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Mucin2 is Required for Probiotic Agents-Mediated Blocking Effects on Meningitic E. coli-Induced PathogenicitiesS
Mucin2 is Required for Probiotic Agents-Mediated Blocking Effects on Meningitic E. coli-Induced PathogenicitiesS
Journal of Microbiology and Biotechnology. 2015. Oct, 25(10): 1751-1760
Copyright © 2015, The Korean Society For Microbiology And Biotechnology
  • Received : February 04, 2015
  • Accepted : June 08, 2015
  • Published : October 28, 2015
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About the Authors
Jing-Yi Yu
Saban Research Institute, Children’s Hospital Los Angeles, University of Southern California, Los Angeles, CA90027, USA
Xiao-Long He
1Department of Microbiology, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou 510515, P.R. China
Santhosh Puthiyakunnon
1Department of Microbiology, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou 510515, P.R. China
Liang Peng
Department of Clinic Laboratory, the Second Affiliated Hospital of Guangzhou Medical University, Guangzhou 510260, P.R. China
Yan Li
1Department of Microbiology, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou 510515, P.R. China
Li-Sha Wu
The First School of Clinical Medicine, Southern Medical University, Guangzhou 510515, P.R. China
Wen-Ling Peng
The First School of Clinical Medicine, Southern Medical University, Guangzhou 510515, P.R. China
Ya Zhang
The First School of Clinical Medicine, Southern Medical University, Guangzhou 510515, P.R. China
Jie Gao
The First School of Clinical Medicine, Southern Medical University, Guangzhou 510515, P.R. China
Yao-Yuan Zhang
The First School of Clinical Medicine, Southern Medical University, Guangzhou 510515, P.R. China
Swapna Boddu
1Department of Microbiology, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou 510515, P.R. China
Min Long
1Department of Microbiology, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou 510515, P.R. China
Hong Cao
1Department of Microbiology, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou 510515, P.R. China
gzhcao@smu.edu.cn
Sheng-He Huang
Saban Research Institute, Children’s Hospital Los Angeles, University of Southern California, Los Angeles, CA90027, USA
gzhcao@smu.edu.cn

Abstract
Mucin2 (MUC2), an important regulatory factor in the immune system, plays an important role in the host defense system against bacterial translocation. Probiotics known to regulate MUC2 gene expression have been widely studied, but the interactions among probiotic, pathogens, and mucin gene are still not fully understood. The aim of this study was to investigate the role of MUC2 in blocking effects of probiotics on meningitic E. coli -induced pathogenicities. In this study, live combined probiotic tablets containing living Bifidobacterium , Lactobacillus bulgaricus , and Streptococcus thermophilus were used. MUC2 expression was knocked down in Caco-2 cells by RNA interference. 5-Aza-2’-deoxycytidine (5-Aza-CdR), which enhances mucin-promoted probiotic effects through inducing production of Sadenosyl-L-methionine (SAMe), was used to up-regulate MUC2 expression in Caco-2 cells. The adhesion to and invasion of meningitic E. coli were detected by competition assays. Our studies showed that probiotic agents could block E. coli -caused intestinal colonization, bacteremia, and meningitis in a neonatal sepsis and meningitis rat model. MUC2 gene expression in the neonatal rats given probiotic agents was obviously higher than that of the infected and uninfected control groups without probiotic treatment. The prohibitive effects of probiotic agents on MUC2-knockdown Caco-2 cells infected with E44 were significantly reduced compared with nontransfected Caco-2 cells. Moreover, the results also showed that 5-Aza-CdR, a drug enhancing the production of SAMe that is a protective agent of probiotics, was able to significantly suppress adhesion and invasion of E44 to Caco-2 cells by upregulation of MUC2 expression. Taken together, our data suggest that probiotic agents can efficiently block meningitic E. coli -induced pathogenicities in a manner dependent on MUC2.
