Introduction
The gastrointestinal tract is the largest immune system of the human body and contains complex microorganisms, of which there are about 1013–1014 [13,55]. These intestinal microorganisms are related to important physiologic functions, such as the regulation of immune responses, secretions of antibacterial agents, and the reinforcement of the tight junction in the gut [61,63].
Although human infants are born with aseptic conditional gastrointestinal tracts, gut microbiota colonize immediately in an infant’s gut [52]. The early settled microorganisms induce the beginning of immune reactions that affect the infant and are influenced by internal and external factors [2,49,58]. The immune system during infancy develops when exposed to pathogen-specific immunologic stimuli, which is also accompanied by the maintenance of immune tolerance to digested food products and the development of normal flora in the gut. [56]. In the intestine of a healthy infant, the predominant normal flora are primarily anaerobic bacteria, such as enterobacteria, enterococci, lactobacilli, streptococci, Bacteroides, bifidobacteria, clostridia, and eubacteria [10]. However, pathologic bacteria that involve Escherichia coli (E. coli), Streptococcus, Enterococcus, Staphylococcus, Klebsiella, and Clostridium species are predominant in unhealthy infants [13,68]. In particular, among the beneficial normal flora, bifidobacteria and lactobacilli are especially predominant in healthy infants and rare in unhealthy infants [11]. The normal flora that develops in infancy is critical because its composition at birth is as complex as that of an adult [45,53] and affects the development of a normal acquired immune system [30,31]. The commensal microbes in infancy maintain the balance between type 1 and type 2 T-helper cell immunity, regulate the immune system maturation, suppress excessive immune response, and allow for immune tolerance [6–8]. However, if the composition and diversity of the intestinal normal flora are disrupted, the infant might develop illnesses. The disruption of the intestinal normal flora sensitizes the infant to gastrointestinal disorders and diseases: intestinal dysfunction, inflammatory bowel disease, metabolic dysfunction, diarrhea, autoimmune response, and necrotizing enterocolitis [7,16,30,32,54]. The probiotics, represented by bifidobacteria and lactobacilli, were effective in the prevention and treatment of severe gastrointestinal tract diseases, according to numerous clinical studies [12,35]. In particular, the association of gut Lactobacillus species with increased health has been reinforced by clinical studies [25,26,31,46]. A recent study reported that Lactobacillus species colonize the intestine at about 10 days after birth, whereas Bifidobacterium species colonize it after one month [20]. In other words, lactobacilli are the first to settle in the gastrointestinal tract and exert a beneficial function in humans. However, there have been few reports that reveal the colonization periods and compositions of microorganisms and the environmental factors for the colonization of gut flora, like in the case of Lactobacillus-like bacteria; these reports explain how they are the first settlers in the gut, preceding pathogenic bacteria.
In a previous study by the author, it was observed that the growth of L. plantarum is increased by co-culture with pathogenic supernatant and the antibiotic activity of L. plantarum against pathogenic bacteria is enhanced [22]. Similar effects were observed for all five strains of Lactobacillus spp., but the effects were particularly pronounced in L. plantarum. Thus, this current study focuses on the pathogenic factor that increases the growth rate and antibiotic activity of L. plantarum.
Recent research has shown that opportunistic pathobionts secrete uracil, which is recognized by the host and elicits an immune response related to reactive oxygen species [42,64]. In contrast, commensal bacteria, including L. plantarum, do not secrete uracil because they coexist with the host [42,64].
The results of this research showed that L. plantarum also recognizes potential pathogenic E. coli-derived uracil and increases its growth rate and antibacterial ability more than the reaction with other nucleobases. Based on the results of the inhibitory assay, the antibiotic substance secreted by L. plantarum is assumed to be a bacteriocin-like substance, and production of the bacteriocin-like substance is increased by uracil. This result implies that L. plantarum protects the intestine through effective recognition of E. coli-specific secretory uracil, which is why it settles as the dominant commensal bacteria. Because E. coli is one of the early pathogenic colonizers in an unhealthy infant gut [68], E. coli-derived uracil is a promising candidate for controlling the physiology of L. plantarum. This result explains why L. plantarum is the main bacterium to first colonize the infant intestine and why it plays an important physiological role in humans.
