Modulatory effect of Vibrio cholerae toxin co-regulated pilus on mucins, toll-like receptors and NOD genes expression in co-culture model of Caco2 and Peripheral Blood Mononuclear Cells (PBMC)

Background: Vibrio cholerae, the causative agent of cholera, as a Gram-negative pathogen tend to colonize the small intestine. The intestinal mucus layer forms mucin physical barrier, consisted from high molecular weight proteins. Regarding the role of toxin–coregulated pilus (TCP) as one of the most important colonization factors of V. cholerae, this experimental study was designed to determine the role of TcpA in induction of mucin production and its regulatory effect on innate immunity molecules including TLRs and NODs using Caco2- PBMC cocultures as an interactive model. Materials and methods: The rTcp protein was expressed in pET-28a-tcpA construct and puried using Ni-column chromatography. The identity of rTcp was conrmed by western immunoblotting analysis using anti-poly-histidine antibody. Nontoxic doses of rTcpA was determined on Caco-2 cell lines. The effects of different concentrations of rTcpA (1, 5, 10 and 50 µg/mL) on the expression of mucin 1,3, 4, toll-like receptors (TLR1, 4), and Nucleotide-binding oligomerization domain-containing proteins (NOD1, 2) genes were evaluated in a co-culture model of human colon carcinoma cell line (Caco-2) and Peripheral Blood Mononuclear cells (PBMCs). Results:The rTcpA protein of V. choleraewas expressed in BL21 E. coli and conrmed by western blotting. The rTcpA showed a statistically signicant effect on the expression of muc genes (MUC3 and MUC4) in a dose-dependent manner.This nding is supposed to facilitate physical adhesion and colonization of V. cholerae in intestinal lumen. The rTcpA moderately stimulated the expression of tlr4 and overexpressed tlr1, both of which are supposed to induce a mucosal protective response against bacterial infection and would help a promising protection in prophylaxis applications. No change in NOD2 expression might be attributed to the non-invasive nature of V. cholerae as an intestinal pathogen. Conclusion: In conclusion, the rTcpA protein of V.


Background
Cholera is an infectious disease which is caused by Vibrio cholerae, a Gram-negative rod in the family Vibrionaceae. V. cholerae strains responsible for cholera belong to O1 and O139 serogroups and tend to live in warm waters which can be transmitted to human due to poor sanitation [1]. This enteric pathogen has to move toward enterocytes and colonizes the small intestine. Consequently, the bacterium must overcome the intestinal physical barrier, including the mucus layer which covers the gastrointestinal tract [2]. The mucus layer mainly includes high molecular weight glycoproteins named mucins [3]. Oligosaccharide side chains of mucins entrap bacteria and immobilize them which preclude bacterial access to the epithelium and subsequent enterocyte damage [4]. Conversely, mucins can mediate an interaction between bacteria and epithelium which leads to intestinal colonization [5]. Mucins are classi ed into two subgroups: i) secreted gel-forming and ii) membrane-bound [6]. The enterocytes represent membrane-bound mucins which make a glycocalyx on their apical surface and consist of the mucins 1,3,4,12,13 and 17 [7]. The MUC 3and MUC 4 mucins are considered as two major components of glycocalyx [3]. The MUC 1 which is broadly distributed among different organs and represents a high level of expression by mucosal tissues [7,8].
Even though cholera is known as a non-in ammatory disease but low-grade in ammation can be seen in the early stage of disease [9]. V. cholerae toxin induces a moderate in ammatory response in intestinal milieu mediated by Pattern Recognition Receptors (PRRs). The PRRs like toll-like receptors (TLRs) and nucleotide-binding oligomerization domain-like receptors(NLR) play a crucial role in microbial identi cation [10]. Epithelial cells also play an important role in defense against pathogenic bacteria by expression of TLR1,2,4,5 andTLR6 [11]. NOD-like receptors are another class of PRRs that are expressed by human cells. The important members of the NLRs family are NOD1 andNOD2 which have been known in macrophages, dendritic, Paneth, and intestinal epithelial cells.NLRs are contributed in the activation of caspase 1and mediates pro-in ammatory cytokines IL-1b and IL-18 secretion [12]. Likewise, innate immune responses are induced with TLRs [13].
V. cholerae TCP which mediates the attachment of V.cholerae to enterocytes and M cells, belongs to type IV pili [14]. TcpA is the main component of toxin co-regulated pilus and has approved a crucial role in V.
cholerae colonization [15]. Regarding the pivotal role of TcpA in bacterial colonization, recognition of its potential role in mucin gene expression is compulsory.
This study aimed to determine the effect of rTcpA on the expression of mucin 1,3, 4, TLRs1, 4 and NOD 1,

