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1 School of Medical Sciences, The University of New South Wales, Sydney, NSW 2052, Australia
2 Mucosal Diseases Program, Mater Medical Research Institute, Mater Misericordiae Hospitals, South Brisbane, QLD 4101, Australia
3 School of Medicine, Sydney, The University of Notre Dame Australia, Darlinghurst, NSW 2010, Australia
Correspondence
George L. Mendz
GMendz{at}nd.edu.au
Received 6 November 2007
Accepted 3 February 2008
Abbreviations: PAS, periodic acid–Schiff reagent.
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTS AND DISCUSSIONREFERENCES |
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The mucosal tissues of the gastrointestinal, respiratory, urinary and reproductive tracts, as well as the surfaces of several organs such as the eye, nose and mouth, are exposed to the external environment. The mucus layer present in these tissues provides protection to underlying mucosal epithelial cells against chemical, enzymic, microbial and mechanical insult (Neutra & Forstner, 1987; Strugala et al., 2003). The major components of the mucus layer are secreted gel-forming mucins, complex glycoproteins that give mucus its viscous consistency.
Most pathogenic bacteria that colonize the gastrointestinal tract subvert the mucus barrier by effective motility through the gel and the use of enzymes capable of degrading mucin carbohydrates and/or mucins. MUC2 is the most common gel-forming mucin secreted by goblet cells of the small intestine and colon. The interactions of bacteria with this mucin elicit responses in them that are not well characterized at the molecular level and are not restricted to the modulation of the expression of enzymes involved in mucus modification. Mucins are major chemoattractants for C. jejuni (Hugdahl et al., 1988) and the bacterium binds to them (McAuley et al., 2007; Szymanski et al., 1995), indicating that mucins carry the physiologically relevant oligosaccharide ligands for the adhesins of this bacterium. A very small number of molecular studies have been conducted to investigate the interactions between C. jejuni and human MUC2, but they used poorly characterized bovine mucin preparations (Hugdahl et al., 1988). Knowledge of the responses of C. jejuni to the mucus layer will be another step in understanding the mechanisms of C. jejuni infection. The aim of this work was to determine the effects of MUC2 on C. jejuni growth and to characterize the changes induced by this mucin in the expression of 20 bacterial genes related to virulence, putative mucin-degrading enzymes, cell morphology, adhesion and motility.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Purification and characterization of human mucin MUC2. Mucus was purified from urine samples obtained from patients with artificial bladders made of intestinal tissue that secretes mucins abundantly (Gerharz et al., 2003; Nabi et al., 2005). Samples were obtained from patients in the first few days following surgery to avoid any change in mucin glycosylation that may occur in response to epithelial changes in the urinary tract microenvironment. The extraction of MUC2 was performed based on a published protocol (Davies & Carlstedt, 2000) with some modifications. Briefly, mucus collected from urine was subjected to repetitive extractions in 6 M guanidinium chloride, 5 mM EDTA and 10 mM sodium phosphate (pH 7.0). The insoluble fraction containing oligomerized MUC2 depleted of contaminating non-covalently attached proteins was reduced in 10 mM DTT, 0.1 M Tris/HCl (pH 8.0) for 24 h and alkylated in 25 mM iodoacetamide at 4 °C for 24 h. Reduction of disulfide bonds disassembles the mucin homo-oligomers, thereby greatly reducing viscosity and solubilizing the mucins. Reduced, purified MUC2 was dialysed against water before addition to bacterial cultures. Mucin purity was verified by 1 % agarose gel electrophoresis for 8 h, transfer onto nitrocellulose membranes and staining with periodic acid–Schiff reagent (PAS) and Western blotting with MUC2 rabbit antiserum primary antibody (LUM 2-3; a kind gift of Professor I. Carlstedt, Lund University, Sweden). PAS identifies mucin oligosaccharides, and a single PAS-positive band was obtained, which co-localized precisely with the results of MUC2 Western blotting, verifying that MUC2 was the mucin present in these preparations. Purified mucin was also subjected to standard SDS-PAGE and staining with Coomassie blue, which revealed only negligible contamination with non-mucin proteins that co-purified with MUC2.
