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Originally published In Press as doi:10.1074/jbc.M710095200 on January 22, 2008

J. Biol. Chem., Vol. 283, Issue 13, 8118-8124, March 28, 2008
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Binding Specificity of Salmonella Plasmid-encoded Fimbriae Assessed by Glycomics*

Daniela Chessa{ddagger}, Caleb W. Dorsey§, Maria Winter{ddagger}, and Andreas J. Baümler{ddagger}§1

From the {ddagger}Department of Medical Microbiology and Immunology, School of Medicine, University of California at Davis, Davis, California 95616-8645 and the §Department of Medical Microbiology and Immunology, College of Medicine, Texas A&M University Health Science Center, College Station, Texas 77843-1114

Received for publication, December 11, 2007 , and in revised form, January 22, 2008.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The Salmonella enterica serotype Typhimurium (S. Typhimurium) genome encodes 12 intestinal colonization factors of the chaperone/usher fimbrial assembly class; however, the binding specificity is known for only one of these adhesins, known as type 1 fimbriae. Here we explored the utility of glycomics to determine the carbohydrate binding specificity of plasmid-encoded fimbriae from S. Typhimurium. A cosmid carrying the pef operon was introduced into Escherichia coli and expression of fimbrial filaments composed of PefA confirmed by flow cytometry and immune-electron microscopy. Plasmid-encoded fimbriae were purified from the surface of E. coli, and the resulting preparation was shown to contain PefA as the sole major protein component. The binding of purified plasmid-encoded fimbriae to a glycanarray suggested that this adhesin specifically binds the trisaccharide Galβ1–4(Fuc{alpha}1–3)GlcNAc, also known as the Lewis X (Lex) blood group antigen. Results from the glycanarray were validated by enzyme-linked immunosorbent assay (ELISA) in which plasmid-encoded fimbriae bound Lex-coated wells in a concentration-dependent manner. The binding of plasmid-encoded fimbriae to Lex-coated wells could be inhibited by co-incubation with soluble Lex antigen. Our results establish glycomic analysis as a promising new approach for determining the carbohydrate binding specificity of bacterial adhesins.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The Salmonella enterica serotype Typhimurium (S. Typhimurium)2 genome contains 12 operons encoding type II secretion systems of the chaperone/usher assembly class (1). These include eight members of the {gamma}-Fimbriae (fim, bcf, sti, sth, lpf, saf, stc, and stb), one member of the β-Fimbriae (stj), two members of the {pi} -Fimbriae (std and stf), and one member of the {kappa}-Fimbriae (pef) (2). However, the binding specificity is known only for type 1 fimbriae, which are encoded by the S. Typhimurium fim operon (3, 4). Type 1 fimbriae mediate mannose-sensitive agglutination of erythrocytes or yeast cells (5), because the FimH fimbrial tip adhesin binds terminal {alpha}-D-mannose residues present in host glycoproteins (6–10). The paucity of data on the binding specificities of chaperone/usher systems is partly due to the fact that, with the exception of type 1 fimbriae, these surface structures are poorly expressed during growth of S. Typhimurium under standard laboratory conditions (11, 12).

The pef operon is located on the virulence plasmid of S. Typhimurium (13). Plasmid-encoded fimbriae cannot be detected after growth under standard laboratory growth conditions (11, 12), because expression is controlled negatively by the histone-like protein (H-NS), the stationary phase sigma factor (RpoS), and the presence of type 1 fimbrial biosynthesis genes (fimAICDHF) (2, 14). Although production of the major fimbrial subunit PefA is not detected under laboratory growth conditions, seroconversion of mice infected with S. Typhimurium (12) and detection of PefA on bacteria recovered from bovineligated ileal loops (11) provide evidence for in vivo expression of the pef operon. Introduction of the cloned pef operon into Escherichia coli results in expression of thin (2–5 nm in diameter) flexible fibrillae composed of the major fimbrial subunit PefA (13, 15). The ability to express plasmid-encoded fimbriae under laboratory growth conditions makes them well-suited for experiments aimed at characterizing their binding specificity. The goal of this study was to explore the utility of using glycanarrays for determining the binding properties of plasmid-encoded fimbriae.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial Strains and Growth Conditions—E. coli strains BL21(DE3) and TOP10 were obtained from Stratagene and Invitrogen, respectively. The E. coli strain ORN172 carries a {Delta}fimBEACDFGH::Km allele and is phenotypically non-fimbriate (16). Bacteria were routinely grown at 37 °C in Luria Bertani (LB) broth (with shaking) or on LB agar plates. For expression of fimbriae, bacteria were grown statically in LB broth at 37 °C overnight.

