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Infection and Immunity, November 2008, p. 5341-5349, Vol. 76, No. 11
0019-9567/08/$08.00+0     doi:10.1128/IAI.00786-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Identification of a Bile-Induced Exopolysaccharide Required for Salmonella Biofilm Formation on Gallstone Surfaces

Robert W. Crawford,¹ Deanna L. Gibson,^2,3 William W. Kay,³ and John S. Gunn^1*

Center for Microbial Interface Biology and Department of Molecular Virology, Immunology, and Medical Genetics, The Ohio State University, Columbus, Ohio 43210,¹ Division of Gastroenterology, University of British Columbia and BC Children's Hospital, Vancouver, British Columbia, Canada,² Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, Canada³

Received 23 June 2008/ Returned for modification 28 July 2008/ Accepted 4 September 2008

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Salmonella enterica serovar Typhi can establish a chronic, asymptomatic infection of the human gallbladder, suggesting that this bacterium utilizes novel mechanisms to mediate enhanced colonization and persistence in a bile-rich environment. Gallstones are one of the most important risk factors for developing carriage, and we have previously demonstrated that salmonellae form biofilms on human gallstones in vitro. Thus, we hypothesize that bile-induced biofilms on gallstone surfaces promote gallbladder colonization and maintenance of the carrier state. A colanic acid/cellulose S. enterica serovar Typhimurium double mutant formed a mature biofilm on gallstones in a test tube assay and in a new, gallstone-independent assay using cholesterol-coated Eppendorf tubes. These data suggest the presence of an unidentified exopolysaccharide necessary for mature biofilm development and demonstrate specific binding affinity between salmonellae and cholesterol. Our experiments indicate that the Salmonella O-antigen capsule (yihU-yshA and yihV-yihW) is a crucial determinant in gallstone and cholesterol biofilms but that expression of this exopolysaccharide is not necessary for binding to glass or plastic. Real-time PCR revealed that growth in bile resulted in upregulation of the O-antigen capsule-encoding operon in an agfD-independent manner. Thus, the O-antigen capsule genes are bile induced, and the capsule produced by the enzymes of this operon is specifically required for biofilm formation on cholesterol gallstones. These studies provide new therapeutic targets for preventing asymptomatic serovar Typhi gallbladder carriage.

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Salmonellae remain pathogens of worldwide concern, particularly in nonindustrialized societies with inefficient food and water sanitation systems. The diverse Salmonella enterica serovars infect a broad range of hosts and are increasingly resistant to commonly administered antibiotics (4). Gastroenteritis and enteric (typhoid) fever are the two most prevalent clinical manifestations, both of which cause significant morbidity and mortality.

Salmonella enterica serovar Typhi is the etiologic agent of typhoid fever in humans, a systemic illness afflicting approximately 20 million people globally each year (9). Bacteria reaching the gallbladder can induce an active local infection (cholecystitis) or exist asymptomatically in a chronic carrier state. Nearly 5% of patients suffering from typhoid fever become Salmonella carriers (21). This condition is frequently associated with abnormalities of the gallbladder, including gallstones, and Salmonella carriage is the largest risk factor for cancer of this organ (12, 18, 19).

During colonization of the intestine and gallbladder, Salmonella spp. interact with bile, a complex, lipid-rich, and protein-poor digestive secretion produced by the liver at nearly 1 liter per day (4). Bile consists of many components, including bile acids, cholesterol, phospholipids, and bilirubin, and serves as a potent antimicrobial agent in the gastrointestinal tract (15). Salmonellae, however, resist its surface-acting, amphipathic, detergent-like properties (41), indicating that bile resistance is an important phenotype associated with Salmonella pathogenesis during both acute and chronic infections. The presence of bile is also known to cause pleiotropic responses that affect production of virulence factors (SPI-I-mediated type III secretion, motility, adhesion, antibiotic and bile resistance, efflux pump expression, and biofilm formation) in S. enterica (26, 28) and in enteric commensals and pathogens, including Vibrio cholerae (3, 7, 16, 17, 30, 36, 44), Campylobacter jejuni (22), Escherichia coli (40), and Listeria monocytogenes (37).

