Diarrheal diseases claim more then 2 million lives per year, 80% of them being children younger than age 2 years (1). Disease and death caused by diarrhea are a global problem; however, it occurs predominantly in developing countries where sanitation remains an unresolved issue and education is limited (2). Although antibiotics are theoretically available for the treatment of these devastating diseases, their use is contraindicated because of the risk of selecting bacterial strains that display antibiotic resistance (3). Vaccines remain unaffordable and are not always effective or available (1). Therefore, there is an urgent need for a safe and affordable treatment to solve this serious public health problem.
Treatment of diarrheal diseases remains a major challenge due to the number of disease-causing pathogens, including pathogenic Escherichia coli(4,5). The primary cause of death from diarrheal diseases is dehydration, which causes extreme thirst, shock, and organ failure. Studies suggested that diarrheal diseases can also have a negative long-term impact on both physical and psychological growth due to reduction in appetite, altered feeding practices, and decreased absorption of nutrients (6).
Probiotics are nonpathogenic bacteria that are claimed to have several beneficial effects related to their capability to prevent the growth of several pathogenic microorganisms, including E coli(7). According to the currently adopted definition by FAO/WHO, probiotics are “live microorganisms which when administered in adequate amounts confer a health benefit on the host” (8). There are many forms of probiotics currently commercially available as both pills and liquid medicine (9). Probiotics are known to have a beneficial effect on diarrheal diseases; however, their mechanism of action has not yet been completely established (10). It is well known that probiotics help in maintaining a healthy intestinal microbiota (11).
There are several theories proposed to explain the antibacterial effects of probiotics, including their capability to compete for nutrients, the establishment of a microenvironment in which pathogenic microorganisms are not able to survive, or the elaboration of toxins lethal for pathogenic bacteria (10). To explore these possibilities, we used a combination of microbiological, biochemical, and genetic approaches that led to the identification for the first time of Lactobacillus GG (LGG)-derived small peptides that retain the antibacterial properties of LGG against both Gram-negative and Gram-positive bacteria.
MATERIALS AND METHODS
Luria Broth Base was purchased from GibcoBRL (Carlsbad, CA); MRS Broth was purchased from Becton Dickinson Company (Franklin Lakes, NJ); MRS agar was obtained from Fluka (Buches, Switzerland); and LB Agar plates were purchased from TEKnova (Hollister, CA). Macro-prep DEAE Support anion exchange resin and Criterion precast gel (4%–20%) were obtained from Bio-Rad (Hercules, CA). Spectrophotometry was performed using a spectrophotometer Beckman Coulter DU530 (Fullerton, CA); cultures were prepared using a Forma Orbital Shakers from Thermo (Waltham, MA).
Lactobacillus GG, enteroaggregative E coli strain EAEC 042, Salmonella typhi, and Staphilococcus aureus strains were obtained from the collection of the Center for Vaccine Development, University of Maryland School of Medicine.
Preparation of LGG Conditional Media
Lactobacillus GG was cultured in 5 mL MRS broth, at 37°C, with shaking at 225 rpm overnight. The following day, 0.1 mL cultured MRS broth was diluted to 10−10, 10−11, 10−12, spread on the MRS agar plates, cultured at 37°C for 24 hours, and then colonies were counted. The 4.9 mL of the cultured mixture was centrifuged at 5000 g for 45 minutes, and conditional media (CM) collected, filtered, and used for the studies described below.
Ion Exchange Chromatography
Three milliliters of LGG CM was added to an anion exchange column (d = 1.5 cm, L = 2.0 cm flow rate 0.1 mL/min). Before loading, washing the column using 12 mL of Tris-HCl (pH 8.0); after loading, the column was washed with 12 mL Tris-HCl (pH 8.0) again and then eluted by ImmunoPure Ig G elution buffer (pH 2.8, Pierce, Rockford, IL). The fractions collected for activity assay and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
Each fraction eluted from anion exchange column was mixed with protein sample buffer (1:1), heated at 95°C for 5 minutes, and then applied to Criterion precast gel (4%–20%), using Tris-Glycine-SDS buffer (Bio-Rad) as running buffer at constant 170 V for 1 hour. The gel was stained by 2.5% Coomassie blue and destained by 10% methonol, 7.5% acetic acid solution.