Generation of Anti-PefD Serum—A DNA region encoding an internal portion of PefD was amplified with primers: 5'-CGGGATCCGGCACGCGTTTTATCTATGAGGAAGG-3' and 5'-GGAATTCTCACTTCAGCGTGTAGTCCTGGGTG-3' and cloned into the plasmid vector pGEX4T-2 (17) (Amersham Biosciences) to generate pCWD41, which encodes a PefD-GST fusion protein. The PefD-GST fusion protein was purified from E. coli strain BL21(DE3) using affinity chromatography as described previously (11). Anti-PefD serum was raised in a female New Zealand White rabbit as previously described (11). Serum was pre-absorbed with E. coli BL21 pGEX4T-2 (expressing GST) using a protocol described previously (18).

Flow Cytometry—Approximately 5 x 108 cells were incubated with an equal volume of 4% paraformaldehyde (EM Science) at room temperature for 20 min. Cells were washed twice with 0.5 ml of 0.02% gelatin in PBS (PBS-gel). To block nonspecific binding, cells were harvested and resuspended in 0.5 ml of filter-sterilized 2% normal goat serum (NGS, Sigma) and incubated at room temperature for 30 min on a tabletop rotator. Polyclonal rabbit anti-PefA serum (11) was added to the cells at a final dilution of 1:250 for detection of PefA, and cells were incubated at room temperature for 60 min on a tabletop rotator. After washing the cells three times in PBS-gel, bacteria were resuspended in 0.5 ml of a solution of 0.04 mM propidium iodide in 2% NGS with secondary antibody (fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG, Jackson ImmunoLabs) added at a dilution of 1:250. The mixture was rotated at room temperature for 1 h in the dark. Samples were washed three times with PBS-gel, and bacteria were resuspended in PBS to a final concentration of ~5 x 106 cells/ml. For each sample, the fluorescence of 10,000 particles (bacterial cells) was measured by flow cytometry (FACSCalibur; Becton Dickinson, San Jose, CA).

Purification of Fimbriae—A non-fimbriate E. coli strain (ORN172) carrying the cloned pef operon (pFB11) (15) or the cloned fim operon (pISF101) (19) was grown in two liters of LB broth, harvested by centrifugation, and resuspended in 10 ml of 0.5 mM Tris, 75 mM NaCl. Plasmid-encoded fimbriae were separated from the cells by mechanical shearing in a blender for three 1-min periods, after which cells and cellular debris were removed by centrifugation (3,500 rpm, 30 min, 4 °C). The supernatant was collected and passed through a 0.45-mm filter (Millipore), and (NH4)2SO4 (60% final concentration) was added to precipitate the fimbriae. Precipitated fimbriae were recovered by centrifugation (14,000 rpm, 30 min, 4 °C). The pellet was resuspended in 50 µl of sterile water and was analyzed by SDS-PAGE, Western blot, and electron microscopy.

Western Blot Analysis—Polyclonal rabbit anti-PefA serum has been previously described (11). The serum was diluted 1:5 in PBS, pH 7.4 containing 0.2% sodium azide and pre-absorbed 8 times (18) with ORN172-carrying plasmid pGEX-4T-2 (17) and 4 times with ADH19 (SR11 {Delta}pefBACDI::Km). Purified fimbriae were disassembled into individual subunits by boiling in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) loading buffer for 10 min. Bacterial cultures were resuspended to A600 to a concentration of ~2 x 108 colonyforming units (cfu)/10 µl in SDS-PAGE loading buffer and boiled for 5 min to generate cell lysates. Cell lysates were then separated by 15% SDS-PAGE, transferred to Immobilon-P (Millipore) membranes using a Trans-Blot semi-dry transfer cell (Bio-Rad), incubated with rabbit anti-PefA serum diluted to 1:500 final, detected with goat anti-rabbit alkaline phosphatase conjugate and the Immun-Star chemiluminescent substrate (Bio-Rad), and visualized with X-Omat Blue Film (Kodak).