The main site of human serovar Typhi carriage is the bile-rich gallbladder, suggesting unique or enhanced bile resistance mechanisms utilized by the bacterium that lead to survival in this environment. The formation of biofilms on gallstones has been hypothesized to be a facilitator for enhanced colonization of and persistence in this organ. A biofilm is defined as a community of microorganisms that adhere to each other and to a biotic or abiotic surface, resulting in stability and protection from antibiotics and other environmental factors mediated in part by a self-initiated exopolysaccharide (EPS) matrix. Bacterial biofilms have been implicated as the causes of many chronic infections in humans and are associated with medical and industrial ecosystems (8). Planktonic cells from this sessile, matrix-bound population are continuously shed, which can result in systemic infection or release of the organism into the environment. Bacteria shed by asymptomatic carriers contaminate food and water and account for much of the person-to-person transmission of serovar Typhi in underdeveloped countries.

As mentioned above, it has been demonstrated that patients with gallbladder abnormalities, such as gallstones, are at a much higher risk for serovar Typhi carriage, yet the progression of infection to the carrier state is undefined. Antibiotic regimens can be ineffective in carriers with gallstones (19), and the cost of surgery to remove gallbladders is prohibitive in developing countries where serovar Typhi is prevalent. Interestingly, bile has been shown to promote biofilm formation of S. enterica serovar Typhimurium and serovar Typhi in an in vitro human gallstone test tube assay (29). To date, the EPS matrix of Salmonella bile-induced biofilms on gallstones remains poorly characterized. A better understanding of the key EPS constituents of Salmonella bile-induced biofilms promises to better characterize the asymptomatic carrier state and elucidate important therapeutic targets for alleviating biofilm formation in chronic cases. Here, we define a bile-induced EPS, the O-antigen (O-ag) capsule, required for biofilm formation specifically on gallstone and cholesterol-coated surfaces.

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Bacterial strains and growth conditions. The Salmonella strains and plasmids used in this study are listed in Table 1. Luria-Bertani (LB) broth and agar were used for bacterial growth, creation of mutants, biofilm assays, and expression studies. When necessary, antibiotics were added at appropriate concentrations: ampicillin, 50 µg/ml; kanamycin, 25 µg/ml; and tetracycline, 15 µg/ml.

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TABLE 1. Bacterial strains, plasmids, and relevant characteristics

Molecular biology and genetic techniques. DNA purification and PCR were completed according to established procedures (using a QIAprep spin mini prep kit) (34). Plasmids were transformed by electroporation as previously described (35). Quantitative real-time PCR was performed using RNA extracted from log-phase (optical density of 0.6 at 600 nm) Salmonella cultures with an RNeasy kit (Qiagen, Valencia, CA). Samples grown in 3% crude ox bile extract (Sigma, St. Louis, MO) were quickly washed three times in 1x phosphate-buffered saline (PBS) before extraction. RNA quality and quantity were measured with the Experion automated electrophoresis system (Bio-Rad, Hercules, CA), and 1 microgram was reverse transcribed to cDNA with Superscript II RNase H^– reverse transcriptase (Invitrogen, Carlsbad, CA). Five nanograms of converted cDNA was added to Sybr green PCR master mix for quantitative real-time PCR with a Bio-Rad iCycler apparatus. Table 2 shows the primers used for expression of the target gene yihU and the housekeeping gene dnaN. Relative copy numbers were calculated according to a published protocol (13).

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TABLE 2. Oligonucleotide primers