Results
Cloning, expression and puri cation of rTcpA The tcpA gene was ampli ed by PCR and the 650 bp product was sequenced and con rmed by NCBI (http://www.ncbi.nlm.nih.gov/). The tcpA-pET28a construct was transformed into E. coli Bl-21, rTcpA was expressed and puri ed. The puri ed protein was viewed on SDS-PAGE with a size about 22 kDa and its identity con rmed by western blotting (Figure1).

Cytotoxicity effect of rTcpA on Caco-2 cells
The cytotoxic effect of rTcpA was evaluated on Caco-2 cells by MTT assay. The rTcpAin concentration of 0.1 µg/mL inhibited 3% of Caco2 cells in 24, 48 and 72 h. Whereas, the inhibitory effect was increased to 5%, 8% and 14%, when the Caco-2 cells were exposedto1µg/mL of rTcpA for 24, 48 and 72h, respectively.
The cytotoxicity effect was determined as 8%, 18%, and 30%, for 24, 48 and 72 h exposure time, respectively,when increasing the rTcpA concentration up to 10 µg/mL. The effect of rTcpA on mucin genes expression in Caco-2/PBMCs co-culture by real-time PCR The expression of mucin1,3 and 4 genes was assessed in Caco-2/PBMCs co-culture by real-time PCR after treatment with different concentrations of rTcpA. The results demonstrated that the expression of mucin 1 was signi cantly (P<0.05) increased to 2.3, 5.5, 7.5 and 8.6 folds in the presence of rTcpA1, 5, 10 and 50µg/mL, respectively compared to the untreated Caco-2/PBMC co-culture as control. Our data revealed a highly signi cant increase (P<0.05) in the expression level of muc4 gene (3.8, 9.5, 14.9 and 185 folds) in the co-culture group which was exposed to1,5,10and 50µg/mL of rTcpA, respectively. With the same concentrations of rTcpA after 24 h exposure, 1.3, 7.6, 12.2 and 76.8 fold changes (P<0.05) was observed in muc3 gene expression compared to untreated Caco-2/PBMC co-culture control group. The expression of all muc genes was statistically increased after treatment with different concentrations of rTcpA ( Table 1).
The TLR1 and TLR4 genes expression of Caco-2/PBMCs co-culture in response to rTcpA The expression level of tlr genes (tlr1 and 4) were evaluated in the co-culture model of Caco-2/PBMCs by real-time PCR when treated with different concentrations of rTcpA, considering untreated cells as control.
The effect of rTcpA on NOD genes expression in Caco-2/PBMCs co-culture by real-time PCR In this study, the expression of nod1 was changed 0.177, 0.303, 0.33 and 0.37 folds in the Caco-2/PBMC co-culture medium after exposure to different concentrations of rTcpA (1, 5, 10 and 50 µg/mL, respectively) which did not show a statistical signi cant difference (P>0.05). The expression of nod2 gene was increased to 0.555, 2.35, 4.36 and 19.904 folds when treated by rTpA 1, 5, 10 and 50µg/mL, respectively. The differences in nod2 gene expression after exposure with different concentrations of rTcpA were statistically signi cant (p<0.05) ( Table 1).