N-terminal Sequencing—Proteins separated by SDS-PAGE were transferred to a polyvinylidene difluoride membrane (Millipore) in 10 mM CAPS, 10% methanol, pH 11.0 using a Trans-Blot SD semi-dry electrophoretic transfer cell according to standard protocols. Following electrophoretic transfer, the polyvinylidene difluoride membrane was washed two times in sterile water. Transferred protein was visualized by staining with Amido Black solution (Sigma Aldrich) for 2 min followed by destaining with 1% acetic acid. N-terminal sequencing of the Pef major subunit was performed by the Protein Chemistry Laboratory at Texas A&M University using automated Edman chemistry in a Hewlett Packard G1000A Automated Protein Sequencer.

Electron Microscopy—For immune-electron microscopy, bacteria were grown in a static culture, washed twice in PBS, and resuspended in EM grade water (EM Science) at a titer of ~1 x 109 colony-forming units/ml. The bacterial culture (10 µl) was allowed to adhere to a formvar/carbon-coated grid (EM Science) for 2 min, and grids were incubated for 20 min in a primary antiserum (rabbit) diluted 1:250 in PBS containing 1% bovine serum albumin (BSA). Grids were washed five times for 1 min in PBS containing 1% BSA. Grids were incubated for 20 min in goat anti-rabbit 10-nm gold conjugate (EM Science), diluted 1:20 in PBS containing 1% bovine serum albumin (BSA). Grids were washed three times for 1 min in PBS containing 1% BSA and three times for 1 min in EM grade water (EM Science) before they were analyzed by electron microscopy.

Purified plasmid-encoded fimbriae (10 µl) were visualized by transmission electron microscopy (TEM) by allowing attachment to formvar-coated copper TEM grids for 1 min. The purified fimbriae were negatively stained for 1 min using 1% aqueous ammonium molybdate. The grids were allowed to dry before they were analyzed by electron microscopy.

Glycanarray Analysis—Glycanarray analysis was performed by the Consortium for Functional Glycomics (CFG). Streptavidin-coated high binding capacity black 384-well plates (Pierce 15513) were coated at saturating density (60 pmol/well) with 99 different biotinylated carbohydrate ligands in triplicate (Glycan-Array version 1.2) and washed 3x (Embla 96/384 Well Washer from Molecular Devices) with 0.1 ml of wash buffer (PBS, 0.05% Tween 20). Purified plasmid-encoded fimbriae (0.05 ml of a solution containing 0.03 g/ml) were added and incubated for 1 h at room temperature. After three washes (washing buffer), plates were incubated with rabbit anti-PefA serum (1:23.5) for 1 h at room temperature. After three washes, plates were incubated with goat anti-rabbit IgG-Alexa488 conjugate (0.025 ml/well of a solution containing 0.05 mg/ml) and incubated for 1 h at room temperature. After three washes, wells were loaded with 0.05 ml of PBS, and fluorescence measured at 485/535 nm using the Core H plate reader (Wallac Victor2 1420 Multi-label Counter from PerkinElmer). The background signal was determined in wells treated with primary (rabbit anti-PefA) and secondary antibody (goat anti-rabbit IgG-Alexa488), but not with plasmid-encoded fimrbiae (control wells). The average reading from three wells coated with one biotinylated carbohydrate ligand was divided by the average reading from three control wells. Binding that was increased at least 2-fold above background levels was considered significant.