LC/MS Analysis and Identification of Peptides
Liquid chromatography/mass spectrometry (LC/MS) analysis of peptides derived from proteins present in the conditional media (CM) was performed on Thermofinnigan LCQ mass spectrometer (Thermofinnigan, San Jose, CA), which was connected to a nanoelectrospray ionizer. Initially the supernatant was prefiltered and concentrated using 10,000 MW cut of membranes (Microcon; Millipore, Billerica, MA). The Surveyor chromatographic system with auto sampler (Thermofinnigan) was used for peptide separation. The LC system was connected to 10.5 cm fused silica reverse-phase C18 column (Pico Frit Column; New Objective, Woburn, MA). The peptides were separated during 90-minute linear gradient of 5% to 90% acetonitrile/water mixture, containing 0.1% formic acid at a flow rate of 300 nL/min. The spectra were accumulated and the acquired MS scans were searched against the Lactobacillus database (IPI) using the SEQUEST search algorithm. Several peptides with different MW distribution were detected and synthesized to check for their antibacterial activity.
Peptide synthesis, purification, and identification were carried by the Biopolymer Laboratory at the University of Maryland School of Medicine. Briefly, the peptides were synthesized on a Symphony peptide synthesizer (PTI Instruments, Boston, MA), using the Fmoc coupling strategy. Peptide purification was performed on a Beckman Gold system consisting of two 110B pumps and a 167 detector (215 nm) using a Dynamax reverse-phase C18 column (8 μL, 25.6 × 250 mm) (Varian, Walnut Creek, CA). Peptide characterization was performed by reverse-phase HPLC and MALDI-TOF.
Antibacterial Activity Assays
E coli Growth Time Course
Ten microliters of culture from E coli strain EAEC 042 (2.16 × 1014 CFU/mL) were added in 1-mL LB broth and incubated in 37°C, shaking at 225 rpm, measuring A600 every 30 minutes.
Measurement of Antibacterial Activity by Culture Spectrophotometry at A600
The assay was performed as previously described (12), with minor modifications. Briefly, 10 μL E coli EAEC 042 (7.7 × 1014 CFU/mL) was added to 1.0-mL LB Broth, mixed, and 100 μL of each LGG-derived synthetic peptide solution dissolved in MRS (for peptide final concentration, see Fig. 6B) was added to the mixture. MRS alone (100 μL) and LGG CM 100 μL (LGG concentration: 19.7 × 1012 CFU/mL) were used as negative and positive controls, respectively. The mixture was cultured for 3 hours with shaking at 225 rpm, 37°C, measuring A600 at the end. The relative inhibition activity was calculated according to the following formula:
Experiments With Peptide NPSRQERR for Staphylococcus or Salmonella (CVD908) Growth
Increased concentrations of peptide NPSRQERR were dissolved in 100 μL MRS and added to 150-μL Staphylococcus culture (44 × 106 CFU/mL) or 150-μL Salmonella culture (38 × 106 CFU/mL) in LB broth. The same Staphylococcus or Salmonella culture conditions without the peptide were used as control. The mixture was cultured at 37°C, 225 rpm for 3 hours. At the end, 100-μL culture mixture was spread onto LB agar plates, cultured overnight at 37°C, and colonies counted the next day. The relative inhibition activity calculation was performed according to the following formula:
Two-tailed Student t tests were used to test differences between 2 groups. Data were paired wherever appropriate. Values of P < 0.05 were regarded as significant.
Effect of LGG on E coli Growth
To determine the effect of LGG on pathogenic bacterial survival, increasing amounts of LGG cultures were added to McConkey petri dishes plated with 10−8 dilution of an overnight culture of E coli EAEC 042 bacteria. Lactobacillus GG caused a dose-dependent negative effect on E coli growth (Fig. 1), suggesting LGG and/or factor(s) secreted by LGG present in the CM exert an antibacterial effect on E coli.