Materials and Methods
Bacterial Strains and Culture Conditions
L. plantarum (CCARM0067) was spread on MRS broth (Becton Dickinson, USA) at 37℃. There were seven species of indicator strains: E. coli K-12 strain (Keio Collection) [2], Salmonella Typhimurium (Sal. Typhimurium; CCARM0125), Staphylococcus aureus (Staph. aureus; CCARM3709), Pseudomonas aeruginosa (P. aeruginosa; ATCC29336), Klebsiella pneumoniae (K. pneumoniae; PTCC1290) [62], Bacillus subtilis (B. subtilis; PTCC1715) [62], and Bacillus cereus (B. subtilis; PTCC1015) [62]. E. coli and Sal. Typhimurium were cultured in LB broth (Becton Dickinson, USA) at 37℃. Staph. aureus, K. pneumoniae, P. aeruginosa, B. subtilis, and B. cereus were cultivated in nutrient broth (Becton Dickinson, USA) at 37℃. All microorganisms were purchased from the Culture Collection of Antibiotic-Resistant Microbes (CCARM) at Seoul Women’s University, the American Culture Collection of Microorganisms (ATCC), and the Coli Genetic Stock Center (CGSC). Uracil mutant E. coli, pyrE-(GCSC8027), was purchased from the Keio Collection of single-gene knockout strains [2,48]. The uracil mutant bacteria were cultured in Luria-Bertani (LB) broth broth with 30 μg/ml of kanamycin at 37℃.
Preparation of M9 Minimal Broth Supernatant of Bacterial Strains
Because the normal culture medium of bacteria contains many trophic factors and offers the necessary conditions for bacterial growth, the M9 minimal media used for the bacterial culture in this work should be modified to exclude this environmental factor. Additionally, this test confirmed that the secretory substance of bacteria is indeed effective. To conduct an experiment that excludes the factors of normal culture medium and to focus on the effect of secretion substances, M9 minimal medium was selected [22]. The details are given below.
L. plantarum-cultured M9 (Lp). L. plantarum was cltured aerobically in M9 minimal broth (MB Cell) at 37℃ for 48 h. After incubation, the bacterial cells were collected by centrifugation at 10,000 ×g for 15 min. The supernatant was adjusted to pH 7 with 10 N NaOH to eliminate the inhibitory effect of organic acids. The cell-free supernatant was filter-sterilized through a 0.22 μm filter (Millipore, USA) and dried by lyophilization. The dry cell weight was 0.5 g/l, and it was made up to a final concentration of 0.1 mg/ml using 5 ml of ultrapure water. This concentration solution is referred to as L. plantarum-cultured M9 (Lp).
Potential pathogenic E. coli-cultured (Ec) M9. E. coli was inoculated in M9 minimal broth at 37℃ for 24 h. The next steps for the preparation of the supernatant are the same as the method for L. plantarum-cultured M9. After preparing the E. coli M9 supernatant, as stated above, it was lyophilized to obtain a powder. The concentrated solutions are called E. coli-cultured M9 (Ec) and pyrE--cultured M9 (Ec Uracilmutant).
Treatment of E. coli-Cultured M9 with L. plantarum
The E. coli-cultured M9 (40 μg) was added to L. plantarum, and then incubated for 48 h at 37℃. The concentrated M9 solution was prepared as described above.
Quantitative Analysis of E. coli-Derived Uracil
In the present study, the concentration of the E. coli-secreted uracil was determined by high-performance liquid chromatographic (HPLC) assay using supernatant of the E. coli M9 culture. HPLC was performed by following a previously reported method with minor modifications [18]. Uracil and the internal standard (IS) 5-fluorocytosine (5-FC) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All reagents were of HPLC grade or equivalent purity. The assay was developed and run using a HPLC instrument from Shimadzu Medical Systems (Torrace, CA, USA). The sample was detected at 205 nm. The columns used for chromatographic separation were a Symmetry Shield RP18 column (5 μm, 4.6 mm × 250 mm; Waters) and an Atlantis dC8 column (5 μm, 4.6 mm × 100 mm; Waters). These columns were isocratically eluted with 10 mM potassium phosphate buffer (pH 3.0). The flow rate of mobile phase was 0.6 ml/min at room temperature. The uracil and 5-FC were dissolved in ultrapure water. The uracil was prepared in minimal M9, and the calibration curve was detected at final concentrations of 5, 10, 20, 40, 80, and 100 ng/ml. M9 medium was used to exclude the measurement of physiological uracil in the culture media. All standard calibration curves were analyzed by HPLC. The ratios of the peak areas for uracil to the 5-FC were plotted against the concentrations of the calibration standards. The calibration curve was linear in all six samples with the weight factor and was tested using t-test. The results analysis was performed with reference to Lang and Bolton [36].
Wild-type E. coli-secreted uracil, pyrE--secreted uracil, and synthetic uracil were quantified using a Shimadzu HPLC system equipped with a UV detector at 270 nm. A Primesep S (4.6 × 150 mm; SIELC) column was eluted with acetonitrile/H2O (90/10) as the mobile phase at a flow rate of 1 ml/min, and the column temperature was maintained at 30℃.