Enzyme-linked Immunosorbent Assay (ELISA)—EvenCoatTM streptavidin microplates (R&D Systems) were coated with Lewis X (Lex)-PAA-biotin, D-mannose-PAA-biotin, or HOCH2-(HOCH)4CH2NH-PAA-biotin (GlycoTech). Streptavidin-coated high binding capacity 96-well plates were coated for 30 min at room temperature with 100 µl per well of 2 µg/liter biotin-conjugated sugar diluted in PBS. Serial dilutions of a plasmid-encoded fimbrial preparation were added and incubated for 30 min at room temperature. After washing with PBS plus 0.05%(v/v) Tween 20 (PBS/T), the plate was incubated for 20 min at room temperature with PBS/T containing 0.25% (w/v) BSA. Then, the plate was incubated for 1 h at room temperature with anti-PefA serum (1:500). After washing, the plate was incubated another hour at room temperature with goat anti-rabbit alkaline phosphatase conjugate (1:1000). The reaction was developed with Sigma 4-nitrophenyl phosphate disodium salt hexahydrate. The resulting color reaction was read at 415 nm with an ELISA microplate reader (Bio-Rad Model 680).

Competition assays for determining binding specificity were performed by coating streptavidin microplates (R&D Systems) with 2 µg/liter Lex-PAA-biotin (GlycoTech) in a 100-ml volume as described above. Wells were incubated with 2 µg/liter of a preparation of plasmid-encoded fimbriae in the presence of increasing concentrations of Lex-PAA or D-mannose-PAA (GlycoTech) in a 100-µl volume for 30 min at room temperature. Binding of plasmid-encoded fimbriae to wells was performed with anti-PefA serum and goat anti-rabbit alkaline phosphatase conjugate as described above.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Expression and Purification of Plasmid-encoded Fimbriae—An E. coli {Delta}fim mutant (ORN172) carrying a cosmid (pFB11) containing the S. Typhimurium pef operon was used to express plasmid-encoded fimbriae (15). Expression of plasmid-encoded fimbriae by ORN172(pFB11) was confirmed using flow cytometry. Bacteria were labeled with propidium iodide for detection of DNA and with rabbit anti-PefA serum and goat anti-rabbit IgG-FITC conjugate for detection of plasmid-encoded fimbriae. After gating for bacteria (i.e. for propidium iodide-positive counts) (Fig. 1A), the gate for detection of PefA was set using E. coli strain ORN172 as a negative control. Cells of E. coli strain ORN172(pFB11) were considered positive for expressing plasmid-encoded fimbriae when their FITC fluorescence intensity exceeded that of all but a small fraction (less than 1%) of the control population of the non-fimbriated parent (ORN172). Using this gate, the expression of plasmid-encoded fimbriae was detected on the surface of 29% of cells in the ORN172(pFB11) culture (Fig. 1B). Expression of plasmid-encoded fimbriae is regulated by phase variation (14), which provides a plausible explanation as to why expression of PefA was only detected in a fraction of cells in the population. A fraction of cells in cultures of ORN172(pFB11) carried thin flexible fibrillae on their surface (Fig. 1C), which were not detected in cultures of the parent strain ORN172 (data not shown). The thin flexible fibrillae expressed by ORN172(pFB11) could be labeled with rabbit anti-PefA serum and goat anti-rabbit 10-nm gold conjugate (Fig. 1D), but no labeling was observed with the ORN172-negative control (Fig. 1E). Furthermore, no labeling was detected in a control experiment detecting surface expression of PefD, a periplasmic chaperone involved in fimbrial assembly, in strain ORN172(pFB11) using rabbit anti-PefD serum and goat anti-rabbit 10 nm gold conjugate (Fig. 1F). Collectively, these data suggested that the thin flexible fibrillae detected on the surface of strain ORN172(pFB11) were plasmid-encoded fimbriae of S. Typhimurium.


Figure 1
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  		FIGURE 1.
Expression of S. Typhimurium plasmid-encoded fimbriae by E. coli strain ORN172(pFB11). A and B, expression of plasmid-encoded fimbriae by strain ORN172(pFB11) was detected by flow cytometry using the DNA stain propidium iodide for gating bacteria (A), followed by detection of PefA expression using rabbit anti-PefA serum and goat anti-rabbit IgG-FITC conjugate (B). A non-fimbriate E. coli strain (ORN172) is shown as a negative control. C–F, electron microscopic examination of E. coli strain ORN172(pFB11) expressing plasmid-encoded fimbriae. Fimbriae were visualized in ORN172(pFB11) by negative staining (C) or by immune-electron microscopy with anti-PefA serum (D) or anti-PefD serum (F). A negative control (ORN172) for immune-electron microscopy with anti-PefA serum is shown in E. The binding of antiserum was detected using goat anti-rabbit 10-nm gold particles.