Keywords
Introduction
E. coli is the most common gram-negative bacterium that causes neonatal sepsis and meningitis (NSM) [27 , 28 , 47] . Most E. coli -induced neonatal bacterial meningitis cases occur via hematogenous spread [19] . It is accompanied by the three hallmark features of bacterial meningitis: NF-κB activation, meningitic E. coli invasion, and polymorphonuclear neutrophil migration through the blood-brain barrier (BBB) [10 , 11 , 13 , 51 , 56] . The E. coli -induced NSM generally occurs when bacterial pathogens adhere to intestinal epithelial cells and then cross the intestinal wall into the bloodstream [31 , 32] . Previous studies of humans and animals indicate that the development of NSM is associated with the magnitude of bacteremia, which is essential for pathogen crossing the BBB [31 , 32] .
In recent years, there has been a raising incidence of early onset E. coli infections in very low birth weight neonates. Widespread use of antibiotic may result in an increasing incidence of neonatal infections caused by antibiotic resistance, which has become an evolutionary and ecological problem originating from the response of bacteria to antibiotics [37] . The ongoing antimicrobial resistance crisis will be certainly enhanced by widespread antibiotic use, leading to the increasing global incidence of infectious diseases that are untreatable with any known antimicrobial agent [20] . Recently, from clinical observations, probiotic microorganisms have been shown to be useful in preventing or treating certain infectious diseases as well as allergic disorders, including vaginitis, inflammatory bowel disease, and atopic dermatitis. Both prophylactic and therapeutic effects have been observed in children and adults [1 , 3 , 15 , 29 , 46] . As probiotics contribute to the maintenance of ecological balance, the use of probiotics for the prophylaxis of early onset neonatal sepsis or meningitis may overcome the major adverse consequence of widespread antibiotic use, which disturbs the normal microbiota [17 , 20] .
The introduction of probiotic microorganisms ( e.g. , probiotic agents) into the human body is quite an attractive rationale for modulating the host immune system and gut microbiota, enhancing the symbiotic homeostasis of the super-organism, and providing protection against pathogens ( e.g. , meningitic E. coli ). Lactic acid bacteria (LAB), nonpathogenic antibiotic-resistant ascospore yeasts, and principally Saccharomyces boulardii are the most commonly used probiotics. A wide range of product types such as lyophilized form and fermented food products containing viable microorganisms with probiotic properties are commercially available [1] . In this study, the commercial probiotic agents used were Live Combined Bifidobacterium and Lactobacillus tablets containing original Bifidobacterium , Lactobacillus bulgaricus , and Streptococcus thermophilus . It is a dynamic combination of probiotic agents that is full of activity and colonization rates, which can quickly add to the intestine so that the number of intestinal probiotics could be restored to a healthy level.
Our previous studies have demonstrated that two subsequent steps, consisting of bacterial adhesion and invasion, are of great importance for pathogen access to the host cells. In order to cause meningitis, bacterial pathogens such as meningitic E. coli K1 must penetrate across the gut immune barrier and the BBB [26] . E. coli K1 binding to and penetration across the gut barrier is a prerequisite for bacteria entering into the blood circulation system (bacteremia) [6 , 28 , 44] . Neonatal bacterial meningitis usually develops as a result of bacteremia, which is one of the two key pathogenesis processes for meningitic pathogens to penetrate the brain. Bacterial traversal of the BBB due to high-level bacteremia is the second essential step for the evolution of bacterial meningitis [28] . To investigate the prophylactic efficacy of probiotics in neonatal bacterial bacteremia and meningitis, we had previously examined the suppressive effect of Lactobacillus rhamnosus GG (LGG) on meningitic E. coli K1 infection by using both the in vitro (Caco-2, a human epithelial cell culture model) and in vivo (the neonatal rat model of E. coli bacteremia and meningitis) models [26] . LGG could block E. coli K1 adhesion, invasion, and transcytosis in a dosedependent manner in Caco-2. The levels of pathogen colonization, E. coli bacteremia, and meningitis were significantly decreased in the LGG-treated neonatal rats when compared with those in the control groups. The underlying mechanism remains unclear, however, how probiotics enhance the host defense against bacterial pathogens.