Growth Rate Test of L. plantarum
The growth rate of L. plantarum was tested by colony count assay. E. coli-cultured M9 (40 μg) was incubated with L. plantarum culturing in M9 broth at 37℃ for 6 h. Uracil and other nucleobases (adenine, cytosine, guanine, and thymine) were added to the culturing M9 broth of L. plantarum at 37℃ for 6 h. The concentration of nucleobases incubated with L. plantarum was 0.001 to 50 nM. The survival rate was detected as the relative percentage of colony-forming units (CFU)/ml after reaction compared with the CFU/ml of the control (100%). In the tests using the uracil mutant (pyrE-) to determine uracil’s growth function, L. plantarum-culturing M9 broth was incubated with a mixture of prepared pyrE--cultured M9 (40 μg) and uracil (0.01–0.1 nM) at 37℃ for 6 h.
Antimicrobial Activity Assays
Microbial viability. The antimicrobial activity was confirmed by counting viable CFU/ml and by well diffusion assay. To detect the CFU, 103 CFU/ml of the indicator strain was inoculated into each tube with antibiotic candidates. The test tubes were incubated at 37℃ for 3 h. Viable cells were evaluated as CFU/ml on the agar plate.
Well diffusion assay. The antibacterial activity of L. plantarum from induced bacteria-cultured M9 (Ec and Ec Uracilmutant) was determined by well diffusion assay. Indicator bacteria were spread on agar plates, and 25 μg of Lp was dropped into the formed wells in the agar plates. M9 medium served as a control, and ampicillin (5 μg/disk) was used as a positive control. Antibacterial activity was detected in the clear zone (mm) of the growth inhibition of the indicator strains, and the data represent the results of three independent determinations. In the tests using the uracil mutant (pyrE-) for detecting the uracil-induced antibacterial activity of L. plantarum, Lp was added to a mixture of pyrE--cultured M9 (Ec Uracilmutant) and uracil (0.01–0.1 nM) and incubated at 37℃ for 6 h. After incubation, Ec Uracilmutant-treated Lp was prepared as a supernatant, as described above, and 25 μg of Lp M9 was applied to the indicator strains.
Effects of Enzymes, Temperature, and pH on Inhibitory Activity of the Antimicrobial Substance Produced by L. plantarum
To determine the effect of heat on the inhibitory activity of the antibacterial substances produced by L. plantarum, the M9 culture of L. plantarum was incubated at different temperatures ranging from 30℃ to 100℃ for 30 min, and then assayed for antibacterial activity. The sensitivity to various pH values was tested by adjusting the pH of the Lp M9 to pH 2–11 with 1 N NaOH and 1 N HCl and by testing it against the indicator strain. To test the tolerance of the antibacterial product for proteinase enzyme, the sample was treated with protease K for 30 min and assayed for antibacterial activity against the indicator strain [65]. All antibacterial activity was detected by well diffusion assay. The untreated Lp M9 was the control (100%), and the results of the residual activity for the treated samples were obtained by measuring the relative value of the control sample. The data represent the results of three independent determinations.
SDS-PAGE and Detection of Antimicrobial Substance
To estimate the molecular mass of the purified antimicrobial substance, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was operated by normal protocols using Tris-glycine buffer [1]. The Lp M9 was concentrated into factions of 3, 10, and 30 kDa using Nanostep concentration filters (Pall-Gelman, USA), and each of the fractions was applied to an antibacterial activity test. To precipitate the proteins in the bacteria-free M9 supernatant, 100 ml cultures of L. plantarum and uracil-induced L. plantarum were precipitated using ammonium sulfate (final concentration 50 mM) [1,50,65]. The natural precipitate was centrifuged for 20 min at 10,000 ×g and 4℃. The pellet was dissolved in 20 ml of 10 mM Tris-HCl at pH 7.4. The resuspended pellet was dialyzed using 140 kDa dialysis tubing (Sigma-Aldrich, USA) for 48 h with 5 mM Tris-MgSO4 buffer.
The dialyzed LP M9 and M9 media were then analyzed using SDS-PAGE (12% Tris-Glycine gels; BioWhittaker, BMA, Rockford, Maine, USA); in addition, dialyzed supernatant treated with protease K was analyzed. The separated proteins were determined using the broad-range molecular standards (Elpis-Biotech, Korea) to compare the weight. The proteins were analyzed using Coomassie blue staining.
Statistical Analysis
The results are expressed as the mean ± standard deviation (SD), and the data were processed using the SPSS 20.0 program (SPSS, USA). Statistical analyses were performed using a two-tailed unpaired Student’s t-test or one-way ANOVA. A p value of <0.05 was considered statistically significant.