 
Fimbriae were removed from the surface of strain ORN172(pFB11) by mechanical shearing and concentrated by ammonium sulfate precipitation. Inspection of the fimbrial preparation by electron microscopy revealed the presence of fimbrial filaments (Fig. 2A). The presence of the PefA fimbrial subunit in the fimbrial preparation was demonstrated by Western blot with anti-PefA serum (Fig. 2B). Analysis of proteins present in the fimbrial preparation by SDS-PAGE revealed the presence of two major bands with apparent molecular masses of 17 and 10 kDa (Fig. 2C). N-terminal sequencing of the 17-kDa band yielded the sequence ANEVTF, which corresponded to residues 22–27 in the PefA primary structure (Fig. 2D). Analysis of the PefA primary structure using the SignalP software (20) predicted the presence of a 21-amino acid signal peptide, whose cleavage would give rise to a mature protein with a molecular mass of 15.3 kDa. N-terminal sequencing confirmed cleavage of the predicted signal peptide and identified the 17-kDa band as the mature form of PefA. The 10-kDa protein band detected in the fimbrial preparation (Fig. 2C) contained the N-terminal sequence PNAQGC, which corresponded to residues 75–80 in the PefA primary structure (Fig. 2D). A truncated fragment of PefA containing amino acids 75–172 has a predicted molecular mass of 9.8 kDa, which corresponded well to the apparent molecular mass of 10 kDa detected by SDS-PAGE. Thus, N-terminal sequencing provided evidence that the 10-kDa protein band represented a truncated form of PefA. Interestingly, the 10-kDa band was not detected by Western blot analysis with anti-PefA serum (Fig. 2B). Collectively, our data suggested that preparations of plasmid-encoded fimbriae were composed predominantly of a single protein, PefA. These results identified PefA as the major fimbrial subunit of plasmid-encoded fimbriae. Like other members of the {kappa}-Fimbriae, the pef operon does not encode a tip adhesin (2).


Figure 2
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  		FIGURE 2.
Characterization of preparations of plasmid-encoded fimbriae. A, fimbrial filaments visualized by electron microscopy. B, Western blot of purified plasmid-encoded fimbriae using anti-PefA serum (right lane). Molecular masses of standard proteins are indicated on the left. C, analysis of purified plasmid-encoded fimbriae (right lane) by SDS-PAGE and Coomassie Blue staining. The left lane contains standard proteins (10, 15, 20, 25, 37, 50, 75, 100, 150, and 200 kDa). D, position of amino acid sequences (bold font) determined by N-terminal sequencing of the indicated protein bands (arrows) in the PefA primary structure are shown.

 
Screening for Carbohydrate Ligands of Plasmid-encoded Fimbriae using Glycanarrays—The preparation of plasmid-encoded fimbriae was allowed to bind to streptavidin-coated 384-well plates in which triplicate wells had each been coated with one of 99 different biotinylated carbohydrate ligands. Carbohydrates present on the glycanarray are shown in Table 1. The binding of plasmid-encoded fimbriae to wells was detected using rabbit anti-PefA serum and goat anti-rabbit IgG-Alexa488 conjugate. The background signal was determined in wells treated with primary (rabbit anti-PefA) and secondary antibody (goat anti-rabbit IgG-Alexa488), but not with plasmid-encoded fimbriae (control wells). For each biotinylated carbohydrate ligand, the average signal from three wells was determined and divided by the average background noise detected in three control wells (average S/N ratio). By analogy to procedures commonly used during analysis of DNA microarrays, we used a 2-fold increase in the average S/N ratio as an arbitrary cutoff for scoring binding to a biotinylated carbohydrate ligand as positive.


