potassium release and monitoring the changes in transmembrane potential (Δψ) and transmembrane pH (ΔpH) of the sensitive cells. Lactosporin caused efflux of potassium ions from M. luteus cells and dissipation of ΔpH in G. vaginalis while it had no effect on the Δψ. The safety of lactosporin was evaluated by using EpiVaginal™ ectocervical (VEC-100) tissue model. Over 80% of the cells in the vaginal tissue remained viable after exposure to lactosporin for 24 hours.

Conclusions

Lactosporin potentially exerts its antimicrobial activity by selective dissipation of ΔpH and/or by causing leakage of ions from the sensitive cells. Safety studies suggest that lactosporin is a non-cytotoxix antimicrobial for vaginal application.

Significance and Impact of the Study

This study revealed that lactosporin is an effective and safe antimicrobial preparation with potential application for control of bacterial vaginosis.

Introduction

Bacteriocins are ribosomally-synthesized antimicrobial compounds of proteinaceous nature produced by virtually all microorganisms that act against closely related species (Klaenhammer 1993). Bacteriocins produced by lactic acid bacteria (LAB) have received great attention in the past decade due to their potential applications as food preservatives (Cleveland et al., 2001). Production of bacteriocins and bacteriocin-like inhibitory substances (BLIS) from the Bacillus genus have been studied by several research groups (e.g., Babasaki et al., 1985; Stein et al., 2004; Shelburne et al., 2007; Sutyak et al., 2007; Sutyak Noll et al., 2011). Many bacteriocins and BLIS kill sensitive cells by a common mechanism of action through the formation of a transient pore in the cytoplasmic membrane resulted in the leakage of small intracellular compounds and dissipation of proton motive force (PMF) component(s). PMF is an electrochemical proton gradient across the cytoplasmic membrane which is composed of two components; membrane potential (Δψ) and pH gradient (ΔpH). PMF is directly involved in a number of biological processes such as ATP synthesis, active ion transport, protein phosphorylation and bacterial motility (Montville and Bruno, 1994; Montville and Chen, 1998). Antimicrobial proteins may deplete either or both components of PMF and cause intracellular ATP depletion by either efflux or intracellular ATP hydrolysis (Chen and Montville, 1995; Montville et al. 1995).

Lactosporin is a novel antimicrobial protein produced by a strain of B. coagualns isolated from a dietary supplement, Lactospore® Probiotic (Sabinsa Corp., East Windsor, NJ, USA). Previous work has studied the characterization of this antimicrobial including inhibitory spectrum, pH, temperature and enzyme sensitivity, isoelectric point determination, antimicrobial activity visualization by SDS and native-PAGE and PCR analysis (Riazi et al. 2009). Lactosporin may have applications in feminine care products due to its antimicrobial activity against G. vaginalis (Riazi et al. 2009), one of the most frequent causative organisms in bacterial vaginosis (BV) (Turovskiy et al., 2011). BV is one the most common infections in women of reproductive age and approximately 10-15% of the female population are affected by this infection (Persaud et al. 2006). Effective BV treatment should avoid a negative impact on the healthy vaginal microflora such as Lactobacillus spp. Conventional treatments of BV with antibiotics such as metronidazole and clindamycin may have negative impact on the growth of vaginal lactobacilli (Aroutcheva et al., 2001) while lactosporin has no inhibitory effects against the healthy vaginal microflora (Riazi et al., 2009).

The aim of this research was to study the mechanism of action of lactosporin against G. vaginalis and to evaluate the safety of lactosporin in-vitro using the EpiVaginal tissue model from MatTek Corporation (Ashland, MA, USA).