To establish whether the LGG antibacterial effect was related to its direct action on E coli or to the secretion of an antibacterial factor(s), LGG CM was used to repeat the experiments described in Figure 1. Figure 2 shows that LGG CM media is responsible for the antibacterial effect observed with LGG. Similar results were obtained when the antibacterial activity was monitored by spectrophotometry, with an average inhibition rate of 95.03% (n = 8).
To establish whether the factor secreted by LGG was thermostable, CM was heated at 95°C and added to EAEC 042 bacterial cultures. When grown at a 10−4 dilution, EAEC 042 growth was quantitated to be 800.5 ± 96.9 CFU/mL. Lactobacillus GG CM inhibited the growth of EAEC 042 either when the culture was heated (24 ± 2.8 CFU/mL, P < 0.00005) or not heated (22.5 ± 4.3 CFU/mL, P < 0.00005) (Fig. 3). Similar results were obtained at higher EAEC 042 culture dilutions (Fig. 3). These results proved that the antibacterial moiety present in LGG CM is heat resistant.
Ion Exchange Chromatography and SDS-PAGE Analysis Results
Five fractions were collected from ion exchange chromatography. On the overnight plates culture assay, only fraction 3 showed antibacterial activity (Fig. 4). However, no protein bands could be detected by SDS-PAGE analysis. These results suggested that the concentration of the active peptide(s) was low, the molecular weight of the protein was too small, or the molecule(s) was not a protein. To address this issue, LGG CM was concentrated by dialysis against phosphate buffered saline by using 1000 Da molecular weight cutoff bags. The dialyzed LGG lost its antibacterial activities and, therefore, attention was paid to searching for molecular peptides smaller than 1000 Da.
LC/MS Analysis Results
The LC/MS spectra of the LGG CM were analyzed and the mass spectrometry sequences of the <1000 Da peptides detected in the media were compared with the Lactobacillus database (IPI) using SEQUEST search algorithm. Many peptides with different molecular weight distributions were detected during the process of LC/MS (Fig. 5). Of the several fragments of ∼1000 Da molecular weight, the following 7 peptides resulted being part of the LGG genome: NPSRQERR, PDENK, YTRGLPM, VHTAPK, LSQKSVK, MLNERVK, and GKLSNK. These peptides were synthesized to 95% to 99% purity and tested for potential antibacterial activity.
Activities Assay Results
The antibacterial activity of these 7 peptides was compared with the linear growth of EAEC 042 over time as determined by spectrophotometry A600 (Fig. 6A) and analyzed at the 180-minute time point. Figure 6B shows the inhibitory effects of the 7 peptides on the growth of E coli in liquid culture. The comparative antibacterial activity of the 7 peptides was NPSRQERR > PDENK > VHTAPK > MLNERVK > YTRGLPM > GKLSNK > LSQKSVK. Only NPSRQERR showed an activity (81.4% E coli growth inhibition) comparable with LGG CM (95% growth inhibition). PDENK had a moderate activity (68.7% growth inhibition), whereas VHTAPK had a mild activity (30% growth inhibition). The remaining 4 peptides had little or no activity.
To establish whether the antibacterial activity of peptide NPSRQERR was specific for E coli, we repeated our biological assay using both S typhi and Staphylococcus aureus as bacterial targets. Although the effect of peptide NPSRQERR on S typhi was similar to that observed in E coli EAEC 042 (Fig. 7), its effect on S aureus was only mild but dose dependent (Fig. 7)
Probiotics were defined by Fuller in 1989 as “live microbial feed supplements that beneficially affect the host animal by improving its intestinal microbial balance” (13). Fuller's definition emphasizes the requirement of viability for probiotics and introduces the aspect of a beneficial effect on the host. Probiotics, which means “for life,” have been used for centuries as natural components in health-promoting foods. The original observation of the positive role played by certain bacteria was first introduced by Russian scientist and Nobel laureate Eli Metchnikoff, who in the beginning of the 20th century suggested that it would be possible to modify the gut flora and to replace harmful microbes by useful microbes (14). Experiments into the benefits of probiotic therapies suggest a range of potentially beneficial medicinal uses for probiotics. However, for many of the potential benefits, research is limited and only preliminary results are available. Among others, probiotics are claimed to protect against pathogens by means of competitive inhibition (ie, by competing for growth) or by improving immune functions (15,16). As concerns infection of the gastrointestinal tract, it has been reported that probiotics present in food or supplements are effective in the treatment and prevention of acute diarrhea; decreasing the severity and duration of rotavirus infections in children as well as travelers' diarrhea in adults (15,16).