Results
E. coli-Derived M9 Increases the Growth Rate and Antibacterial Activity of L. plantarum
L. plantarum is one of the bacteria that settle in the intestine in the earliest period of life, and an infant intestine colonized by L. plantarum can combat pathogenic infection [20,24]. Therefore, it is assumed that L. plantarum is able to recognize pathogens and start the defense mechanism accordingly. To confirm this fact, the change in the growth rate of L. plantarum in response to a pathogen was observed by co-culturing the potential pathogenic E. coli with L. plantarum (Fig. 1A). E. coli is one of the early colonizers of an unhealthy infant gut as a potential pathogen [68]. Concentrated E. coli-cultured M9 was added to L. plantarum and then cultured at 37℃ for 6 h. In comparison with the control (-) treated with M9 only, the relative growth rate of the L. plantarum group treated with E. coli-cultured M9 (Ec) showed an increase of 436 ± 85.04%. Interestingly, L. plantarum-cultured M9 (Lp) had no effect on the growth of the L. plantarum itself. This result implies that L. plantarum recognizes the existence of the pathogen and increases its proliferation accordingly.
Fig. 1.E. coli supernatant induces biomass and antimicrobial activity of L. plantarum. The tests were conducted after preparing a supernatant of minimal M9 broth cultured with E. coli and treating it with L. plantarum (A). An aliquot of 40 μg of each M9 (-), E. coli-cultured M9 (Ec), and L. plantarum-cultured M9 (Lp) was treated in L. plantarum. After incubation at 37℃ for 6 h and spreading on an agar plate, the CFU of L. plantarum was observed. Ec increased the growth rate of L. plantarum. Next, 25 μg of this Lp was treated with E. coli (B) and Sal. Typhimurium (C) to observe the antimicrobial activity. This Lp was prepared after treating with 40 μg of E. coli-cultured M9. Antimicrobial activity was analyzed by counting the viable CFU log/ml. (B) Microbial viability of E. coli was measured by treating with L. plantarum-cultured M9 only (LP: second bar graph), L. plantarum-cultured M9 that reacted with E. coli-cultured M9 (Ec + Lp), and ampicillin (5 μg) against E. coli. M9 only (-) was the control. (C) Antimicrobial activity was measured by treating M9 with Sal. Typhimurium. Values represent the mean ± SD. The p value was determined using a two-tailed unpaired Student’s t-test. **p < 0.01, ***p < 0.001 compared with M9 (-)-treated sample. N.S.: not significant.
A following test was conducted to determine if E. coli-cultured M9 (Ec) can also influence the antimicrobial activity of L. plantarum (Figs. 1B and 1C). To observe the antibacterial ability of L. plantarum, different preparations of Lp were treated with the indicator pathogens. The preparation conditions of Lp are prepared M9 only (-); L. plantarum-cultured M9 (Lp); Lp co-cultured with 40 μg of Ec (Ec + Lp); and ampicillin as an antibiotic against E. coli bacteria (Fig. 1B) and Sal. Typhimurium (Fig.1C) . Because the growth rate of L. plantarum is increased by the reaction of E. coli-cultured M9, to exclude the antibacterial ability regarding the increased cell number, an antibacterial test was set equally to the dry cell weight of 25 μg and treated in the indicator strains (E. coli and Sal. Typhimurium). The antibacterial activity was detected by the measurement of bacterial CFU log/ml. The initial cell count of the indicator strains was approximately 3 log/ml. The resulting value of the control (-) group corresponds to that of the indicator strains treated only in culture medium M9. L. plantarum-cultured M9 treated with Ec M9 (Ec + Lp) showed a higher increase of antibacterial ability against E. coli (Ec + Lp = 1.31 ± 0.16 log/ml) than Lp M9 only (2.67 ± 0.5 log/ml). This antibacterial ability was observed to be similar to that of 5 μg of ampicillin (1.32 ± 0.06 log/ml). The antimicrobial activity of Ec + Lp against Sal. Typhimurium was also observed to have results similar to those above. This suggests that L. plantarum can recognize E. coli and promote the secretion of antimicrobial substances.
Quantitative Analysis of Uracil Secretion from E. coli
I set out to ask a fundamental question, based on the above results, about the essence of the E. coli-derived substance that L. plantarum recognizes. A recent study examined pathogen-derived uracil and the host immune response [42]. According to the report, pathogen-derived uracil is a pathogen-specific secretory substance. The uracil is secreted in a small quantity or is not observed inside the intestinal commensal bacteria [42]. To further investigate whether E. coli releases uracil, quantitative analysis of uracil was performed in the supernatant of in vitro cultured E. coli by HPLC. The identity of the putative E. coli-secreted uracil peak was established by comparison against the peak for the uracil reference standard. The peak appeared at the retention time of 13.8 min, which corresponded to the references tandard uracil in M9 media (Fig. 2B). The result showed that E. coli secreted a significant amount of uracil (~45 ng/107 cell/40 μg of dried E. coli M9) (Fig. 2C).