Materials and Methods

Bacterial strains and growth condition

Producer strain B. coagulans ATCC 7050 was grown aerobically at 37 °C in Difco Lactobacilli MRS broth (Becton, Dickinson) with aeration for 24 hours. The indicator strains Micrococcus luteus ATCC 10420 was cultured aerobically in tryptic soy broth (Becton, Dickinson) (enriched with 6 g l-1 yeast extract and 2.5 g l-1 glucose) at 30 °C. M. luteus is frequently used as an indicator microorganism in bacteriocin detection assay (Li et al. 2005; Wirawan et al. 2006; Riazi et al. 2007). G. vaginalis ATCC 14018 was grown on a selective human blood Tween bilayer agar medium at 37 °C, anaerobically. The cultures used in this study were maintained as frozen stocks at -80 °C in a bio-freezer.

Production and inhibitory activity of lactosporin

The production of lactosporin was carried out as described by Riazi et al. (2009). For the mode of action and safety assays reported in this paper, the same batch (lot# 2/3) of lactosporin preparation was used (previously used for lactosporin characterization studies). Lactosporin samples were further concentrated by lyophilization (Freeze-dryer 4.5, Labconco) for both mode of action and safety experiments. The antimicrobial activity of lactosporin was confirmed by a well diffusion assay against M. luteus ATCC and G. vaginalis, as described by Cintas et al. (1995).

In all experiments nisin A was used as a positive control. The commercial preparation (Sigma, St Louis, MO, USA) contains 2.5% nisin A in denatured milk solids and was solubilized using nisin diluent (HCl solution at pH 1.7).

Measurement of arbitrary units of activity (AU)

An arbitrary unit is defined as the reciprocal of the highest dilution showing inhibition against the indicator microorganism (Kojic et al., 1991). Ten 2-fold dilutions of lactosporin were made and tested for activity in well diffusion assay against M. luteus. AU ml-1 was calculated based on the highest dilution with antimicrobial activity.

Measurement of potassium release

The measurement of potassium efflux was performed as described by Orlov et al. (2002) with some modifications. A potassium-selective electrode (Phoenix Electrode Company, TX, USA) was used to measure the K+ ion release from the M. luteus ATCC 10420 after treatment with lactosporin or nisin. M. luteus was grown in trypticase soy broth enriched with 2.5 g l-1 glucose and 6 g l-1 yeast extract. Twenty ml culture was incubated aerobically at 37 °C until an optical density (OD600) of 1-1.2 was reached. The cells were washed three times (5000 g, 10 min, 4 °C) with 20 ml of a 10 mM Tris acetate buffer solution containing 100 mM NaCl, pH 7.4. The washed cells were resuspended in 5 ml of the same buffer and kept on ice until use. The electrode was calibrated with 0.01, 0.1 and 1 mM solutions of KCl in 100 mM NaCl before each experiment and the measurements were done in small (10 ml) pyrex beakers (Fisher Scientific) that contained a small magnetic stirrer. A 500 μl of a 20 times concentrated (320 AU) and dialyzed lactosporin or a 500 μl of a 10 mg ml-1 nisin A solution was added to each beaker contacting 5 ml of the cells and the potassium release was measured every minute for 5 minutes. Nisin diluent and MRS medium were used as controls for this experiment.

Dissipation of transmembrane potential (Δψ)

The changes in transmembrane potential (Δψ) of intact G. vaginalis cells were determined according to the protocol outlined by Sims et al., (1974) and modified by Turovskiy et al. (2009). Briefly, G. vaginalis cells were grown anaerobically at 37 °C in BHI medium supplemented with 3% horse serum to an OD600 of 0.6. Twenty ml of the culture were harvested by centrifugation (5000 g,10 min, 22 °C) followed by a wash step with 20 ml of fresh growth medium and resuspension in 200 μl of the same medium at room temperature. A Perkin Elmer LS-50B spectrofluorometer (Perkin Elmer Inc, Boston, MA) was utilized to evaluate the changes in the Δψ of the cells with excitation and emission wavelengths of 643 and 666 nm, respectively, and a slit width of 10 nm.