Construction of bcsE wcaA, yihO, and yihP mutants. The serovar Typhimurium cellulose/colanic acid double mutant (bcsE wcaA) was created by P22 HT105 int-102-mediated, generalized transduction between strains carrying single mutations in bcsE or wcaA (35). The Red mutagenesis method of gene disruption was used to create a deletion of yihO in serovar Typhimurium and of yihP in serovar Typhi (11). Primer pairs JG1281/JG1282 and JG1643/JG1644 (Table 2) were designed to amplify yihO'-kan-'yihO and yihP'-kan-'yihP, respectively, using pKD4 as a template. The kanamycin cassettes and surrounding regions of homology were exchanged into the chromosome of serovar Typhimurium or serovar Typhi, generating strains carrying yihO::kan in serovar Typhimurium and yihP::kan in serovar Typhi. Correct placement of these constructs in the Salmonella genome was confirmed by PCR, using primers JG1283 and JG1284 for serovar Typhimurium and primers JG1647 and JG1648 for serovar Typhi. The kanamycin cassettes were then resolved by introducing pCP20, and the resulting strains were named JSG2897 (carrying yihO) and JSG2950 (carrying yihP) (Table 1). For complementation, the yihO gene from serovar Typhimurium was amplified and cloned into the HindIII and EcoRI sites of vector pUC18 (GenScript Corporation, Piscataway, NJ) and expressed from the lac promoter. The resulting construct was introduced into JSG2897 by electrotransformation.

Gallstone tube assay. Uniform gallstone samples composed primarily of cholesterol and retrieved from a single patient were used in this study. Salmonella strains were grown overnight in LB broth with or without 3% crude ox bile extract. Each overnight culture was diluted 1:10, added to LB broth in test tubes containing human gallstones (with or without bile), and incubated in a rotating drum at 37°C with aeration. To attempt to mimic normal gallbladder physiology, the medium and bacteria were removed from the tubes every 24 h, the gallstones were washed three times in LB by light vortexing, and fresh medium (with or without bile) was returned. The optimal growth period was 12 days, as described previously (29).

Tube biofilm assay (TBA). Chromatography-grade cholesterol (product number C8667; Sigma, St. Louis, MO) was dissolved in anhydrous ether (J. T. Baker, Phillipsburg, NJ) at a concentration of 10 mg/ml and delivered in 100-µl aliquots to siliconized Eppendorf tubes (Fisher Scientific, Pittsburgh, PA). The ether was allowed to evaporate, consistently leaving tubes with a 1-mg coating of cholesterol to which 100 µl of bacterial culture could be added. Salmonella strains were grown overnight in LB broth with or without 3% crude ox bile extract, diluted 1:100 the following day, and subsequently grown to an optical density at 600 nm of 0.6. These cultures (100 µl) were incubated in cholesterol-coated Eppendorf tubes at room temperature on a Nutator shaker (Labnet International, Edison, NJ). Every 24 h, the medium was removed, the tubes were washed three times with LB, and fresh medium (LB with or without 3% bile) was added. Each strain was examined in triplicate, and the experiment proceeded for a period of 6 days.

Plastic/glass binding assay. Round, Chromerge-cleaned glass coverslips were placed in plastic 24-well plates (Fisher Scientific, Pittsburgh, PA). Liquid cultures of S. enterica diluted 1:100 following overnight growth were allowed to reach an optical density at 600 nm of 0.6 in the presence or absence of 3% crude ox bile extract and then added to each coverslip-containing well in 2-ml volumes and incubated at room temperature on a Nutator shaker. The assay continued for 6 days, with removal of medium, washing, and addition of fresh medium occurring every 24 h. Each strain was tested with triplicate wells. Biofilms on the plastic 24-well plates and on the glass coverslips were examined as described below.

Crystal violet quantification of biofilms. Following bacterial growth on cholesterol-coated tubes in the TBA or on glass/plastic in binding assays, samples were washed three times in LB and incubated at 60°C for 1 hour to fix the cells. A solution of 0.1% crystal violet (gentian violet in isopropanol-methanol-1x PBS [1:1:18]) was then added to stain cells for 5 minutes at room temperature. Specimens were washed thoroughly with 1x PBS until the liquid ran clear. The dye was extracted using 33% acetic acid and then quantified with optical density readings at 570 nm to determine the amount of dye retained by the biofilm cells.