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  		TABLE 1
Carbohydrates present on the glycanarray

 
Glycanarray analysis revealed that binding of plasmid-encoded fimbriae to wells scored positive for only a single biotinylated carbohydrate ligand, Galβ1–4(Fuc{alpha}1–3)GlcNAc-O(CH2)2NH(O)(CH2)5NHC(O)(CH2)5NHC(O)-Biotin (average S/N ratio = 2.41) (Fig. 3). These data suggested binding to the Galβ1–4(Fuc{alpha}1–3)GlcNAc moiety, also known as the Lewis X (Lex) blood group antigen, because various carbohydrates present on the glycanarray were conjugated to biotin via the same linker as Lex (-O(CH2)2NH(O)(CH2)5NHC(O)-(CH2)5NHC(O)-). Although binding of plasmid-encoded fimbriae to the remaining sugars scored below the cutoff value, it should be mentioned that the biotinylated carbohydrate ligand producing the greatest average S/N ratio was tri-Lewis X-biotin (average S/N ratio = 1.65) (Fig. 3). Results from the glycanarray experiments thus suggested that Lex is a carbohydrate ligand specifically bound by plasmid-encoded fimbriae.

Confirmation of Glycanarray Results—We next wanted to confirm data from our glycomics screen by performing more detailed binding studies for those interactions that scored positive in the glycanarray. Increasing concentrations of plasmid-encoded fimbriae were added to 96-well plates coated with Lex-PAA-biotin, and binding was detected with anti-PefA serum and goat anti-rabbit alkaline phosphatase conjugate (Fig. 4A). The binding of plasmid-encoded fimbriae to Lex-PAA-biotin-coated wells was concentration-dependent, and saturation was reached with a fimbrial preparation containing 2 µg of protein per liter. Half-maximal binding was observed at concentrations between 0.1 and 0.01 µg/liter of plasmid-encoded fimbriae. No binding of plasmid-encoded fimbriae to wells coated with {alpha}-D-mannose-PAA-biotin or HOCH2(HOCH)4CH2NH-PAA-biotin was observed. These data further supported the idea that plasmid-encoded fimbriae specifically bind the carbohydrate ligand Lex, thus confirming results from the glycanarray analysis.

To determine the specificity of Lex binding by plasmid-encoded fimbriae, we tested the ability of Lex-PAA to inhibit binding of plasmid-encoded fimbriae to Lex-PAA-biotin-coated wells in a solid phase binding assay (Fig. 4B). Plasmid-encoded fimbriae (2 µg/liter) were incubated in wells coated with Lex-PAA-biotin in the presence of increasing concentrations of Lex-PAA. Soluble Lex-PAA blocked binding of plasmid-encoded fimbriae to Lex-PAA-biotin-coated wells in a concentration-dependent fashion. Inhibition was observed at Lex-PAA concentrations of >2 µg/liter (which corresponds to concentrations of Lex-polyacrylamide repeat units of >1.8 nM). In contrast, no inhibition of binding was observed when plasmid-encoded fimbriae (2 µg/liter) were incubated in wells coated with Lex-PAA-biotin in the presence of increasing concentrations of {alpha}-D-mannose-PAA. Collectively, solid-based binding studies supported the idea that plasmid-encoded fimbriae specifically bind the Lex blood group antigen, thereby validating results from our glycanarray analysis. These data demonstrate that glycanarrays can be an effective tool in determining the carbohydrate binding specificity of intact bacterial fimbrial filaments.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The human blood group antigens are defined by the presence of glycosphingolipids on the surface of erythrocytes, which carry characteristic terminal ends in their saccharide chains. In addition to their presence on erythrocytes, blood group antigens are abundant in epithelia of skin and mucosal surfaces (histo-blood group antigens), where they are present at the terminal ends of saccharide chains of surface-localized glycoproteins or glycosphingolipids (21). Thus the heterogeneity of blood group antigens within the human population reflects the heterogeneity of glycans that cover the surface of the intestinal tract. Pathogenic microbes colonizing the intestinal tract can bind to blood group antigens (22), and it has been speculated that their role as ligands for microbial adhesins represents one of the driving forces for evolving the structural heterogeneity found in the glycans that cover the surface of the digestive tract. In other words, the human blood group polymorphism may have developed in part through the selective pressures exerted by pathogenic microorganisms (23). At the same time, the heterogeneity of glycans found in a particular host species may have selected for the presence of a diverse array of different adhesins in microbes colonizing its intestinal tract. For example, the S. Typhimurium genome contains 12 operons encoding chaperone/usher-type fimbriae (11, 24) and it is tempting to speculate that the presence of this large repertoire of adhesins may in part be explained by the necessity for the pathogen to deal with the heterogeneity of carbohydrate receptors encountered either during its transmission between different individuals within a host species and/or during its transmission between different host species.