The intestinal epithelial cells are involved in the formation of a physicochemical barrier and possess innate protective strategies against pathogenic challenge [33] . Others have indicated that intestinal tract mucins, located on the surface of the intestinal epithelium, acted as a protective layer against microbial damage and inhibit bacterial translocation [21 , 24 , 41] . MUC2, the main secreted mucin in the small and large intestines, is considered to be the major structural gel-forming component of the mucus gel [16 , 23 , 41] . Studies indicated that MUC2 mucin is an important regulatory factor in the gut immune systems, and selected lactobacilli may be able to induce the upregulation of MUC2 gene expression [53] . It has been shown that mucin-mediated gut barrier functions and beneficial effects of probiotics are enhanced by S-adenosyl-L-methionine, whose production is up-regulated by 5-aza-2-deoxycytidine (5-aza-CdR) [5 , 14 , 43] . As mentioned before, enteric bacterial pathogens crossing the gut barrier is the first critical step to cause bacteremia and then meningitis. In this report, the in vitro and in vivo model systems of the gut barrier and the BBB were used to examine whether MUC2, a protective layer against microbial damage, could play an active role in the host defense against bacterial translocation. Indeed, our studies on down- and up-regulation of MUC2 expression have shown that MUC2 is required for probiotics to efficiently block E. coli -induced bacteremia and meningitic infections.
Materials and Methods
- Bacterial Strains and Culture Conditions
Live Combined Bifidobacterium and Lactobacillus tablets (Trade Name: Golden Bifid; 0.5 g/tablet, containing no less than fifty million living Bifidobacterium , five million living Lactobacillus bulgaricus and Streptococcus thermophilus ) were generously provided by Inner Mongolia ShuangQi Pharmaceutical Co. Ltd. The three active components of the tablets were produced according to authorized manufacturing production and identifying procedures. The probiotic agents were grown in Lactobacilli MRS Broth (MRS Hardy Diagnostics, Santa Maria, CA, USA) at 37℃ for 24 h without agitation. To examine the molecular identities of the commercial probiotic agents containing Bifidobacterium , Lactobacillus bulgaricus , and Streptococcus thermophilus , PCR analysis and DNA sequencing were performed [48 , 52] . The bacteria genomic DNAs were extracted with a bacteria genomic DNA extraction kit (TIANGEN, China) according to the manufacturer’s instructions. The 16S RNA gene sequences of Bifidobacterium , Lactobacillus bulgaricus , and Streptococcus thermophilus were obtained from GenBank, and the corresponding PCR primers of the specific 16S RNA gene were designed using Primer 5.0 software (Table S1). PCR amplification was performed with a Biometra PCR amplification system (UNOII-Themoblockt, Germany) and DNA fragments were amplified as follows: initial heating at 94℃ for 2 min, followed by 30 cycles of denaturation at 94℃ for 20 sec, annealing at 55℃ for 40 sec, extension at 72℃ for 30 sec, and a 7 min final extension step at 72℃. The PCR products were electrophoresed on a 1.5% agarose gel containing ethidium bromide and visualized by UV-induced fluorescence. In this analysis, mean values of three separate experiments were calculated Results showed that the PCR products were of the expected length (Fig. S1).
E. coli K1 strain E44, a clinic E. coli RS218 (O18:K1:H7) isolate from the CSF of a newborn infant with meningitis, is a rifampicinresistant strain. E44 was grown in brain heart infusion (BHI) broth with rifampicin (50 μg/ml) at 37℃ overnight without shaking [26 , 55] .
- Cell Culture Conditions and DNA Methylation Inhibitor Treatment
Human colonic carcinoma cell line Caco-2 (ATCC, Rockville, MD, USA) was maintained in 25 mmol/l glucose-Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 4 mmol/l glutamine, 1% nonessential amino acids, and 1% penicillin/streptomycin at 37℃ in a humidified atmosphere with 5% CO 2 . Cells were used within 14 days of seeding or within 5 days of confluence [21] .