Immunofluorescence microscopy of tube biofilms. After completion of the TBA as described above, cells were visualized using immunofluorescence microscopy for the production of O-ag capsule EPS in the presence or absence of 3% crude ox bile extract. Three vigorous washes with 1x PBS were followed by 4 hours of incubation with 5% bovine serum albumin (Sigma, St. Louis, MO). Cells were washed and then exposed to capsule-specific primary antibody (diluted 1:2,000 in PBS) at room temperature for 2 hours. This polyclonal antibody was generated in rabbits and showed no cross-reactivity with commercially purified lipopolysaccharide (LPS) from S. enterica serovar Enteritidis (14). The tube contents were washed three additional times and incubated with a 1:5,000 dilution of fluorescein-conjugated, goat affinity-purified antibody to rabbit immunoglobulin G (ICN/Cappel, Aurora, OH) in the dark for 2 hours. Biofilm samples were washed, scraped gently from the tubes by using a microspatula, diluted in 1x PBS, transferred to glass slides, and visualized on a BX51 Olympus fluorescence microscope.

Tube biofilm ELISA. Cells from the TBA were measured for production of O-ag capsule EPS by using a modified enzyme-linked immunosorbent assay (ELISA) approach. After 6 days in the TBA, the tubes were washed three times with 1x PBS and incubated with 5% bovine serum albumin for 4 hours. After three more washes with 1x PBS, capsule-specific antibody (diluted 1:2,000 in PBS) was added to the tubes at room temperature for 2 hours. Another set of three washes was followed by a 2-hour incubation period with a goat anti-rabbit immunoglobulin G (H+L) horseradish peroxidase (HRP) conjugate diluted 1:5,000 in PBS (Bio-Rad, Hercules, CA). Measurements of O-ag capsule expression in biofilms were obtained using a Bio-Rad HRP substrate kit according to the manufacturer's specifications. Reactions were performed with the TBA Eppendorf tubes, and the reaction products were transferred to 96-well plates for optical density readings at 415 nm.

Scanning electron microscopy (SEM). Following sample growth for 12 days, gallstones were washed twice with LB. Specimens were either fixed immediately in 2% glutaraldehyde or air dried overnight in a Laminar flow hood before being fixed. All samples were dehydrated postfixation using a Cressington critical point drier at The Ohio State University Campus Microscopy and Imaging Facility. The samples were sputter coated with a Pelco Model 3 and viewed using a FEI Nova NanoSEM.

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A serovar Typhimurium colanic acid/cellulose double mutant forms mature gallstone biofilms. The extracellular matrix produced by bacterial biofilms is frequently composed of polysaccharides and has been well characterized for organisms such as E. coli, V. cholerae, and Pseudomonas aeruginosa (10, 17, 31). As described previously, the EPS of bile-induced Salmonella biofilms on gallstones can be preserved and visualized by SEM (29). Individual mutants unable to produce the known Salmonella EPS components cellulose and colanic acid and the ViAg capsule (serovar Typhi) were previously shown to maintain the ability to form a biofilm on gallstones (27). To extend these results, a bcsE wcaA double mutation was created in serovar Typhimurium. Mutant and wild-type strains were incubated in the presence of 3% crude ox bile extract and a human gallstone under established assay conditions. Gallstones were fixed immediately in 2% glutaraldehyde and examined by SEM (Fig. 1). Biofilm formation and the presence of EPS (flocculent and stringy material) were equally robust for wild-type and mutant strains, suggesting involvement of an as-yet-unidentified EPS.

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FIG. 1. The loss of colanic acid and cellulose does not affect Salmonella gallstone biofilm formation. (A) SEM micrograph showing the multilayered, heterogeneous surface of a cholesterol gallstone incubated in LB broth without bacteria. (B) A serovar Typhimurium wild-type (14028s) strain exposed to a human gallstone for 12 days in the presence of 3% bile forms a mature biofilm. (C) A serovar Typhimurium bcsE wcaA mutant strain exposed to a human gallstone for 12 days in the presence of 3% bile also forms a mature biofilm. The web-like strands or flocculent material is indicative of EPS in the biofilm ECM.

S. enterica binds to and forms biofilms on cholesterol-coated Eppendorf tubes. Though regional differences in diet and other environmental and genetic factors can alter gallstone development, human gallstones are primarily composed of cholesterol or calcium bilirubin. To eliminate dependence on human gallstone samples and to allow gallstone constituents to be analyzed individually for their abilities to interact with salmonellae, siliconized Eppendorf tubes were coated with 1 mg of cholesterol to mimic gallstone surfaces.