In quartz cuvettes with a 10 mm light path, 5 μl of the fluorescent probe 3,3′-dipropylthiadicarbocyanine iodide (DiSC3 (5)) (Molecular Probes, Eugene, OR) was added to 2 ml of fresh BHI broth supplemented with 3% horse serum followed by addition of 20 μl of the cell suspension, which caused a substantial decrease in fluorescence intensity. After the signal was stabilized, 2 μl of 5 mM nigericin (Sigma) was added to convert the ΔpH to Δψ. After the signal was equilibrated, either lactosporin, nisin (positive control), lactosporin diluent (MRS medium) or nisin diluent (negative controls) were added to each cuvette. This step was followed by an addition of 2 μl of 2 mM valinomycin (Sigma) to dissipate the remaining Δψ.

Dissipation of the pH gradient (ΔpH)

The changes in transmembrane pH (ΔpH) of G. vaginalis cells caused by lactosporin were studied according to the protocol outlined by Molenaar et al. (1991) and the modifications made by Turovskiy et al. (2009). Briefly, G. vaginalis was inoculated in 20 ml of fresh BHI medium supplemented with 3% horse serum and grown anaerobically at 37 °C to an OD600 of 0.6. The cells were harvested by centrifugation (5000 g, 10 min, 22 °C), washed twice with 50 mM potassium phosphate buffer (PPB, pH 6.0) and resuspended in 200 μl of PPB. BCECF-AM (MP biomedicals, Inc., Solon, OH), a pH sensitive fluorescent probe was added to the cell suspension for 5 minutes at room temperature. After the probe was incorporated into the membrane, the cells were washed twice with 1 ml of 50 mM phosphate buffer saline (PBS, pH 6.0) and resuspended in 200 μl of the same buffer. To monitor the dissipation of ΔpH, 2 ml of PPB (pH 7.0) and 10 μl of the cell suspension were added to quartz cuvettes and the fluorescence was measured in a Perkin Elmer LS-50B spectrofluorometer with slit widths of 5 nm for excitation and 15 nm for emission, and wavelengths of 502 and 525 nm, respectively. Once the signal had stabilized, the cells were energized with 4 μl of 2.2 mM glucose. The addition of glucose caused an increase in intracellular pH, therefore a noticeable and gradual increase in the fluorescence intensity was observed. After the signal stabilized, 2 μl of 5 μM valinomycin was added to convert the Δψ to ΔpH. After the addition of valinomycin, the cells were treated with lactosporin, nisin (positive control), lactosporin diluent or nisin diluent (negative control). The last step included adding 2 μl of 2 μM nigericin to dissipate the remaining ΔpH.

Determination of safety of lactosporin using EpiVaginal™ ectocervical tissue model

The EpiVaginal™ (VEC-100) ectocervical tissue model (MatTek Corporation, Ashland, MA, USA) was utilized to evaluate the safety of lactosporin for vaginal application. To determine the cytotoxic effects of lactosporin on EpiVaginal™ tissue, the protocol outlined by MatTek Corp and Dover et al. (2007) was followed. Briefly, tissues were placed in a 6-well plate containing 900 μl of DMEM-based DC-100 MM medium (MatTek Corporation) prior to exposure to the test compounds. The plate containing the tissues was placed in an incubator with 5% CO2 for 1 hour at 37 °C to pre-equilibrate the tissues. After incubation for 1 hour, the media was removed and replaced with 900 μl of VEC-100-MM medium and then 83μl of lactosporin was applied on VEC-100 tissues topically in triplicate for 24, 36 and 48 hours. For the 48 hour exposure time, two washers were used to airlift the tissue inserts and 5 ml of the assay medium was placed in each well. Two negative controls were used for the cytotoxicity assay; sterile distilled water and an antifungal cream containing 4% miconazole nitrate (Monistat-3, Ortho McNeil Pharmaceutical, Inc., Raritan, NJ, USA) that is known to be a nontoxic preparation for vaginal application (Sawyer et al., 1975; Ozyurt et al., 2001; Ayehunie et al., 2006). The viability measurements for these two negative controls were performed at 24, 36 and 48 hours similar to lactosporin.