Fig. 2.Validation of uracil determination from the E. coli supernatant. (A) Representative chromatograms of a blank M9 media sample. (B) The M9 medium sample spiked with synthetic uracil (concentration is 100 ng/ml) and internal standard 5-FC (concentration is 500 ng/ml). (C) The E. coli-cultured M9 medium sample spiked with E. coli-derived uracil, spiked with IS 5-FC (concentration is 500 ng/ml). The E. coli-derived uracil is equivalent to culture supernatant secreted from the ~45 ng/107 cells of E. coli. Values represent the mean ± SD at each calibration level calculated using data obtained from three serial runs.
E. coli-Derived Uracil Is a Specific Inducer of the Growth Rate and Antibacterial Activity of L. plantarum
Through previous experiments, the physiological concentration of E. coli-secreted uracil was observed in culture media in vitro. This current work also involved an experiment to determine whether L. plantarum recognizes E. coli-released uracil and reacts to it (Fig. 3). First, uracil was treated with L. plantarum according to each concentration to compare its growth rate (Fig. 3A). L. plantarum was treated with uracil at concentrations of 0.001, 0.01, 0.1, 1, 10, and 50 nM and it was found that L. plantarum’s growth rate increased in a uracil-dose-dependent manner. The dose-dependent analysis showed that uracil can promote the growth rate of L. plantarum most effectively in the range of 0.01-50 nM. The second step involved a test to determine if other nucleobases can influence the growth rate of L. plantarum (Fig.3 A). The other nucleobases used were adenine, cytosine, guanine, and thymine. It was found that only uracil was capable of stimulating the growth rate of L. plantarum. Based on the previous results, when the effects of uracil were tested, a uracil concentration of 0.1 nM was used. Furthermore, the uracil-induced increase in the antibacterial ability of L. plantarum (Fig. 3B) was verified. As a result, in comparison with the Lp-only treatment (Lp), Lp cultured with uracil (Lp + Uracil) accelerated the antibacterial activity against E. coli and Sal. Typhimurium.
Fig. 3.Uracil stimulates the growth rate and antimicrobial activity of L. plantarum; however, the other nucleobases do not. (A) Uracil promoted the growth of L. plantarum in a dose-dependent manner. Uracil induced the most proliferation of L. plantarum at a concentration of 0.1 nM. Uracil was the only stimulating factor for the growth of L. plantarum; the other nucleobases had no effect. The nucleobases (adenine, cytosine, guanine, and thymine) were treated with 0.001–50 nM of L. plantarum, and the growth rate was observed. Values represent the mean ± SD. The p value was determined using a two-tailed unpaired Student’s t-test. ***p < 0.001 compared with untreated sample (uracil 0 nM). N.S.: not significant. (B) Uracil induced the antimicrobial activity of L. plantarum against pathogens. For M9 only (M9), 25 μg of L. plantarum-cultured M9 (Lp) was treated with E. coli or Sal. Typhimurium, and 25 μg of pre-stimulated L. plantarum-cultured M9 with uracil (0.1 nM) (Lp + Uracil) was treated with E. coli or Sal. Typhimurium. Values represent the mean ± SD. The p value was determined using a two-tailed unpaired Student’s t-test. **p < 0.01 compared with M9 sample.
Confirmation of Uracil’s Role by Using Uracil Biosynthesis Mutant Bacteria Strain
HPLC analysis was carried out to verify the actual secretion of uracil in wild-type E. coli-cultured M9 (EcWT) (Fig. 4A). At the same time, HPLC analysis was also carried out on E. coli uracil mutant-cultured M9 (Ec Uracilmutant), whose uracil biosynthesis ability is lost. For this purpose, the uracil mutant bacteria, pyrE-, was purchased from the Keio Collection of single-gene knockout strains. The results showed wild-type E. coli secreted high amounts of uracil, whereas the E. coli uracil mutant did not (Fig. 4A, blue box).