As shown in Fig. 2A, Eppendorf tubes required a coating of cholesterol to support Salmonella biofilm formation, suggesting that salmonellae bind to cholesterol. The presence of bile in this TBA significantly enhanced biofilm formation on cholesterol as it did for human gallstones, further promoting the hypothesis that bile in the gallbladder helps to establish biofilm formation on cholesterol gallstones (Fig. 2A and B). It was also observed that bacteria preincubated with bile (grown overnight in 3% bile) formed more-robust biofilms and formed them earlier than bacteria encountering bile for the first time upon addition to cholesterol-coated tubes (data not shown). Additionally, tubes coated with bilirubin supported significantly less biofilm than those coated with cholesterol, though bile still induced bilirubin-coated-tube biofilm formation (data not shown). Therefore, bile is an important environmental signal for salmonellae that enhances biofilm formation on cholesterol-coated surfaces.

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FIG. 2. Eppendorf tubes coated with 1 mg cholesterol support Salmonella biofilm formation in the TBA. The TBA spanned 6 days, with washings every 24 h to mimic emptying of the gallbladder. (A) Salmonella biofilms from the TBA were stained with crystal violet and photographed using the BioChemi system (UVP BioImaging). (B) Crystal violet-stained TBA biofilms were extracted with acetic acid, and absorbance was measured for at 570 nm. *, statistical significance (P < 0.005) based on a two-tailed Student t test. OD570, optical density at 570 nm.

The Salmonella O-ag capsule is necessary for binding to cholesterol versus binding to glass or plastic. In addition to cellulose and colanic acid, E. coli expresses a variety of chemically and serologically distinct capsules (43). One such EPS is the O-ag capsule, the composition of which is similar to that of the LPS O polysaccharides (32). Recently, Gibson and colleagues identified an O-ag capsule associated with the serovar Enteritidis extracellular matrix that was shown to be important for environmental persistence (14). Assembly and translocation of the conserved O-ag capsule involve the enzymes produced by two operons, yihU-yshA and yihVW. Mutations in several genes of these operons, including yihO, have been shown to eliminate expression of this capsule.

To determine the role of the O-ag capsule in biofilm formation on cholesterol-coated surfaces, deletions of yihO in serovar Typhimurium and of yihP in serovar Typhi were created by lambda Red-mediated recombination and were examined in the TBA. The data show that the O-ag is a necessary component of Salmonella biofilms on cholesterol-coated Eppendorf tubes in serovar Typhimurium, serovar Typhi, and serovar Enteritidis (Fig. 2A and B). Real-time PCR experiments confirmed that the serovar Typhimurium yihO deletion did not have a polar effect on downstream genes (data not shown). Thus, we performed complementation experiments with plasmid-borne yihO. These studies resulted in amounts of bile-induced biofilm formation on cholesterol of the yihO mutant similar to those for wild-type serovar Typhimurium (Fig. 2B).

The O-ag capsule was originally identified by White et al. (42) in association with the AgfD-regulated extracellular matrix. AgfD (CsgD) is an important regulator of Salmonella biofilm formation (5, 33) and has been demonstrated to control O-ag capsule biosynthesis (14). To determine the role of AgfD in bile-mediated biofilm formation on gallstone/cholesterol-coated surfaces, a serovar Typhimurium agfD mutant was added to the TBA. As shown in Fig. 2B, loss of AgfD had no effect on biofilm formation in these assays.

To examine whether the O-ag capsule was important for biofilm formation on other surfaces, wild-type and yihO and yihP deletion strains were assayed in the presence of bile for their abilities to bind to glass or plastic coverslips. As shown in Table 3, wild-type salmonellae formed mature biofilms on glass, plastic, or cholesterol-coated surfaces. However, absence of the O-ag capsule eliminated biofilm formation on cholesterol-coated tubes but not on glass or plastic coverslips. Furthermore, a bcsE wcaA double mutant formed poor biofilms on glass and plastic surfaces but formed a biofilm similar to that of the wild-type strain on cholesterol-coated tubes (Table 3). These data suggest specificity of binding to cholesterol mediated directly or indirectly by the O-ag capsule.