One day before treatment, Caco-2 cells were seeded in a 24-well plate (10 5 cells/well) and grown to confluence. The cell monolayers were treated with 5 × 10 -6 mol/l 5-Aza-CdR (freshly prepared) respectively for 72 h [22 , 42 , 49] . The medium was changed with the same concentration every 24 h. The cells without 5-Aza-CdR treatment were the control group, which was treated with an equal volume of medium instead of 5-Aza-CdR culture medium. The wells were washed twice with sterile PBS and 0.5 ml/well experimental media without antibiotics was added before bacterial treatment/infection.
- Neonatal Rat Model ofE. coliK1 Strain E44 Meningitis
The probiotic agents-induced blocking effects on meningitic pathogens were examined in the neonatal rat model of E. coli K1 strain E44 meningitis. All procedures involving laboratory animal use were in accordance with the guidelines of the Instituted Animal Care and Use Committee of Southern Medical University (SMU). All protocols were submitted and validated by the Animal Care Ethics Committee of SMU (No. 2012–055). A total of 20 pathogen-free Sprague Dawley rats (5 pups/group) were pooled together and randomly distributed into four groups (I: probiotics; II: probiotics + E44; III: E44; and IV: PBS). The 2-day-old pups were given probiotic agents (10 10 CFU/pup/day) by feeding using an FB Multiflex tip for 3 days. Control group and E44 group rats were fed PBS only [26] . At 5 days old, pups in the probiotics + E44 group and E44 group received E44 (10 9 CFU/pup), and pups in the probiotics group and control group received the same volume of PBS only. Intestine, blood, and CSF samples were plated onto LB agar plates with rifampin (50 μg/ml) after E44 inoculation for 48 h. Intestinal colonization, bacteremia, and meningitis were defined as a positive intestine culture, a positive blood culture, and a positive CSF culture, respectively. Colon tissue samples were washed with PBS and stored in RNAlater Storage Solution (Sigma) for RT-qPCR.
- RNA Extraction and Reverse Transcription PCR Analysis in Rat Colon Tissues
In order to determine the role of MUC2 in the probiotic agentsinduced preventing and blocking effects on meningitic E. coli ., total RNA in colon tissues and Caco-2 cells were extracted with Trizol reagent (Invitrogen, USA) according to the manufacturer’s recommendations, and then reverse transcribed in a 20 μl reaction system using the RT kit (Takara, Japan) according to the manufacturer’s protocol. For RT-qPCR experiments, the forward and reverse primers corresponding to MUC2 and β-actin were designed using Primer 5.0 software ( Table 1 ). This was performed with an Mx3000P Real-Time PCR system and SYBR Premix Ex Taq II (Perfect Real Time) Kit (Takara, Japan) under the following PCR conditions: 95℃ for 30 sec; 40 cycles od 95℃ for 5 sec, 60℃ for 30 sec, and 72℃ for 5 min. The MUC2 mRNA expression levels relative to β-actin were calculated by the 2 -ΔΔCt method [36] . In this study, data from three separate experiments were averaged.
Target gene primers corresponding to MUC2 and β-actin.
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Target gene primers corresponding to MUC2 and β-actin.
- Silencing Vector Construction and Generation of Transient Transfectant
We used the short hairpin RNA (shRNA) from the pGPU6/GFP/Neo vector containing the human U6 promoter (GenePharma, Shanghai, China) to knock down the MUC2 gene in Caco-2 cells. The scrambled shRNA was defined as a negative control (marked as “NC” in the figure). The recombinant constructs were verified through analyzing the fragments generated from digestion with Bam HI and by DNA sequencing. The interference efficiency was analyzed by RT-qPCR.
Caco-2 cells were seeded in a 12-well plate and grown to 85%-90% confluence. Cells were incubated in the transfection mixtures of shRNA plasmids and Lipofectamine 2000 reagent (Invitrogen, USA) for 48 h according to the manufacturer’s protocol. Transfected cells were observed via fluorescence microscopy to estimate the transfection rate. Cells transfected with siRNA oligos containing no homology with any human gene were used as the negative control [18] .