Fig. 4.Potentially pathogenic E. coli releases uracil, and uracil induces the growth rate and antibiotic activity of L. plantarum. (A) Detection of E. coli-derived uracil by HPLC analysis. It was detected that E. coli wild type-cultured M9 (EcWT) included high amounts of uracil, whereas E. coli uracil mutant, pyrE--cultured M9 (Ec Uracilmutant), did not. The positive control was the synthesized uracil. The peaks of uracil are indicated by asterisks. The peak of interest from the E. coli uracil mutant, pyrE--cultured M9, is represented by the blue box. (B, C) Ec Uracilmutant did not stimulate the growth rate or the antibacterial activity of L. plantarum. The lost activity of pyrE--cultured M9, Ec Uracilmutant, was dramatically recovered by the introduction of synthetic uracil. (B) Control (-) was M9-only treatment with L. plantarum. EcWT was treated with L. plantarum (EcWT). Uracil mutant pyrE--cultured M9 (Ec Uracilmutant) was treated with uracil (0, 0.01, and 0.1 nM) to L. plantarum. After each M9 was treated with L. plantarum, the CFU of L. plantarum was detected. Values represent the mean ± SD. The p value was determined using a two-tailed unpaired Student’s t-test. **p < 0.01, ***p < 0.001 compared with M9 (-)-treated sample. N.S.: not significant. (C) The control was a sample treated with non-stimulated M9 only (-) to E. coli . EcWT-treated L. plantarum (EcWT + Lp) had antibacterial activity against E. coli. In contrast, Ec Uracilmutant -treated L. plantarum lost its antimicrobial activity (Ec Uracilmutant + Lp + Uracil 0 nM). This lost antimicrobial activity was overcome through the mutual treatment of uracil (Ec Uracilmutant + Lp + Uracil (0.01 or 0.1 nM)). These results demonstrate the dose-dependence of uracil. Values represent the mean ± SD. The p value was determined using a two-tailed unpaired Student’s t-test. **p < 0.01, ***p < 0.001 compared with M9 (-)-treated sample. N.S.: not significant.
The next step involved a test to determine if the E. coli-derived uracil exerts an influence on the growth of L. plantarum and the secretion of antimicrobial substances. Fig. 4B shows the significantly different growth rates of L. plantarum treated with EcWT and L. plantarum treated with Ec Uracilmutant. As expected, the growth rate of L. plantarum treated with EcWT showed an increase, whereas Ec Uracilmutant was not influenced. However, interestingly, after adding uracil (0.01 and 0.1 nM) to the Ec Uracilmutant test group, the growth rate of L. plantarum was recovered. The same findings were made in the test of antibacterial activity (Fig. 4C).
In short, the test results based on Ec Uracilmutant strongly support the idea that E. coli-derived uracil is one of the substances that is recognized by L. plantarum to increase the growth rate and antibacterial ability of L. plantarum.
Analysis of Antibacterial Activity of L. plantarum Induced by E. coli-Derived Uracil
Spectrum analysis was carried out by applying the antibacterial ability of L. plantarum increased by E. coli uracil to diverse pathogens (Table 1). The indicator strains used for the test are as follows: E. coli, Sal. Typhimurium, Staph. aureus, P. aeruginosa, K. pneumoniae, B. subtilis, and B. cereus. The evaluation of antibacterial ability was confirmed based on the method of well diffusion assay by preparing M9 of a corresponding condition and treating each M9 with the indicator bacteria. Antimicrobial activity was measured and analyzed by the growth inhibitor zone (mm).
Table 1.The control was only treated with M9 in the indicator strains, and a growth inhibition zone was observed. Lp was treated with L. plantarum-cultured M9 in the indicator strains. EcWT + Lp was treated in the indicator strains with the prepared Lp reacting EcWT. Ec Uracilmutant + Lp was treated in the indicator strains with prepared Lp after E. coli uracil mutant-cultured M9 (Ec Uracilmutant) was treated with L. plantarum. Ec Uracilmutant + Uracil + Lp was treated in the indicator strains with prepared Lp after adding 0.1 nM of uracil simultaneously when Ec Uracilmutant reacted with L. plantarum. The concentrations of each of the M9 supernatants and ampicillin were 25 μg/disc and 5 μg/disc, respectively. The expressed growth inhibition zone included the size of the disk. The data represent the results of three independent determinations. Values represent the mean ± SD. The p value was determined using a two-tailed unpaired Student’s t-test.
Lp had equal antibacterial ability against all the bacteria. It was observed that the antibacterial ability of EcWT + Lp was higher than that of Lp only. On the other hand, the antibacterial ability of Ec Uracilmutant + Lp was similar to the activity of Lp only. However, through adding uracil, the antibacterial activity recovered to the antibacterial ability of EcWT + Lp.
Based on the findings so far, it is observed that L. plantarum’s growth rate and production of antimicrobials are increased by exposure to E. coli-derived uracil.