Despite major research efforts aimed at finding an effective treatment, diarrheal disease remains a human plague claiming millions of lives every year (1). EAEC 042 is among the leading enteric pathogens in pediatrics, causing prolonged diarrheal diseases in children (4). The discovery and characterization of a small peptide elaborated by LGG opens unexplored horizons for an effective treatment of diarrheal diseases affordable for third world countries.
Our stepwise approach was initially focused on the effect of LGG on E coli growth and on the realization that the antibacterial activity was related to a moiety secreted by LGG in the CM. These results confirmed previous reports of an antimicrobial substance elaborated from lactobacilli (17). However, the nature of this inhibitory substance has never before been characterized. For the first time, we report in this article the identification and characterization of this moiety as a molecule(s) that is heat resistant and small in size. Anion exchange chromatography showed that this factor(s) is peptide in nature with an approximate molecular weight of less than 1000 Da. MS/MS analysis identified 7 peptides elaborated by genes present in the LGG genome, 3 of them showing variable antibacterial activity (see Fig. 6B). Interestingly, probiotics also elaborate peptides that regulate intestinal cell survival and growth (18,19), suggesting that their beneficial action on infective gastroenteritis can be the combined effect of both direct antibacterial activity and intestinal mucosal protection.
The use of small peptides as antidiarrheal drugs is novel and would offer several advantages over current treatments (20). Being small peptides, it would be easy and cheap to produce them in large amounts and would be virtually devoid of the side effects experienced with current antidiarrheal remedies (drug resistance for antibiotics, immune reaction for vaccines) (21). Due to the fact that these peptides are elaborated by probiotics found in common food (such as yogurt), is it conceivable to hypothesize that their use would be safe for the treatment of children and adults experiencing diarrhea. However, proper clinical trials are necessary to confirm the safety of these peptides from their clinical use. Of the 7 peptides isolated and characterized, the peptide NPSRQERR showed the highest antibacterial properties, both on Gram negative and Gram positive. We note that each of the peptides is predicted to carry a net positive charge at neutral pH by virtue of the preponderance of basic amino acids. Such cationic peptides may act similarly to cationic antibacterial peptides produced by mammalian species, although this hypothesis remains to be tested.
Notably, the small LGG-derived peptides are also thermostable, a characteristic that would be advantageous for the transport and usage of the drug in developing countries. The synthesis of this small peptide would not require sophisticated equipment. Therefore, developing countries could produce their own LGG-derived peptide at relatively low, affordable costs. Nevertheless, peptide formulation for proper protection against degradation and delivery to specific gastrointestinal regions are pitfalls that need to be addressed before possible clinical applications to decrease the burden of diarrheal diseases in developing countries.
The object of this article may far reach beyond the development of novel treatment of diarrheal diseases. There are a growing number of reports suggesting that human-associated microbes influence human health and vice versa. The estimated 10 to 100 trillion microorganisms that inhabit the human intestine actually outnumber the body's own cells by a factor of 10. The human microbiome is the genetic sum of this community of microorganisms living in symbiosis with their host. Therefore, it is conceivable to hypothesize that the metabolic activities of the bacterial population in the colon can be manipulated to promote health. Unfortunately, this hypothesis has not been rigorously challenged because of several shortcomings. First of all, the complexity of the colonic biota (flora) is vast and also largely undefined. Consequently, many studies have lacked the proper scientific stringency necessary for meaningful readouts on the impact of probiotics on gut microflora. Another important issue is to establish the role of microbial biota in the pathogenesis of several gastrointestinal (eg, inflammatory bowel diseases) and extraintestinal (eg, autoimmune diseases) diseases. A third and related issue is defining conditions that may be ameliorated by probiotic therapies. Therefore, it is possible to conceptualize the use of probiotics or probiotic-derived peptides for the treatment of these other conditions in which manipulation of the intestinal microbiome can represent a novel and yet unexplored alternative approach.
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