Growth inhibition studies. The growth response of C. jejuni as a function of MUC2 concentration was determined. Frozen cultures were thawed to room temperature and 20 µl bacterial suspension was spread on CSA plates and incubated at 37 °C under microaerobic conditions. After 24 h incubation, bacteria were harvested in 2 ml brain heart infusion (BHI) medium, transferred to 10 ml BHI medium in 25 ml vented flasks with 0.2 µm filter caps and grown for 12 h under the same conditions. Bacteria were harvested by centrifuging the cultures for 5 min at 5000 g at room temperature. The pellets were resuspended in 50 ml fresh sterile BHI medium and the suspension was adjusted to an OD600 of 0.5. Three millilitres of bacterial suspension was dispensed into each well of Cellstar six-well plates (Greiner Bio One) and cultures were supplemented with MUC2 at concentrations of between 0.01 and 2 mg ml–1. The growth rates of C. jejuni were determined spectrophotometrically by measuring the OD600 of cell suspensions and by colony counts (c.f.u. ml–1) at 0 and 12 h incubation. Thirty microlitres of serial dilutions of 10–2 to 10–9 were plated on CSA plates and incubated microaerobically, and the number of colonies was counted in triplicate for each dilution after incubation for 1 day under microaerobic conditions. Growth rates were measured for each MUC2 concentration by the difference in growth at both time points in three independent cultures (n=3).
To compare the growth characteristics of C. jejuni in the presence or absence of MUC2, growth curves of the bacterium were measured at a mucin concentration that caused a measurable decrease in bacterial growth but was not lethal to the cells. Bacterial suspensions were prepared and dispensed in six-well plates, and cultures were supplemented with 0.05 mg MUC2 ml–1, or left without mucin, and incubated microaerobically. At 0, 3, 6, 9, 12, 15, 18 and 24 h, the densities of viable bacteria were determined in duplicate by counting c.f.u. ml–1 of serial dilutions of the cultures. The experiment was repeated twice with independent cultures (n=2).
Sampling and total RNA extraction from C. jejuni liquid cultures.
C. jejuni was grown microaerobically in liquid cultures at 37 °C in shaking vented flasks containing 10 ml BHI medium for 12 h. The cell suspensions were centrifuged at room temperature for 5 min at 5000 g and the pellets were resuspended in 10 ml BHI medium to an initial OD600 of 0.3. MUC2 was added to the cultures at a concentration of 0.05 mg ml–1. Cultures with or without MUC2 were incubated microaerobically at 37 °C and harvested at 12 h. For total RNA isolation, 10 ml bacterial suspension was mixed with 2 ml RNAprotect Bacterial Reagent (Qiagen). Bacterial cells were pelleted by low-speed centrifugation for 10 min at 5000 g at room temperature. The pellets were resuspended in 200 µl DEPC-treated lysozyme in TE buffer consisting of 30 mg chicken lysozyme ml–1 (
50 000 U mg–1; Sigma-Aldrich), 10 mM Tris/HCl (pH 8.0), 1 mM EDTA and 10 µl proteinase K (600 mAU ml–1; Qiagen). Total RNA was extracted using Qiagen RNeasy mini columns and DNase treatment to remove traces of genomic DNA, following the manufacturer's instructions. Total RNA samples were resuspended in 20 µl RNase-free DEPC-treated water; 6 µl total RNA was set aside for the immediate assessment of RNA quantity and quality, and the rest was stored at –80 °C for further experiments.
RNA purity and the presence of genomic DNA were assessed following the protocol published in Qiagen's RNeasy Handbook. Using 2 µl aliquots of the samples, the RNA quality and quantity were also measured with an ND-1000 NanoDrop UV-Vis spectrophotometer (NanoDrop Technologies). Values of A260/A280 >2.0 and A260/A230 >2.0 are indicative of no protein and solvent contamination, respectively.
Reverse transcription: cDNA synthesis. Total RNA was reverse-transcribed using a SuperScript III First-Strand Synthesis System for quantitative RT-PCR according to the manufacturer's instructions (Invitrogen). The purity and quantity of cDNA were assessed using an ND-1000 NanoDrop UV-Vis spectrophotometer as above.
Quantitative real-time RT-PCR.
Primers were designed based on the published C. jejuni NCTC 11168 genome sequence (GenBank accession no. AL111168; Parkhill et al., 2000) using Primer3 (Rozen & Skaletsky, 1998). The sequences of the primers used in this study are listed in Table 1
. They all had the following properties: melting temperatures of between 61 and 63 °C, length 19–23 nt, GC content 50–80 mol% and expected product size of 300–310 bp. BLAST searches (Altschul et al., 1997) were performed against other bacterial genomes to determine the specificity of the primers.
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The relative expression ratio of genes was calculated based on the Ct comparative threshold cycle method (Livak & Schmittgen, 2001). Primer efficiencies in this study were assumed to be 2, as they were between 85 and 90 % (Livak & Schmittgen, 2001). Statistical analyses were performed on the ratio R using the pair-wise randomization test of the REST software (Pfaffl et al., 2002). Genes were considered to be significantly regulated when the gene expression normalized to the 16S rRNA reference gene differed significantly from the control (P <0.05).