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TABLE 3. Summary of biofilm formation on cholesterol-coated, glass, and plastic surfaces

The extracellular-matrix-associated O-ag capsule is a critical component of Salmonella gallstone biofilms. To evaluate the role of the O-ag capsule in bile-induced Salmonella gallstone biofilms, O-ag mutants were examined by electron microscopy for biofilm formation on gallstone surfaces. As shown in Fig. 3, wild-type serovar Typhimurium, serovar Typhi, and serovar Enteritidis formed mature biofilms on human gallstones after 12 days of incubation with 3% bile. The extracellular-matrix material was preserved in a more natural state by air drying samples overnight before fixation in 2% glutaraldehyde. This extra step accounted for the visual differences in Salmonella gallstone biofilm ECM composition observed in Fig. 1 (samples subjected to immediate fixation) and Fig. 3 (samples air dried before fixation). Unlike the wild-type strains, none of the three O-ag capsule mutant strains formed detectable biofilms on gallstones (Fig. 3B, D, and F). These results implicate the extracellular-matrix-associated O-ag capsule as a crucial component of bile-mediated biofilm formation on gallstones.

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FIG. 3. Mutations in the O-ag operon affect Salmonella gallstone biofilm formation. Human gallstones were incubated with salmonellae and 3% bile for 12 days, washed in LB, air dried overnight, fixed in glutaraldehyde, and visualized by SEM. (A) Serovar Typhimurium wild type. (B) Serovar Typhimurium yihO mutant. (C) Serovar Typhi wild type. (D) Serovar Typhi yihP mutant. (E) Serovar Enteritidis wild type. (F) Serovar Enteritidis yihO mutant.

Bile increases Salmonella O-ag capsule production in biofilms. The presence of bile has been shown to alter protein production, antibiotic and bile resistance, virulence, and EPS synthesis for enteric bacterial groups, including Shigella flexneri (24, 25), Helicobacter pylori (39), Campylobacter spp. (22, 38), and V. cholerae (3, 17; for a review, see reference 2). To examine the effect of bile on capsule expression in biofilms grown on cholesterol-coated surfaces, antibody to Salmonella O-ag was used in a quantitative TBA ELISA. Growth in 3% bile significantly enhanced O-ag capsule induction for wild-type serovar Typhimurium, serovar Typhi, and serovar Enteritidis as well as the serovar Typhimurium colanic acid/cellulose double mutant (Fig. 4A). This was not seen with strains deficient for O-ag production.

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FIG. 4. Effect of bile on Salmonella O-ag capsule expression. (A) Modified ELISA in the TBA. Salmonellae were incubated with or without bile in cholesterol-coated Eppendorf tubes for 6 days, with washings every 24 h. Samples in the tubes were then blocked with BSA, exposed to O-ag capsule antisera and the appropriate HRP-conjugated secondary antibody, and developed. *, statistical significance (P < 0.005) based on a two-tailed Student t test. OD415, optical density at 415 nm. (B) Immunofluorescence microscopy of TBA biofilms gently removed from the tubes. Primary O-ag capsule antibody was used with a fluorescein-conjugated secondary antibody for visualization with an Olympus fluorescence microscope.

Because some of the differences observed in Fig. 4A may be due to the reduction in biofilm in the absence of bile, fluorescence microscopy of Salmonella biofilm material removed from the TBA was also used to measure O-ag capsule expression (Fig. 4B). As with the modified ELISA, the images show that production of the O-ag capsule was increased in biofilms for all wild-type strains and the bcsE wcaA mutant when bacteria were grown in bile. Quantification of signal intensity indicated that bile mediated a 166 to 278% fluorescence increase in the Salmonella strains examined. The yihO and yihP mutants of serovar Typhimurium, serovar Typhi, and serovar Enteritidis showed little O-ag expression in the presence or absence of bile (Fig. 4B). In addition, there was no significant difference in thickness of biofilm material between samples from tubes with and without bile (data not shown). Furthermore, a fluorescence increase was also observed on a single-cell basis for bacteria grown in the presence of 3% bile versus those grown without bile (data not shown). These results further implicate the importance of the O-ag capsule in Salmonella biofilm ECM and demonstrate that its production is bile induced.