Discussion
The effect of TCP as an important virulence factor of V.cholerae on the expression of muc, tlr and nod genes was considered in this study.The MUC4 mucin, as the membrane-bound subgroup of mucins, play a critical role in enterocyte protection and indirect immune responses against infections [16,17]. Our results showed that the expression of this protein was strongly stimulated by rTcpA in a co-culture model. It seems that TcpA protein probably facilitates physical adhesion of V. cholerae by augmentation of MUC4 mucin production by inducing intestinal enterocytes, which is supposed to facilitate bacterial colonization. Vieira et al.(2010) demonstrated that atypical Enteropathogenic E. coli strains (aEPEC) caused up-regulation of muc4 expressionand increased its secretion followed by increased attachment and colonization of bacteria at the apical surface of HT29-MTX cells [18].
The expression of muc3 gene was induced by rTcpA in the present study. It seems that this increase is also important in the adhesion stage of V. cholerae to Caco-2 cells. Bhowmick (2008) established that the chitin binding protein (GbpA) enhances both muc3 expression and mucin secretion that leads to the initial colonization of V. cholerae [5]. Therefore, it can be concluded that these two binding factors, synergistically, empower bacteria for adherence to intestinal cells.
The critical role of MUC1 as a receptor for Salmonella adhesion (SiiE) has been con rmed in apical invasion of Salmonella into enterocytes [19] . On the other hand, it was demonstrated that overexpression of mucin1 gene occurs in response to invasive intestinal bacterial infections, including Campylobacter jejuni which plays a serious role in limiting their pathogenesis [20]. The expression level of muc1 was up-regulated by rTcpA, but it was affected less than MUC3 and MUC4. Considering the toxin producing and extracellular lifestyle of V. cholerae infection, the limited expression of MUC1in response to tcpA can be attributed to non-invasive nature of bacterium.
It has been suggested that MUC protein interacts with bacterial adhesins through mucin oligosaccharide side chains; however, it entraps bacteria and prevents enterocyte from damage when overexpressed [4,7,21]. Therefore, it seems that the cooperative or restrictive role of mucin in bacterial pathogenesis is dosedependent and relies on different stages of bacterial infection. As an enteric pathogen, V. cholerae possesses several strategies to overcome the physical mucus barrier of intestinal tract and induce the immune system. It seems that V. cholerae has a dual function in its interaction with mucins, i) the bacterium penetrates to intestinal mucus layer by agellum, cleaves mucin proteins and facilitates bacterial movement probably via HA/P (hemagglutinin/protease) and contribute to bacterium dissemination, ii) promotes microcolony formation by induction of increased MUC2 and MUC3 mucins secretion . 2,5 However, the production of mucinase by bacteria which helps to overcome the restrictive role of mucin should not be ignored.
TLR4 induces in ammatory cascade, through NF-κBpathway, in response to LPS of Gram-negative bacteria and plays essential role in host defense through production of TNF-α and iNOS by immune cells [22,23]. In the current study, rTcpA moderately stimulated expression of tlr4 gene in Caco-2 model in a dose-dependent manner which is supposed to play amucosal protective role against bacterial infections in baseline concentration and may trigger an abandoned in ammatory response when overproduced by lamina propria mononuclear and intestinal epithelial cells [24]. This proposes the TcpA protein as a promising candidate for prophylaxis applications.
In this study, the expression of tlr1 was up-regulated by rTcpA. TLR1 recognizes peptidoglycanand triacyllipoproteinsin concert with TLR2(as a heterodimer) in Gram-negative and Gram-positive bacteria and promotes the activation of NF-B, and cytokines production by DCs and in ammatory IFN-gamma T-cells (Th1). IFN-gamma enhances antigen processing and presentation and displays immunomodulatory effects, boosting the anti-microbial responses [25,26] . It seems that TcpA induces tlr1 overexpression and probably improves Caco-2 cell protection.
No signi cant increase was observed in NOD2 expression in our study which may be one reason for moderate grade in ammation of cholera. NOD1 and NOD2 are specialized NOD-like receptors that contribute to the recognition of some pathogenic microorganisms that can invade and multiply intracellularly. Considering the extracellular nature of V. cholerae, no change in NOD2 can be attributed to bacterial lifestyle [27] . Ampli cation of tcpA using PCR technique Genomic DNA was extracted from V. cholerae (ATCC 14035) and used for tcpA gene ampli cation by forward and reverse primers speci cally chosen according to whole-genome sequence of V. cholerae N16961 (Genebank accession number CP028827) Forward: 5'-GCTGGATCCATGACATTACTCGAAGTGATCATC-3' and Reverse: 5'-GCTCTCGAGGCTGTTACCAAATGCAACGCCGAA-3'. PCR conditions were as follows: initial denaturation at 95˚C for 5 min, secondary denaturation at 95˚C for 30sec, annealing at 53˚C for 30sec, extension at 72˚C for 1min and a nal extension at 72C º for 2 min. The ampli cation was performed for 35 cycles.