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RESULTS AND DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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shows the changes in bacterial growth given as the number of c.f.u. ml–1 for five different concentrations of MUC2; growth inhibition was measured at mucin concentrations from 0.05 to 2.00 mg ml–1 without becoming lethal to the bacterium (Table 2). There were no significant changes in bacterial morphology as determined by light microscopy. Under the experimental conditions used, this decrease in C. jejuni growth may have resulted from the adhesion of mucin oligosaccharide ligands to the bacterium (Hugdahl et al., 1988), creating a physical barrier that limited its capability to obtain nutrients. Another explanation could be that MUC2 itself has antimicrobial properties, as several mucin oligosaccharides have these properties. For example, the O-linked oligosaccharides of the human gastric mucin MUC6 contain an 
1,4-linked N-acetylglucosamine able to inhibit the biosynthesis of cell-wall components of Helicobacter pylori (Kawakubo et al., 2004). Also, the 20-mer of MUC7 mucin has direct antifungal properties against Candida albicans via a histatin domain at its N-terminus (Bobek & Situ, 2003). It is unlikely that increased viscosity was responsible for the altered growth, as reduced individual MUC2 subunits were used in these experiments. The rheological properties of mucus are attributable mainly to the polymerization of secreted mucins. In vivo, MUC2 is homo-oligomerized into complexes polymerized via disulfide bonds. The mucin aggregates give mucus its viscous properties and help retain many molecules, including antimicrobial ones, secreted by the mucosa. This study used low concentrations of MUC2 with reduced disulfide bonds and complexes disassembled into individual subunits. Reduction solubilizes the mucin and only at high concentrations (>10 mg ml–1) do suspensions become more viscous. Thus the mucin concentrations used in these experiments did not change the viscosity to any significant extent. This study did not attempt to duplicate the environment of mucus but rather to investigate the response of the bacteria to MUC2, the major constituent of human intestinal mucus. A 0.05 mg ml–1 concentration of MUC2 produced measurable inhibition of C. jejuni growth and was chosen to compare the characteristics of bacterial growth in the presence and absence of this mucin, and to investigate the transcriptomic regulation induced by its presence.
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). These results are in good agreement with previous data (Sampathkumar et al., 2006). There were no differences in the duration of the exponential growth phase between bacteria grown with or without MUC2 at this concentration.
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Degradation of mucin carbohydrates exposes the protein core to proteases, resulting in disruption of the macromolecular complexes and, consequently, the function of mucins in the mucus. For example, Vibrio cholerae HapA, which has both mucinolytic and cytotoxic activities, is induced by mucin and is required by the bacterium to traverse through mucin-containing gels (Silva et al., 2003). Several putative mucinolytic enzymes have been identified in the genome of C. jejuni strain NCTC 11168: the sialoglycoprotease Cj1344c, the murein lytic glycosylase Cj0843c and the sulfoglycolases Cj0256 and Cj1055c. Consistent with their functions, these four genes were upregulated by MUC2.
A determinant of host colonization by C. jejuni is the motility of the bacterium. A major component of its flagellar apparatus is the flagellin encoded by flaA and a minor component is the flagellin encoded by flaB (Sommerlad & Hendrixson, 2007). The transcription of both flagellins is under the regulation of different sigma factors, 
28 for flaA and 54 for flaB (Hendrixson et al., 2001). Fully motile, wild-type C. jejuni bacteria express flaA, but expression of flaB is not required for motility (Wassenaar et al., 1993). MUC2 upregulated the expression of flaA but did not change that of flaB, suggesting that mucin serves as an environmental signal to enhance motility.
Adhesion to host cells is necessary to allow C. jejuni to remain in the intestine and invade the epithelium. Adherence involves different types of lectin-like molecules attaching to their corresponding glycosylated receptors on the epithelial cells (Guerry et al., 2002). Major outer-membrane proteins such as the putative rod-shape-determining protein MreB, the putative periplasmic protein MreC and C. jejuni lipoprotein JlpA play important roles in bacterial adhesion (Ashgar et al., 2007; del Rocio Leon-Kempis et al., 2006; Jin et al., 2003; Kakuda & DiRita, 2006; Karlyshev et al., 2000; Konkel et al., 1997; Linton et al., 2000; Mamelli et al., 2006; McSweegan & Walker, 1986; Monteville & Konkel, 2002; Moser et al., 1997; Pei & Blaser, 1993). The genes encoding these three proteins were upregulated in response to MUC2.