Transcription of O-ag capsule genes is upregulated by bile in an AgfD-independent manner. Bile has been shown to influence the expression of microbial virulence factors at the level of gene transcription. For example, eukaryotic cell invasion by serovar Typhimurium is repressed by bile, as evidenced by transcriptional downregulation of genes within Salmonella pathogenicity island I (26). To examine the effect of bile on transcription of O-ag genes, real-time PCR was performed using wild-type serovar Enteritidis, serovar Typhimurium, and serovar Typhi and an agfD mutant of serovar Typhimurium, all grown in the presence or absence of bile (Fig. 5). Transcription of yihU, a putative oxidoreductase located in the O-ag operon, was enhanced 18-fold for serovar Enteritidis, 24-fold for serovar Typhimurium, 11-fold for serovar Typhi, and 23-fold for serovar Typhimurium agfD when strains were grown in the presence of bile, suggesting that bile is a signal resulting in increased transcription of the O-ag cluster independently of AgfD.

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FIG. 5. Real-time PCR measuring O-ag capsule transcription for serovar Enteritidis, serovar Typhimurium, serovar Typhi, and agfD of serovar Typhimurium grown in the presence or absence of bile. Primers target yihU, a putative oxidoreductase located in the O-ag capsule-encoding operon. *, statistical significance (P < 0.005) based on a two-tailed Student t test.

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Bacterial biofilms are increasingly associated with serious illnesses in humans and animals. We hypothesize that the Salmonella chronic carriage state is frequently if not always a result of biofilm formation on gallstones. In limited studies, a high correlation was shown to exist between those individuals who became carriers and those who have gallstones (12, 19), and the protection afforded the bacteria within the biofilm would help explain their resistance to killing by bile and antibiotics. In this work, we describe a bile-induced EPS necessary for and specific toward biofilm formation on gallstones and cholesterol-coated surfaces.

ECM material within bacterial biofilms is thought to be heterogeneous and to provide both rigidity to the biofilm and protection to bacteria embedded within these biofilms. It was previously shown that the serovar Typhi Vi antigen did not contribute to the establishment of gallstone biofilms (27). Furthermore, serovar Typhimurium and serovar Enteritidis do not produce this EPS but form biofilms on gallstones that are indistinguishable from those of serovar Typhi. To further examine the role of EPSs in Salmonella gallstone biofilms, a double mutation in the known Salmonella ECM constituents colanic acid and cellulose was created. When assayed by SEM for biofilm formation on human gallstones, robust biofilms and ECM material were still clearly present. These data suggested the presence of an undetermined EPS in the Salmonella gallstone microenvironment.

Previous work performed by Gibson et al. (14) identified a conserved serovar Enteritidis EPS termed the O-ag capsule. This substance was similar in structure to the LPS O-ag of serovar Enteritidis and was encoded by two divergent operons (yihU-yshA and yihV-yihW) (14). Despite having similar repeating sugar units, the O-ag capsule and the LPS O-ag differ in size, charge, substitution patterns, and immunoreactivity (42). In addition, this capsule is linked to the membrane via a lipid anchor. O-ag capsule expression was found to be necessary for serovar Enteritidis biofilm formation leading to enhanced plant colonization (1). To examine the role of the O-ag capsule in Salmonella gallstone biofilms, a yihO deletion was constructed in serovar Typhimurium and yihP was deleted in serovar Typhi. Lack of O-ag capsule production proved to be critical, as these strains failed to form a biofilm on gallstone surfaces. Similar results were observed for an O-ag serovar Enteritidis mutant, though they were not as exaggerated as those observed for serovar Typhimurium or serovar Typhi.