Conclusions
Puri cation of PCR products and cloning PCR product from tcpA ampli cation was puri ed using the Spin Combo kit (Biotech, Chinese) from agarose gel and digested with BamHI and XhoI (Thermo Fisher Scienti c, USA). The expression vector, pET-28a, was phosphorylated by rSAP (Thermo Fisher Scienti c, USA) and ligated with the puri ed tcpA gene using T4 DNA ligase (Thermo Fisher Scienti c, USA). The tcpA-pET-28a construct was transformed into E.coli Bl-21 and colony PCR and plasmid extraction was performed to con rm the ligation. The ligated vector was sequenced (BIONEER company, South Korea) to con rm the identity of cloned gene.
Expression and puri cation ofrTcpA E.coli Bl-21 was transformed with pET-28a for the production of rTcpA. Bacteria were grown in the LB broth (Thermo Fisher Scienti c, USA) containing 50µg/mL kanamycin for overnight at 37 º C and then diluted into new LB broth and incubated again at 37 ºC with shaking at 230 rpm until the achievement to OD=0.6 at λ=600 nm.Bacterial culture was treated with 0.01 mM IPTG for induction of tcpA expression and further grown at 30 ºC with shaking at 230 rpm (the concentration of IPTG, incubation temperature and shaking speed were set up in separate assays).
Subsequently, bacterial cells were lysed by sonication and supernatant was collected for analysis by SDS-PAGE [28]. The rTcpA protein waspuri edusing Ni-column chromatography according to worksheet protocol (CMSephadex C-25GE; Healthcare Life Science). The concentration of puri ed protein was determined using BCA protein assay kit (Thermo Fisher Scienti c, USA).

Con rmation of rTcpA identity by western immunoblotting
The identity of the puri ed rTcpA was analyzed by western blotting (WB) assay. Brie y, the SDS-PAGE gel was electroblotted onto polyvinylidene di uoride (PVDF) membrane and the blotted PVDF was blocked with 1% skim milk for overnight at 4 ºC. After washing, membranes were soaked in 1:1000 diluted antipolyhistidineantibody (Abcam, Cambridge, USA) at 4 ºC for 24 h. After incubation with HRP conjugated rabbit anti-IgG (Abcam, USA) at room temperature (RT), WB-enhanced chemiluminescence (ECL) substrate was added to the membrane for chemiluminescence detection. The membrane was then lifted and the photoluminescence recorded by ECL camera.
Evaluation of rTcpA cytotoxicity on Caco-2 cell line Human colon carcinoma cell line (Caco-2 cells) were purchased from Pasteur Institute of Iran and cultured in Dulbecco's Modi ed Eagle Media (DMEM) F12(Gibco, Thermo scienti c, USA) containingLglutamine and high glucose, supplemented by 10% fetal bovine serum (Gibco, Thermo scienti c, USA) and 1% penicillin-streptomycin. The cell culture asks were incubated at 37 ºC with 5% CO 2 in a moist atmosphere [29] . The Caco-2 cells start to polarize when con uence and macromolecules are sorted and maintained between apical and basolateral surfaces of con uent cells. Moreover, markers of colonocytes are also present in Caco-2 cells [30].
The Caco-2 cells were trypsinated when reached 90% con uency and seeded in 96-well plates (SPL life science, South Korea) at a density of 2×10 4 cells per well and incubated at 37 ºC with 5% CO 2 . The con uent monolayer was treated with different concentrations of rTcpA (0.1, 1 and 10 µg/mL) for 24, 48 and 72 h. Subsequently, 50 µL of the medium was removed and replaced with MTT (Methyl Thiazolyl diphenyl-tetrazolium bromide) reagent. After 4 h incubation, 100 µLDMSO was added, mixed well and the absorbance was read at 570 nm and the cell viability was measured by the following formula: Cell viability (%)= OD of sample/OD of control × 100 [31].
Determination of TLRs, NODs, and mucin related genes expression in Caco-2/PBMCs co-culture treated with rTcpA by real-time PCR