Effects of Temperature, pH, and Enzymes on the Inhibitory Activity of Antibiotic Substances Released from L. plantarum
For characterization, the effects of enzymes, heat, and pH on candidate antibiotics released from L. plantarum were determined. The sample Lp induced by Ec was observed to be sensitive to protease K (Table 2). The susceptibility of the antimicrobial compounds to protease K also represents the properties of the bacteriocin. Numerous researchers have shown that the activity of bacteriocin is lost by treatment with proteolytic enzymes [43]. This result showed that the antimicrobial substance of L. plantarum is proteinaceous as a bacteriocin. The inhibitory activity test of heat was not effective at 30℃ for 60 min, and approximately 50% of activity remained at 70℃, but its activity was completely lost at 90℃ and 100℃. The antibiotics of L. plantarum were completely stable at pH 6, 7, and 8, and approximately 50% of activity remained at the pH values between 4–5 and 9, but its activity was lost at all other pH values (Table 2). Taken together, the above results suggests that the antibacterial substance is a protein and partially has the properties of a bacteriocin. These findings have already been demonstrated in several studies, which reported that L. plantarum species produce bacteriocins as antimicrobial substances [1,50,65,66]. These results provide a basic knowledge of the chemical properties of the antimicrobial substance of E. coli-induced L. plantarum.
Table 2.To determine the effect of heat on the inhibitory activity of antimicrobial compounds produced by L. plantarum, the M9 of L. plantarum was exposed to different temperature ranges, pH, and protease K for 30 min. Pre-treated L. plantarum-cultured M9 was adjusted to the indicator strain E. coli. All antibacterial activity was detected by well diffusion assay. The untreated L. plantarum-cultured M9 was the control (100%), and the results of residual activity for the treated samples were obtained by measuring the relative value of the control sample. The data represent the results of three independent determinations. Values represent the mean ± SD. The p value was determined using a two-tailed unpaired Student’s t-test.
SDS-PAGE and Detection of Uracil-Induced Antimicrobial Substance
The fractionated L. plantarum M9 supernatant (Lp) was dialyzed and analyzed using SDS-PAGE (Fig. 5). The gel showed a distinct band at approximately 140 kDa. This band’s intensity was increased in Lp treated with uracil (0.1 nM). The 140 kDa band disappeared upon treatment with protease K (Lane 4), suggesting that it may responsible for the antibacterial activity by Lp. These data show that uracil increases production of the bacteriocin-like substance of L. plantarum.
Fig. 5.Protein analysis of antimicrobial substance from L. plantarum supernatant. The L. plantarum M9 medium supernatant (Lp) was analyzed by standard protocols using Tris-glycine PAGE after concentration using ammonium sulfate and dialysis. Lane 1: Lp only; Lane 2: Lp reacted with uracil (0.1 nM); Lane 3: broad molecular weight standard; Lane 4: Lp treated with protease K after reacting with uracil. The arrows indicate the antibacterial substance at approximately 140 kDa.
Discussion
The intestinal commensal flora is essential for regulating the physiologic process, and the adequate composition of gut flora is critical for preserving a healthy condition [60]. Accumulated evidence shows strong associations between the gut normal flora and risks of immune-related diseases in infancy [24,67]. In general, as mentioned previously, reducing the diversity of beneficial microorganisms and increasing the number of pathogenic bacteria have a meaningful correlation. Therefore, understanding the factors that affect the consortium of gut microbiota would improve the efficacy of clinical treatment.
This work aimed to determine how the beneficial intestinal bacteria settle faster and better than pathogenic bacteria. It was found that the cultured supernatant of potential pathogenic E. coli increased not only the growth rate, but also the antimicrobial activity of L. plantarum.
According to a recent report, pathogenic bacteria secrete considerable amounts of uracil, whereas commensal bacteria secrete small amounts or none [42]. Uracil is a specific factor that differentiates pathogens from commensal bacteria [42,64]. Based on this research, the present study demonstrates that uracil acts as a microbe-derived factor that induces the growth rate and antimicrobial activity of L. plantarum. These results are confirmed by the E. coli uracil synthesis mutant strain.