Different factors and determinants may be expressed at various stages of infection to help the adaptation of a bacterium to different conditions. Some virulence factors help bacterial pathogens to attach themselves to cell surfaces, to invade particular host cells and potentially to modify cells or tissues to their advantage, and they can cause collateral damage at the same time (Chen et al., 2005). The gene ciaB encoding the Campylobacter invasion antigen CiaB was upregulated by MUC2; CiaB plays an important role in internalization of C. jejuni into mammalian cells (Konkel et al., 1999). The genes neuB1 and cadF were downregulated by MUC2. NeuB1 is the main component of N-acetylneuraminic acid synthetase involved in sialylation of lipo-oligosaccharides; C. jejuni lipo-oligosaccharides mimic human gangliosides and may serve to disguise the bacterium following bacterial infection (Linton et al., 2000). The gene cadF encodes a protein that helps C. jejuni to bind to host fibronectin (Krause-Gruszczynska et al., 2007). NeuB1 and CadF are important for immune shielding and adhesion to the extracellular matrix, respectively. Thus it can be hypothesized that these proteins are not required to penetrate the mucus barrier, but their expression may be upregulated when the bacterium enters the epithelial microenvironment.
Toxins are considered major C. jejuni virulence factors contributing to cellular or tissue damage and disease induction. Cytolethal distending toxin (Cdt) is a secreted holotoxin present in many non-enteric and enteric pathogens (Lara-Tejero & Galan, 2001; Lee et al., 2003). It comprises three subunits, CdtA, CdtB and CdtC, which act together to block cell division by performing cell-cycle arrest (Lara-Tejero & Galan, 2001): CdtA and CdtC bind to the cell surface helping to deliver the active subunit CdtB, which uses its DNase I-like activity to cleave dsDNA molecules during the G1 or G2 phase (Whitehouse et al., 1998). The gene cluster cdtABC and the gene vacB encoding a vacuolating toxin were significantly upregulated by MUC2.
The genes encoding the multidrug efflux system CmeAB also were upregulated by MUC2. This efflux system is involved in resistance of C. jejuni against bile and other antimicrobial agents (Lin et al., 2002, 2005). Thus MUC2 may act as a cue to the bacterium to enhance its defences against host antimicrobial factors.
For the first time, we have shown here that C. jejuni has evolved an ability to respond to the presence of the human MUC2 intestinal mucin by modulation of expression of genes known to be important for colonization and pathogenicity (Fig. 2
). Transcriptional activation of genes encoding cell-morphology proteins, motility proteins, adhesins and virulence factors upon entering intestinal mucus would facilitate C. jejuni colonization of mucus and attachment to and invasion of the underlying mucosal epithelial cells.
The response of C. jejuni to MUC2 may not be specific to MUC2 or even to human mucins. It is likely to be mediated through oligosaccharide structures that could also be present on intestinal cell-surface mucins, secreted mucins from other regions of the gastrointestinal tract and other mucosal tissues, or intestinal mucins from other mammalian and avian species (Karlsson et al., 1997; Robbe et al., 2004; Schulz et al., 2002). However, MUC2 is virtually the sole secreted human intestinal mucin, and this work aimed to understand changes in the transcriptome of C. jejuni when it infects the human intestine and interacts with a mucin found there. MUC6 is secreted in the stomach but not in the intestine, and its concentration in intestinal mucus is bound to be very small. Shed extracellular domains of intestinal cell-surface mucins such as MUC12, -13 and -17 can be found within mucus at low concentrations. Small amounts of these mucins could have been present in our preparations, but PAS staining of blots showed only a single mucin band that coincided with MUC2, indicating that any contamination with other mucins was at a very low level.
The observation that C. jejuni is responsive to MUC2 raises the question of the identity and nature of the bacterial MUC2 receptor, which could possibly be a lectin-binding oligosaccharide. Oligosaccharides vary throughout the gastrointestinal tract, among different mucosal tissues and among species. Moreover, because of inherited differences in the glycosyltransferases that build these structures, inter-individual differences in mucin glycosylation within individual mucosal tissues occur within human populations. In the case of gastric mucins and the related bacterium H. pylori, these host differences influence their susceptibility to various strains of the bacterium (Aspholm-Hurtig et al., 2004; Lindén et al., 2008). In future work, it will be of interest to investigate which intestinal mucin oligosaccharides trigger C. jejuni responses, and whether differences in the expression of these structures among individuals and species underlie differential susceptibilities to pathology following infection. If this were the case, the different glycosylation of MUC2 in various mammalian and avian species could modulate the expression of different C. jejuni genes in the intestinal microenvironments of those hosts and explain the differential pathogenicity of C. jejuni to them. Identification of the bacterial receptors could thus provide new molecular therapeutic targets against C. jejuni infection.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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and triggers signalling pathways leading to the activation of NF-
B and p38 MAP kinase in epithelial cells. Cell Microbiol 5, 165–174.[CrossRef][Medline]
CT method. Methods 25, 402–408.[CrossRef][Medline]
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, Bacterial growth in the absence of MUC2; 
, bacterial growth in the presence of 0.05 mg MUC2 ml–1. The results are shown as means±SE from duplicates of two independent cultures.