- Western Blot Analysis
To detect MUC2 protein expression in Caco-2 cells, western blot analysis was performed. For the preparation of lysates, the cells were washed with ice-cold PBS and lysed in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, and 0.1% SDS) supplemented with protease inhibitors. Cells were scraped and centrifuged at 4℃ for 15 min. The supernatant was obtained, and the protein concentration was determined using the Bradford method (Bio-Rad). Samples were then prepared by mixing with 4× SDS sample buffer and boiling for 5 min, and then subjected to 10% SDS-PAGE and electrophoretically transferred to PVDF membranes (Millipore, USA) [12] . The membranes were blocked with 5% nonfat dry milk for 1 h at room temperature and incubated with primary antibody (anti-MUC2 antibody purchased from Abcam) at 1:500 under constant agitation overnight at 4℃. After washing with PBST, the membranes were incubated with HRP-conjugated secondary antibody (anti-rabbit, 1:5,000) for 1h at room temperature under constant agitation. Proteins were developed using an enhanced chemiluminescence system (ECL; Amersham Biosciences, USA).
- Adhesion and Invasion Assays
The bacterial adhesion and invasion assays were performed as described previously to assess exclusion of E44 strain by probiotics agents [6 , 28] . For the competition assay, Caco-2 cells were seeded in a 24-well plate and then pre-incubated with 1 × 10 8 CFU of probiotic agents and 1 × 10 7 CFU of E44 in the EM (experimental medium) at 37℃ for 1.5 h to allow adhesion and invasion to occur.
For adhesion assays, the cells were washed three times with PBS to remove unattached bacteria. The bacteria bound to intestinal mucus were released and lysed with 200 μl of 0.5% Triton X-100 for 8 min, followed immediately by the addition of 150 μl of sterile water. The lysate samples were diluted with a double dilution method and cultured on LB agar plates containing rifampin to determine the total number of colonies (CFU/ml) recovered from the lysed cells. Each experiment was repeated for three times. Results were indicated as relative adhesion (adhesion% as compared with the adhesion of the control E44 group).
For invasion assays, the cells were washed three times with PBS, and incubated with EM containing gentamicin (100 μg/ml) for 1 h at 37℃ to eliminate the extracellular bacteria for the determination of the number of intracellular bacteria. Each experiment was repeated for three times. Results were indicated as relative adhesion (adhesion% as compared with the adhesion of the control E44 group).
- Statistical Analysis
Data analysis was performed as described previously [9] . ANOVA and covariates followed by a Student-Newmann-Keuls test for a multiple comparison test were performed to determine the significant difference between the control and experimental groups. P < 0.05 was considered as statistically significant.
Results
- Probiotic Agents Enhance MUC2 Gene Expression in Neonatal Rats
The intestinal mucus layer and gut microbiota are strongly intertwined and this would be conducive to the maintenance of the intestinal epithelial barrier and immune intestinal homeostasis [45] . Our in vivo studies showed that probiotic agents could block E. coli -caused intestinal colonization, bacteremia, and meningitis in a rat model of NSM ( Figs. 1 A, 1 B, and 1 C). We tested the effect of the probiotic agents and E44 on baseline mucus expression in rats and assessed the regulation of the MUC2 gene in the presence of probiotics and E44 bacteria. MUC2 expression was based upon the relative expression as compared with β-actin. As had been expected, the mRNA levels of MUC2 mucin were up-regulated in the probiotics group. In rats with E44, MUC2 gene expression was lower than in the control group. Conversely, no changes in MUC2 were detected in rats fed intragastrically by probiotic agents and E44 ( Fig. 1 D). These results indicated that the MUC2 gene expression in rat colon could be selectively stimulated by probiotic agents, and these rats may up-regulate MUC2 mucin synthesis as a defense mechanism.
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Blocking effects of probiotic agents on intestinal colonization, bacteremia, meningitis, and MUC2 expression in rat colon tissues after receiving probiotics and E44. A total of 20 rat pups were randomly divided into four groups; (I: probiotics group; II: probiotics + E44 group; III: E44 group, IV: control (PBS) group). For groups I and II, probiotic agents (1010 CFU/pup/day) were given orally to 2-day-old rats for 3 days before E44 infection (109 CFU/pup) for 2 days. At 7 days old, the intestine, blood, and CSF samples were obtained and used for bacterial culture in LB agar plate with rifampin (50 μg/ml) for displaying the intestinal colonization (A), bacteremia (B), and meningitis (C), respectively. (D) The total RNAs of intestine samples were isolated and the expression of MUC2 was analyzed with RT-qPCR. In contrast to the control group, the mRNA levels of MUC2 were up-regulated in the probiotic agent-treated group. MUC2 expression was based on the relative value as compared with β-actin. **P < 0.01 vs. control group.