A spermicidal cream containing 4% Nonoxynol-9 (Ortho Options CONCEPTROL vaginal Contraceptive Gel, Advanced Care Products, Skillman, NJ, USA) was used as the positive control due to its known toxicity to vaginal tissues for 2, 4 and 6 hours. At the end of each exposure time, tissue viability was determined using MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The effective time for each product to reduce the tissue viability to 50% (ET-50) was then calculated.

The viability of ectocervical cells was determined by measuring the breakdown of the yellow tetrazolium to purple formazan as only viable cells are able to carry out this reaction (Mosmann, 1983). After completion of each exposure time for LP and the controls, the liquid in the inserts was removed and the tissues were washed with Dulbeccos phosphate buffer saline solution (DPBS). Test compounds were removed by gently swabbing the surface of the tissue with a sterile polyester fiber tipped swab (Thermo-Fisher, Waltham, MA, USA). Tissues were placed in a 24-well plate and each well was filled with 300 μl of MTT solution that was prepared in culture medium (1 mg ml-1). After 3 hour incubation at 37 °C with 5% CO2, the tissue inserts were removed and placed in a new 24-well plate and 1660 μl isopropanol (MatTek Corporation) was added to each well to extract the formazan. The plate was incubated at room temperature in the dark for 24 hours. Then, 200 μl of this mixture was transferred to a 96-well plate and measured spectrophotometrically in triplicates at 570 nm using a plate reader (MRX revelation, Dynex Technologies, VA, USA). The percentage of tissue viability was calculated using the following equation; %viability = OD570 (treated tissue)/OD570 (control tissue).

Results

Antimicrobial activity of lactosporin

The Lactosporin preparation used in the mode of action study was active against M. lutues (indicator strain) and G. vaginalis when tested in a well diffusion assay as reported in our previous publication (Riazi et al. 2007).

Measurement of potassium ion release

We evaluated the pore-forming activity of lactosporin against selected indicator strain M. luteus using a potassium-sensitive electrode. Figure 1 demonstrates the effect of antimicrobials used in our experiment on potassium release from M. luteus cells. Both lactosporin and nisin caused rapid efflux of K+ ions from the membrane of the indicator organism. After addition of MRS medium, one of our negative controls, a slight raise in K+ ion concentration was observed that can be attributed to the presence of potassium in the formulation of this medium.

Figure 1.

Figure 1

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Impact of lactosporin and nisin on the integrity of the cytoplasmic membrane of Micrococcus luteus was evaluated by monitoring the efflux of K+ ion after addition of lactosporin, nisin and the negative controls. Antimicrobials were added after 20 seconds after the first mesurement, and potassium release was monitored with a potassium-sensitive electrode. Both nisin (Inline graphic) and lactosporin (Inline graphic) caused leakage of intracellualar potassium ions. The experiment was performed twice creating two replicates. The values represented in this figure are the calculated means of the two replicates.

Lactosporin does not dissipate the transmembrane potential (Δψ) of G. vaginalis

The fluorescent probe 3,3′-dipropylthiadicarbocyanine iodide (DiSC3 (5)) was used to qualitatively assess the changes occurred by lactosporin on the transmembrane potential (Δψ) of the G. vaginalis cells.

An increase in the fluorescent signal of the probe after addition of the antimicrobial is indicative of the transmembrane potential collapse. The increase in the fluorescent signal is due to the depolarization of the cell membrane by the antimicrobial compound. In contrast to nisin that caused an immediate collapse of Δψ, lactosporin did not dissipate this component of the PMF (Fig. 2). The ionophore valinomycin was subsequently added to the cells to deplete the remaining transmembrane potential. Addition of valinomycin to the cells that were exposed to nisin did not cause any further depletion of the Δψ but caused complete dissipation of Δψ in cells treated with lactosporin, nisin diluent and lactosporin diluent (Fig. 3). It can be concluded that nisin causes an instantaneous collapse of the Δψ in G. vaginalis cells unlike lactosporin that has no effect on this component of the PMF.