Some of the fundamental questions raised are why E. coli releases uracil and how L. plantarum recognizes E. coli-derived and/or host cell-released uracil in the human gut. First, E. coli entering the stationary phase accumulates endogenous and exogenous nucleobases [57] and especially secretes uracil and xanthine. There was not a significant amount of other nucleobases, nucleosides, and nucleotides in the culture media of E. coli, except for a small amount of cyclic AMP [57]. The excretion of nucleobases of E. coli is always connected to a decline in their growth rate [57]. Interestingly, it was discovered that uracil increases quorum sensing and promotes the secretion of virulence factor and the formation of biofilm in Pseudomonas aeruginosa [64]. These findings suggest that uracil may serve as a regulator for virulence phenotypes, including E. coli [64]. In this context, bacterial symbionts may evolve to reduce uracil excretion in the gut [42], and this mechanism may be the basis for commensal bacteria coexisting in the gut of the host. Next, under certain conditions – for example, the inflammation and infection of pathogens – several cell types release nucleotides, particularly ATP and/or UTP, from the intracellular to the extracellular space [14,15,28,69]. Other studies have confirmed that the secretion of uridine nucleotides, such as UTP, UDP, and UDP-glucose, is increased during cystic fibrosis [38]. Indeed, many reports have directly confirmed that various cells secreted a nanomolar amount of UTP [39–41]. According to these studies, it is suggested that cells under an inflammatory disease condition release nucleotides into the extracellular space. The P2X/P2Y receptors are activated by ATP and/or UTP. Recent studies suggest that the cellular signal transduction pathways of P2X/P2Y had a significant relationship with the inflammatory reaction that defends against pathogens and regulates the emergence of tumors [15,28]. Similarly, damaged cells secreted UTP and induced the expression of P2Y6R, usually in inflammatory bowel syndrome and during the phagocytosis process [33]. Although these studies do not directly report uracil release in the gut, they show that uracil nucleotides could be released as extracellular signaling molecules. In other words, cell lysis (e.g., owing to inflammation or infection of the gut) is likely to lead to the leakage of a small amount of uracil nucleotides (~5 nM) [41] and its degradative products, such as uracil, to extracellular parts. If L. plantarum senses extracellular uracil in the gut, L. plantarum may induce the growth rate and secretion of antimicrobial substances. In addition, L. plantarum has the potential to further enhance the tight junction of the intestinal barrier, which protects against pathogen invasion in the gut [61]. Therefore, it is important that L. plantarum senses uracil released from E. coli and the intestinal epithelium cells, and this mechanism promotes immune homeostasis and maturation. Because of this, the mechanism is restricted by cellular damage, apoptosis, and specific inflammatory condition [9].
In previous reports, bacteriocin production and growth in L. plantarum spp. have been shown to be activated by co-culturing with specific bacterial strains in olive fermentation [47]. These strains are Enterococcus faecium 6T1a-20 and Pediococcus pentosaceus FBB63, which have the ability to produce bacteriocin. These results showed that the two heat-killed bacterial strains (Enterococcus faecium 6T1a-20 and Pediococcus pentosaceus FBB63) retain their ability to induce growth and bacteriocin production when co-cultured with L. plantarum [47]. Viable bacteria are not strictly necessary for the induction of bacteriocin by L. plantarum. Therefore, L. plantarum recognizes either heat-stable inducing molecules or the presence of inducing strains. These results demonstrate the physiological mechanisms of L. plantarum that can enhance its viability and the production of antimicrobial substances. Thus, these results show that uracil is one of the inducers of L. plantarum.
Many studies have reported the following influential factors in the development of infant gut microbiota: intrauterine colonization, gestational age (preterm vs. term), delivery type (vaginal vs. cesarean delivery), feeding type (breast vs. formula), maternal nutritional status (pre-or probiotics), antibiotics, and host genetics [16,24]. Alternation of the gut microbiota by environmental factors may result in the incidence of immune-related disorders in infants. In light of these factors, the initial colonization of intestinal normal flora in infants could be affected by delivery modes. A baby born by cesarean section has fewer beneficial gut microflora, which raises the prevalence of atopic diseases [5,10,30,37,51,60]. This suggests that allergic children are colonized with Lactobacillus less often than non-allergic children [21]. The difficulty of Lactobacillus settling in the gut can be explained as follows: maternal antibiotics are administered, and cesarean-delivered babies lack probiotic supplementation by taking infant formula, which causes the delay of the colonization of Lactobacillus. Thus, the pathogen eventually settles as a dominant species [5]. On the other hand, Lactobacillus settles swiftly in infants born by normal delivery and forms a resistance to pathogen infection.
The application of prebiotics has been another target of research on the regulation of gut flora. A prebiotic is “an ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of bacterial species already resident in the colon” [20]. Several studies have indicated that prebiotics may effectively reduce the infection rate of pathogens in infants [44] and the adequate administration of probiotics may protect infants against necrotizing enterocolitis [4]. Based on these notions, uracil can be classified as a prebiotic in favor of L. plantarum according to the results of this study. Thus, crosstalk between uracil and L. plantarum promotes immune homeostasis and maturation. In addition, it could contribute to future clinical applications of probiotics and increase the effectiveness of treatment.
To conclude, this study has significance, as it was found that L. plantarum recognized a pathogen, E. coli, thereby inducing the growth and enhancement of antibacterial ability, and E. coli-derived uracil was discovered as an inducer. Since L. plantarum secretes antibiotic substances, such as bacteriocin, as symbiotic bacteria that first inhabit the gut of an infant, its antibacterial ability is excellent. Based on its ability, it can be assumed that L. plantarum will play a defensive role at the forefront against pathogen infection. Thus, finding factors for improving the growth and antibacterial ability of L. plantarum will provide basic knowledge of medical and pharmaceutical areas related to intestinal immunity.
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