- MUC2 Expression was Down-Regulated in Caco-2 Cells Transfected with MUC2-shRNA Interference
To gain further insight into the particular function of MUC2 in Caco-2 cells, we employed an RNAi module to selectively down-regulate MUC2 expression. A total of four plasmids were constructed and named as pGPU6/GFP/Neo/MUC2-1, pGPU6/GFP/Neo/MUC2-2, pGPU6/GFP/Neo/MUC2-3, and pGPU6/GFP/Neo/MUC2-4, respectively. Proper construction of the plasmid was confirmed by cutting with restriction enzymes. Positive clones were verified by DNA sequencing (data not shown). The state of transfected cells was evaluated by fluorescence microscopy, which showed the transfection efficiency was approximately 90%. The mRNA levels of MUC2 were tested by RT-qPCR. As indicated in Fig. 2 A, mRNA expression of MUC2 in Caco-2 cells transfected with pGPU6/GFP/Neo/MUC2-1, pGPU6/GFP/Neo/MUC2-2, pGPU6/GFP/Neo/MUC2-3, and pGPU6/GFP/Neo/MUC2-4 were reduced by 78%, 41%, -14%, and 62%, respectively, in comparison with the control cells. To determine the inhibition efficiency of MUC2 protein expression, western blot analysis was done. Similar to the results of RT-qPCR analysis, pGPU6/GFP/Neo/MUC2-1 exerted the strongest effect in suppressing MUC2 expression ( Fig. 2 B). Values for the fold-increase are presented as mean ± SEM of three independent experiments as quantitated by densitometry ( Fig. 2 C). Therefore, we selected pGPU6/GFP/Neo/MUC2-1 for the further experiments.
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MUC2 knockdown efficiency in Caco-2 cells. Caco-2 cells were cultured in 12-well plates. The cells reaching 85-90% confluence were incubated with both mixtures of shRNA plasmids and Lipofectamine 2000 reagent for 48 h. The transfection rate was estimated with a fluorescence microscope and MUC2 knockdown efficiency was detected by RT-qPCR (A) and western blotting (B-C). β-Actin was used as a loading control, and the expression level of MUC2 mRNA was normalized with β-actin (n = 3, means ± SD). The densitometric analysis of MUC2/β-actin ratio was performed using the Gel-Pro Analyzer 4.0 software.
- MUC2 Is Required for Probiotics-Induced Blockage of E44 Adhesion to and Invasion of Caco-2 Cells
We hypothesized that probiotic agents could suppress bacterial penetration across the gut barrier and bacteremia/meningitis through enhancing host MUC2 defence functions. In order to investigate the role of MUC2 in probioticsmediated blocking effects on meningitic E. coli -induced pathogenicity, the competitive exclusion assay was perform. We examined the ability of probiotic agents to intervene in the adhesion and invasion of meningitic E. coli E44 to Caco-2 cells. As shown in ( Fig. 3 ), probiotic agents were able to emulously suppress E44 invasion and adhesion in Caco-2 cells (* p < 0.05, ** p < 0.01). In contrast, after MUC2 silencing, the inhibition by probiotics was significantly lower than the untreated group. The relative adhesion rate of E44 was 63.64% ( Fig. 3 A), and the relative invasion rate of E44 was 70.33% ( Fig. 3 B). The invasion and adhesion rates of E44 at the zero concentration of probiotic agents were assigned as 100% and the effects of probiotic agent pre-incubation were compared with this control level.