Figure 2.

Figure 2

Figure 2

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Addition of lactosporin caused an immediate decrease in the fluorescent signal indicating complete dissipation of the transmembrane pH gradient (ΔpH) of G. vaginalis cells (A). Nisin caused a more gradual decrease in the fluorescent signal that indicated a slower dissipation of the ΔpH component of the PMF. There was no additional depletion of the ΔpH after addition of nigericin (A, B). Both negative controls had no effect on the pH gradient of G. vaginalis (A, B).

Figure 3.

Figure 3

Figure 3

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Lactosporin (A) did not cause any changes in the fluorescent signal indicating that it does not dissipate transmembrane electric potential (Δψ) in G. vaginalis cells. Addition of nisin (B) caused an increase in the intensity of the fluorescent signal, due to release of the DiSC3(5) probe and dissipation of Δψ. Addition of valinomycin to dissipate the remaining Δψ did not cause any further increase of fluorescence in nisin-treated sample (B) since nisin caused a total depletion of the Δψ. Negative controls in both samples (A, B) had no effects on the Δψ of G. vaginalis cells.

Lactosporin causes dissipation of the transmembrane pH (ΔpH)

The pH sensitive fluorescent probe BCECF-AM was used to track the changes in the transmembrane pH of the G. vaginalis cells. Ionophore valinomycin was utilized to convert the transmembrane potential to transmembrane pH before addition of the antimicrobial compounds. Lactosporin and nisin both caused an immediate decrease in the fluorescent signal of the BCECF-AM probe (Fig. 4), indicating a decrease in the internal pH of the cells. In contrast, nisin diluent and lactosporin diluent (MRS) did not have such an effect on the interacellular pH of G. vaginalis cells. Nigericin was added to deplete any remaining ΔpH in the cells. Addition of this ionophore to lactosporin and nisin treated cells indicated that almost all ΔpH was already depleted and did not cause any further dissipation of this component of the PMF.

Figure 4.

Figure 4

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Lactosporin is a non-cytotoxic antimicrobial for human vaginal cells as tested in EpiVaginal tissue model (VEC-100). This figure presents % viability of vaginal cells at different time points following treatment with nonoxynol 9, miconazole and lactosporin. The experiment was performed twice in triplicate and the error bars represent the standard deviation.

Lactosporin is a safe and non-cytotxic compound for vaginal application

The EpiVaginal™ ectocervical tissues were utilized to study the cytotoxic effects of lactosporin on vaginal tissues. The tissue viability was approximately 66% after a 48 hour exposure to lactosporin (Fig. 6). The viability of the tissues treated with the negative control, 4% miconazole nitrate, after 48 hours was about 41% while the positive control, Nonoxynol-9, reduced the viability to 28% in 6 hours. The ET-50 value for lactosporin could not be calculated since the tissue viability was not less than 50% at any of the time points used in this experiment. The calculated ET-50 values for Nonoxynol 9 and miconazole were approximately 4 and 23 hours, respectively.