RNA extraction and cDNA synthesis
The Caco-2/PBMCs co-culture was treated by different concentrations of naked rTcpA (1, 5, 10 and 50 µg/mL). The Caco-2/PBMCs co-cultures without treatment were used as control. The treatment was performed on co-culture model for 24h at 37 ºC in CO 2 atmosphere and Caco-2 cells were collected and lysed by trizol reagent (Sigma Aldrich, Germany) for RNA extraction. Accordingly, chloroform was added to the cell lysate and centrifuged(15min,1200g and 4 ºC), afterward the aqueous phase was mixed with 2propanol and centrifuged. The isolated total RNA was washed twice with ethanol 70% and after drying was solubilized in diethyl pyrocarbonate (DEPC) treated water. The purity of RNA was determined by absorbance at OD260/280 and only samples with a ratio of 1.8±2.0 were subjected for cDNA synthesis by cDNA Synthesis Kit (YektaTajhizAzma, IRAN). According to manufacturer protocol, 100 ng template RNA, 50 µM Random hexamer, 50 µM Oligo dT and DEPC treated water were mixed and incubated for 5 min at 70ºC, after which M-MLV (Moloney Murine Leukemia Virusreverse transcriptase) (10,000 U), dNTP 10mM, RNasin (40u/µl), 5X rst-strand buffer were added and the mixture was incubated for 60 min at 37 ºC. The reaction was terminated by heating at 37 ºC for 5 min.

Real-time PCR analysis
Real-time PCR was performed to determine the probable role of rTcpA in the modulation of the TLRs, NODs, and mucin related genes expression (muc1,3and4) using the Applied Biosystems 7500 (Thermo Fisher Scienti c, USA). The qPCR was performed according to the following conditions: Pre-incubation at 95˚C for 5 min, 45 cycles consisting of denaturation at 95˚C for 30 sec, annealing at 60˚C for 30 sec, extension at 72˚C for 30 sec. The melting pick analysis was performed in 95 ˚C for 5 min. Each PCR reaction contained 10μL 5x Real-time PCR Master Mix (Takara, Japan), 2 μL cDNA template, 0.8μL of each primer and 6.4μL distilled water in a total reaction volume of 20 μL. The nucleotide sequence of the forward and reverse primers used for ampli cation of muc (muc1, 3and 4), tlr (tlr1, 4) and nod genes (nod1, nod2) are depicted in Table 2. In each reaction, the same amounts of RNA were converted into cDNA and pipetting error was removed. The expression of GAPDH gene was considered as the internal control for each sample and the ΔCT(CT target−CT reference) of each sample and expression fold change were calculated. Each real-time PCR reaction was carried out in triplicate and results were presented as Mean±SD.

Statistical Analysis
The Kruskal-Wallis test was used to perform statistical analysis (using SPSS software,ver. 16) to compare the gene expression between treated Caco-2/PBMC co-culture cells groups with untreated cells and a p-value <0.05 was considered as signi cant. The correlation coe cient between therTcpA concentration and the different genes expression level was determined by Spearman test. Tables Table 1 The expression of muc, tlr and nod genes when treated by different concentration of rTcpA (µg/mL) ' T  T  G  T  A  A  T  T  T  G  T  T  G  T  -3 '