Human gallstones in North America are primarily cholesterol but also contain calcium bilirubin and other components. Interaction of salmonellae with cholesterol has been described to occur in host cells, as cholesterol is recruited to the Salmonella-containing vacuole and esterified by the SPI-2 effector SseJ (6, 23). Because of such known cellular associations with cholesterol and because cholesterol is the primary constituent of the human gallstones used in our assays, we examined Salmonella biofilm formation on Eppendorf tubes coated with cholesterol (TBA). The results of these experiments mimicked those of the experiments performed with gallstones in test tubes for all wild-type and mutant strains examined, suggesting that S. enterica binds to cholesterol. Biofilms on gallstones were previously shown to often occur in patches (29), which, given this new knowledge, may reflect the local distribution of cholesterol on the gallstone surface. Furthermore, bile was shown to enhance biofilm formation on cholesterol as it did for human gallstones. This model has advantages over that of human gallstones as it is more efficient, is amenable to large-scale mutagenesis screens, avoids retrieval of patient materials, and will allow other gallstone constituents to be analyzed for Salmonella binding in the future.

It has previously been shown that Salmonella biofilm-associated factors are not uniformly required for biofilm formation on all surfaces (27). Interestingly, serovar Typhimurium, serovar Typhi, and serovar Enteritidis O-ag capsule mutants were all shown to be deficient in biofilm formation on cholesterol-coated surfaces and human gallstones but produced robust biofilms on glass and plastic coverslips when grown in the presence of bile. Cellulose and colanic acid mutants had the opposite phenotype, as they did not form biofilms on glass or plastic coverslips but formed mature biofilms on human gallstones and in the TBA. Interestingly, cellulose and colanic acids have been shown to play a role in biofilm formation on biotic as well as abiotic surfaces, as mutations in either wcaM or yhjN have been shown to disrupt serovar Typhimurium biofilm formation in flowthrough chambers on HEp-2 cells (20). However, in the presence of cholesterol and bile, cellulose and colanic acid are not a factor in biofilm formation.

A modified ELISA and immunofluorescence microscopy were used to determine if bile enhanced O-ag capsule expression in Salmonella biofilms on cholesterol. Both experiments showed that bile increased not only biofilm formation but also production of the O-ag capsule in all wild-type strains as well as the serovar Typhimurium bcsE wcaA double mutant. Real-time PCR analysis indicated that bile-mediated O-ag capsule induction occurred at the level of transcription of the yih genes. Gibson and colleagues showed that AgfD, an important regulatory factor of biofilm formation (5, 33), controls expression of the yih operons (14). However, loss of AgfD had no effect on biofilm formation in the TBA. Furthermore, real-time PCR revealed that bile upregulated transcription of the yih operon in an agfD-independent manner, suggesting that another bile-responsive transcription factor is involved in yih gene expression.

We have identified a critical EPS in the extracellular matrix of Salmonella bile-induced biofilms on gallstones and a new method of examining Salmonella-gallstone interactions (the TBA). It could be hypothesized that bile induction of the O-ag capsule has evolved to aid chronic carriage of S. enterica specifically in the gallbladder environment. This new understanding of how bile mediates Salmonella gallstone biofilm formation via O-ag capsule expression provides a therapeutic target for potentially alleviating carriage in the gallbladder and subsequently much of the human-to-human transmission that makes this pathogen a relevant global concern.

 
We thank Brian Kemmenoe of the Ohio State University Campus Microscopy and Imaging Facility for technical support with SEM work.

This study was supported by NIH grant AI066208 (J.S.G.) and a graduate education fellowship from Ohio State University Public Health Preparedness for Infectious Diseases (R.W.C.). W. W. Kay was supported through grants from the NSERC, while D. L. Gibson was supported by postgraduate fellowships from the NSERC and postdoctoral fellowships from the Canadian Association of Gastroenterology, AstraZeneca, the Canadian Institutes of Health Research, and the Michael Smith Foundation for Health Research.

 
* Corresponding author. Mailing address: The Ohio State University, Center for Microbial Interface Biology, 460 W. 12th Ave., Biomedical Research Tower, Room 1006, Columbus, OH 43210. Phone: (614) 292-6036. Fax: (614) 292-5495. E-mail: gunn.43{at}osu.edu

Published ahead of print on 15 September 2008.

Editor: F. C. Fang

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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Infection and Immunity, November 2008, p. 5341-5349, Vol. 76, No. 11
0019-9567/08/$08.00+0     doi:10.1128/IAI.00786-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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