Discussion

In this study, we investigated the mechanism of action of lactosporin against G. vaginalis cells in order to determine whether this antimicrobial preparation was safe for vaginal application. Our mode of action data indicated that lactosporin instantaneously dissipated the transmembrane pH gradient (ΔpH) of the G. vaginalis cells while it had no effect on the transmembrane electric potential (Δψ), in contrast to nisin that fully depleted both components of the PMF. Similar to lactosporin, the selective dissipation of one of the portions of the PMF has been reported for a number of other bacteriocins. For instance, Herranz et al. (2001) indicated that enterocin P did not cause any changes in the pH gradient of the Enterococcus faecium T136 cells while it caused full depletion of the transmembrane electric potential and the release of intracellular ATP from the target cells. Subtilosin, a bacteriocin produced by Bacillus amyloliquefaciens, acts similar to lactosporin by depleting the transmembrane pH gradient and has no effect on the transmembrane electric potential of the G. vaginalis cells (Sutyak Noll et al., 2011). Majority of bacteriocins have amphiphilic and cationic characteristics. They act by permeabilization of the membrane of the sensitive cells, forming transient pores and the leakage of intracellular materials such as small ions, amino acids or ATP and dissipation of proton motive force that may result in secondary effects such as inhibition of protein, DNA, RNA and peptidoglycan synthesis (Montville and Bruno, 1994). Our previous research indicated that lactosporin is an anionic antimicrobial protein that potentially targets the membrane of sensitive cells (Riazi et al., 2009).The ability of lactosporin to form pores in sensitive cells of M. luteus was observed by rapid K+ efflux from these cells. The K+ assay requires bacterial cells to be washed with Tris acetate buffer solution and kept on ice throughout the experiment to slow down the cells metabolism. These conditions worked well for the indicator organism M. luteus but the fastidious G. vaginalis could not survive the assay's condition. Therefore, only the K+ efflux results for M. luteus cells are presented. As reported by Van Kuijk et al., (2011) the action of antimicrobial proteins can be species-specific; therefore it cannot be concluded that lactosporin exerts its antimicrobial properties on G. vaginalis cells in a similar manner. The aim of this investigation was to study the effects of lactosporin on G. vaginalis cells and the reference strain M. lutues was only used when an experiment could not be performed using G. vaginalis cells.

Together, PMF and K+ leakage data suggest that lactosporin may exert its antimicrobial activity by targeting the cytoplasmic membrane of sensitive cells and forming transient pores followed by leakage of small ions and dissipation of proton motive force. Our previous data indicated that lactosporin had antimicrobial activity against the vaginal pathogen G. vaginalis but did not have any effects on the healthy vaginal lactobacilli (Riazi et al., 2009). The MatTek EpiVaginal™ tissue model was utilized to evaluate the cytotoxic effects of lactosporin on vaginal and ectocervical epithelial cells. Our data revealed that lactosporin is a safe preparation for vaginal application to control bacterial vaginosis. The EpiVaginal™ in vitro test is gradually replacing the traditional in vivo models to study vaginal irritation due to its higher reliability and reproducibility. A prior study conducted in our laboratory evaluated the effects of an antimicrobial peptide, lactocin 160, on vaginal tissues using the EpiVaginal™ in vitro model and the in vivo rabbit vaginal irritation (RVI) system (Dover et al., 2007). This study confirmed that the used in vitro model is a reliable alternative for animal testing and provides higher reproducibility. In a different study, EpiVaginal™ tissue system was used to evaluate safety of an antimicrobial peptide subtilosin (Sutyak Noll et al., 2011).

Natural antimicrobial proteins produced by friendly bacteria have received a great deal of attention in the last decade due to the potential health problems associated with the use of chemical antimicrobial agents in foods and personal care products, and because of the rapidly growing microbial resistance to conventional antibiotics (Abee et al. 1995; Cleveland et al. 2001). Antimicrobial resistance has become a major problem that is associated with increased negative impacts on human health and healthcare costs (Cohen, 1992). The bacterial resistance issue can be addressed by using a multiple-hurdle approach. In hurdle technology different synergistic antimicrobial agents with different mechanisms of action are utilized to more efficiently inhibit the growth of target microorganisms (Cleveland McEntire et al., 2003). In our future research we will explore the synergistic activity of lactosporin with other natural antimicrobial agents against G. vaginalis. This information will be used to design and formulate more effective products for control of bacterial vaginosis.

Acknowledgements

This research was sponsored in part by NIH grant “Natural antimicrobials against bacterial vaginosis” NCCAM NIH R21AT002897-01 and by Sabinsa Corporation (East Winsor, NJ).

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