Figure 3
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  		FIGURE 3.
The binding of purified plasmid-encoded fimbriae to glycanarrays containing wells coated with different biotinylated carbohydrates (x-axes). A dashed line indicates the 2-fold increase in the average S/N ratio, which was used as an arbitrary cutoff for scoring binding to biotinylated carbohydrate. The identities of carbohydrates producing the greatest average S/N ratio are indicated.

 
The above considerations suggest that some of the 12 chaperone/usher-type fimbrial operons present in the S. Typhimurium genome may encode adhesins that bind human blood group antigens. Our finding that plasmid-encoded fimbriae bind the Lex blood group antigen represents the first evidence supporting this concept for the human pathogen S. Typhimurium. The Lex antigen is defined by the presence of a terminal Galβ1–4(Fuc{alpha}1–3)GlcNAc-R moiety on saccharide chains of glycoproteins or glycosphingolipids. In the human intestine, the Lex histo-blood group antigen is expressed mainly by crypt epithelial cells (21). Studies in bovine-ligated ileal loops show that S. Typhimurium initiates infection by invading epithelial cells at the tips of absorptive villi and in the follicle-associated epithelium of Peyer's patches (25, 26). However, these surfaces rapidly become unavailable, because the ensuing inflammatory reaction leads to necrosis of the uppermost mucosa, while the crypt epithelium remains intact (27). Abundant crypt abscesses are commonly found in S. Typhimurium patients (28, 29), thus raising the possibility that the pathogen may bind to human crypt epithelium at later stages of infection. Our finding that S. Typhimurium possesses an adhesin that binds a crypt-specific histo-blood group antigen may thus be relevant to the pathogenesis of human infections.


Figure 4
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  		FIGURE 4.
Binding of purified plasmid-encoded fimbriae to Lex. A, ELISA plates were coated with equal amounts of biotinylated Lex (Lex-PAA-biotin, closed circles) or control carbohydrates (mannose-PAA-biotin, open circles, or HOCH2(HOCH)4CH2NH-PAA-biotin, open squares). The binding of increasing amounts of purified plasmid-encoded fimbriae to wells was detected with rabbit anti-PefA serum and goat anti-rabbit alkaline phosphatase conjugate. Data are shown as averages ± S.E. from three independent experiments. B, binding of purified plasmid-encoded fimbriae (100 µl/well of a preparation containing 2 µg protein/liter) to Lex-PAA-biotin-coated wells in the presence of increasing concentrations of the inhibitors Lex-PAA or D-mannose-PAA. Data are shown as averages ± S.E. from three independent experiments.

 

   FOOTNOTES
 
* This work was supported in part by Public Health Service Grants AI040124, AI044170, and AI065534. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Medical Microbiology and Immunology, School of Medicine, University of California at Davis, One Shields Ave., Davis, CA 95616-8645. Fax: 530-754-7240; E-mail: ajbaumler{at}ucdavis.edu.

2 The abbreviations used are: S. Typhimurium, Salmonella enterica serotype Typhimurium; BSA, bovine serum albumin; FITC, fluorescein isothiocyanate; Lex, Galβ1–4(Fuc{alpha}1–3)GlcNAc-R; PAA, polyacrylamide; CAPS, 3-(cyclohexylamino)propanesulfonic acid; PBS, phosphate-buffered saline; S/N, signal to noise ratio. Back


   ACKNOWLEDGMENTS
 
We thank Manuela Raffatellu for help with immunogold labeling and Grete Adamson and Robert Droleskey for help with the electron microscopy. The authors would like to acknowledge Core H of The Consortium for Functional Glycomics funded by the NIGMS GM62116 and Richard Alvarez, Director, for the glycanarray analysis.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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