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MUC2 is required for probiotic agents-induced blocking effects on E44 adhesion and invasion. Caco-2 cells were cultured into a 24-well plate and grown as a monolayer, and 1 × 108 CFU of probiotics mixtures and 1 × 107 CFU of E44 were added to incubate for 1.5 h. Adhesion (A) and invasion (B) assays were performed as described in the Materials and Methods section. The results are presented as relative adhesion% and invasion% compared with that of the control group without probiotics. *P < 0.05, **P < 0.01.
- Re-Expression of MUC2 in 5-Aza-CdR-Treated Caco-2 Cells
In order to investigate whether 5-Aza-CdR could enhance the biological activity of probiotics through up-regulation of MUC2, which may be activated by SAMe, we first analyzed and compared the changes of MUC2 expression before and after adding with 5-Aza-CdR. We used 5-Aza-CdR to selectively up-regulate MUC2 expression in Caco-2 cells. RT-qPCR analysis showed that the promoter region of MUC2 exhibited a demethylation state and induced MUC2 re-expression. In the 5-Aza-CdR and probiotics group, MUC2 expression was significantly up-regulated. However, MUC2 expression was much lower in the E44 group (** p < 0.01, Fig. 4 ).
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Comparative analysis of the effect of 5-Aza-CdR and probiotic agents on MUC2 expression in Caco-2 cells. Caco-2 cells were seeded at a density of 105 cells/well in a 24-well plate to become confluent, 5 × 10-6 mol/l 5-Aza-CdR (freshly prepared) was stimulated with the cells for 72 h, and the total mRNA was then collected. RT-qPCR was carried out and the expression of MUC2 mRNA was normalized with β-actin (n = 3, means ± SD). **P < 0.01 compared with control group.
- Effects of 5-Aza-CdR on E44 Adhesion to and Invasion of Caco-2 Cells
Next, we tried to test if probiotic effects could be enhanced through 5-Aza-CdR-mediated demethylation of the MUC2 gene in Caco-2 cells. The ability of 5-Aza-CdR and probiotic agents to interfere with the pathogen E44 adhesion and invasion in Caco-2 cells was examined by competitive exclusion assays after treating with 5-Aza-CdR for 72 h. As shown in Fig. 5 , 5-Aza-CdR was able to strongly inhibit E44 penetration of Caco2 cells. The relative adhesion rate of E44 was 10.33% ( Fig. 5 A) and the relative invasion rate of E44 was 29.45% ( Fig. 5 B). The result of the probiotics group was similar to 5-Aza-CdR treatment (** P < 0.01). The adhesion and invasion rates of E44 at the zero concentration of probiotics and 5-Aza-CdR were assigned as 100% and the effects of 5-Aza-CdR and probiotics preincubation were compared with this control level.
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Blockage of E44 penetration across Caco-2 cells by 5-Aza-CdR and probiotic agents. Caco-2 cells seeded in 24-well plates were incubated with 1 × 108 CFU probiotic agents and 1 × 107 CFU bacteria (E44) for 1.5 h, after treating with 5-Aza-CdR (5 × 10-6 mol/l) for 72 h. Adhesion (A) and invasion (B) assays were performed as described in the Materials and Methods section. The results were presented as relative adhesion% or invasion% compared with that of the control group without probiotic agents or 5-Aza-CdR. **P < 0.01.
Discussio
The use of probiotic microorganisms for the prevention and treatment of disease has become increasingly popular over the past few decades because of increased scientific studies that demonstrate the favorable effects of probiotics on human health [1] . The most frequently used probiotic agents is LAB, such as Lactobacillus rhamnosus GG, which has been examined for its efficacy in prevention of neonatal sepsis in both animal models and humans caused by enteric bacterial pathogens [2 , 26 , 39 , 40] . Probiotic LGG supplement in neonates, including very low birth weight newborns, are confirmed to be microbiologically safe as well as clinically well tolerated. Moreover, Lactobacillus and Bifidobacterium are the most commonly used probiotics. In our studies, the probiotic we used was the commercially Live Combined Bifidobacterium and Lactobacillus Tablets, which could inhibit E. coli K1 strain E44 intrusion into the intestinal epithelial cells in vitro and be able to reduce the incidence of bacteremia and meningitis in vivo . It was indicated that the significant reduction of pathogens across the gut barrier results in the release or elimination of bacteria access into the bloodstream, which eventually blocks pathogens from crossing the BBB to cause meningitis.
The mucin-containing cells in the surface of the gastrointestinal tract provide a physical protection and an interface between the internal environment and the dynamic external challenge from food-derived antigens and microbes [26] . Pathogens and benign microbes are discriminated by the gut mucosal immune system, which induces protective immunity without disrupting the integrity of the gut mucosa. Some studies have shown that the intestinal mucin gene MUC2 was constitutively expressed by the confluent human intestinal epithelial (Caco-2) cells [16 , 34 , 38] . In this study, we investigated the role of MUC2 in probiotics-mediated blocking effects on meningitic E. coli -induced pathogenicity. We hypothesized that probiotic agents could suppress bacterial penetration across the gut barrier and bacteremia/meningitis through enhancing host MUC2 defence functions. Probiotic agents induce up-regulation of epithelial MUC2, which in turn inhibits bacterial invasion and bacteremia/meningitis. We found that Caco-2 cells treatment with probiotic agents could be induced to present higher expression of MUC2. We previously demonstrated that Lactobacillus rhamnosus GG could inhibit pathogen penetration in vitro and in vivo [26] . In this report, we further demonstrated that probiotic agents could block bacterial invasion, bacteremia, and meningitis through up-regulation of MUC2 production.
Numerous factors, such as the imbalance of nutrition, antibiotics use, immunosuppressive therapy, and other means of treatment, might induce alteration both in the composition and effects of the human normal microbiota that may influence the efficacy of probiotics [25] . Previous studies demonstrated that S -adenosyl-L-methionine is a key nutritional factor for mucosal foraging and fitness of probiotic microorganisms and the prominent human gut symbiont, Bacteroides thetaiotaomicron [5 , 7 , 43] . Most interestingly, a synergistic anti-breast cancer effect can be achieved by a combined treatment with the methyl donor SAMe and the DNA methylation inhibitor 5-Aza-CdR [14] . 5-Aza-CdR could increase production of SAMe and sialyl Lewis X on MUC1 by activation of the gene coding for β-galactoside, α2,3-sialyltransferase 6, via inhibition of DNA methylation [8 , 35] . Based on these findings, we hypothesized that 5-Aza-CdR could enhance the biological activity of probiotics through up-regulation of MUC2, which may be activated by SAMe. To test this hypothesis, we tried to investigate if probiotic effects could be enhanced by 5-Aza-CdR-mediated demethylation of the MUC2 gene in Caco-2 cells. By analyzing and comparing the changes in MUC2 expression before and after treatment with 5-Aza-CdR, we speculated that MUC2 mRNA expression is associated with the methylation of the 5’CpG island of the MUC2 promoter. The changes in methylation of the MUC2 gene may be highly correlated with the inhibition of MUC2 transcription. Research has shown that 5-Aza-CdR could reverse CpG island methylation and restore the expression of multiple tumor suppressor genes [4 , 50] , which is consistent with our present study. This provides us with a new approach to study the relationship among probiotics, pathogens, MUC2, fitness factors ( e.g. , SAMe), and the specific mechanism of probiotics and its role in pathogensis. The underlying mechanisms responsible for up-regulation of MUC2 and SAMe by 5-Aza-CdR remain to be defined.
In conclusion, probiotic agents represent a promising host-directed antimicrobial agent that can be developed as a novel therapeutic intervention through enhancing host defence against infections, neonatal sepsis, and bacterial meningitis. Additionally, our data suggest that probiotic agents can efficiently block meningitic E. coli -induced pathogenicity in a manner dependent on MUC2.
Acknowledgements
This study was partially supported by the National Natural Science Foundation of China (No. 81370740 to S.H. Huang), Public Health Service grants R21NS083967 (S.H. Huang), the Grant from School of Public Health and Tropical Medicine of Southern Medical University, China (No. GW201248), and Guangdong Province Undergraduate Innovative Experiment Program (No. 1212112016 & No.1212112017).
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