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1 Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia V8W 3P6, Canada
2 Department of Microbiology and Infectious Diseases, University of Calgary, Calgary, Alberta T2N 4N1, Canada
Correspondence
W. W. Kay
wkay{at}uvic.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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agfC mutant displayed an abundance of 20 nm fibres, in addition to native Tafi (5–7 nm), and had an increase in cell surface hydrophobicity. Purified 20 nm fibres were depolymerized under exceptionally stringent conditions to release what proved to be AgfA subunits. This revealed that the 20 nm fibres represented a different form of Tafi. The role of AgfC in Tafi assembly was investigated further using an antibody-capture assay of isogenic agf mutants. A soluble antibody-accessible form of AgfA was captured in wild-type (wt), agfB and agfF strains, in support of the extracellular nucleation–precipitation pathway of Tafi assembly, but not in agfC or agfE mutants. This indicates that AgfC and AgfE are important for AgfA extracellular assembly, facilitating the synthesis of Tafi.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Duguid et al., 1966; Low & Woude, 1996). Four general assembly pathways for different fimbriae have been proposed: chaperone–usher, alternate chaperone, general secretion and extracellular nucleation–precipitation (ENP) (Soto & Hultgren, 1999). In the first three pathways, fibre assembly occurs proximally: in the case of the chaperone-dependent pathways, subunits interact with a chaperone protein that caps interactive surfaces and prevents aggregation in the periplasm, or in the case of the general secretion pathway, subunits pass through a putative cell-envelope-spanning scaffold (Burrows, 2005). The ENP pathway deviates from these systems in that fibre growth occurs extracellularly (Hammar et al., 1996).
Thin aggregative fimbriae (Tafi) are the only fimbriae dependent on the ENP pathway. Tafi were identified in Salmonella Enteritidis by Collinson and co-workers, and the operon was termed agf (Collinson et al., 1991), whereas in Escherichia coli, the homologue discovered was named curli, and the operon termed csg (Collinson et al., 1992; Arnqvist et al., 1992). Tafi were later renamed curli using the csg nomenclature in Salmonella (Romling et al., 1998), but will be referred to by the original Salmonella nomenclature of agf here. Tafi are known as curli because, in the absence of extracellular polysaccharides (EPSs), their morphology appears curled; however, when expressed with EPS, their morphology appears as a tangled amorphous matrix (White et al., 2003). Together, Tafi and EPS form the extracellular matrix that results in a colony morphotype that appears red, dry and rough (rdar) on Congo red agar (Romling et al., 2000; White et al., 2003), which is highly conserved in most Salmonella and E. coli strains (Arnqvist et al., 1992; Collinson et al., 1991, 1992; Doran et al., 1993; Gerstel & Romling, 2003; White et al., 2006; Zogaj et al., 2003). Tafi are essential for the formation of the extracellular matrix (White et al., 2006), which is involved in multicellular aggregation (Romling et al., 2000), pellicle formation (Collinson et al., 1993), biofilm formation (Austin et al., 1998; Gerstel & Romling, 2003; Prigent-Combaret et al., 2000; Vidal et al., 1998), environmental persistence (White et al., 2006), and adherence to plant tissues (Barak et al., 2005). Tafi also have pathogenesis-related properties in that they accelerate amyloidosis in mice (Lundmark et al., 2005), bind fibronectin (Arnqvist et al., 1992; Collinson et al., 1993; Olsen et al., 2002), are proinflammatory (Bian et al., 2000, 2001; Persson et al., 2003; Tukel et al., 2005), and enhance adherence and invasion of eukaryotic cells (Dibb-Fuller et al., 1999; Kim & Kim, 2004; La Ragione et al., 2000; Sukupolvi et al., 1997).
The genes involved in Tafi production are organized into two adjacent divergently transcribed operons, agfBAC and agfDEFG (Collinson et al., 1996). Both operons are required for biosynthesis and assembly (Collinson et al., 1993). agfA and agfB encode the major and minor fimbrial subunits, respectively (Bian & Normark, 1997; White et al., 2001). agfD encodes a transcriptional regulator that positively regulates the expression of Tafi (Romling et al., 1998), cellulose (Romling et al., 2000), O-Ag capsule (Gibson et al., 2006) and serine hydroxymethyltransferase (Chirwa & Herrington, 2003), and negatively regulates factors that inhibit biofilm formation (Prigent-Combaret et al., 2001). The agfGFEC gene products are homologous to csgGFEC in E. coli, where csgG encodes an outer-membrane lipoprotein required for fimbrin secretion and stabilization (Chapman et al., 2002; Loferer et al., 1997), and assembles into an oligomeric complex that interacts with the products of csgEF (Robinson et al., 2006). CsgE and -F have uncharacterized roles in assembly, but CsgE at least has been called a chaperone (Chapman et al., 2002). The agfC (csgC) gene has not been previously characterized, and has been referred to as orfC due to the lack of evidence for transcription or a distinct phenotype accompanying inactivation (Collinson et al., 1996; Hammar et al., 1995).
In the ENP model of Tafi formation, surface-localized AgfB subunits nucleate the polymerization of AgfA subunits into insoluble surface fibres, which elongate from the free distal end (Hammar et al., 1996). This model relies on the demonstration of intercellular complementation between donor and recipient cells in LPS O-polysaccharide- and cellulose-deficient E. coli K-12 (Hammar et al., 1996). In Salmonella, intercellular complementation has been demonstrated, but only in LPS O-polysaccharide-deficient mutants (White et al., 2003). Therefore, the significance of intercellular complementation in Tafi-expressing strains with wild-type (wt) LPS and other EPSs has been questioned (White et al., 2003).
The purpose of this study was to investigate Tafi assembly, through examination of the minor assembly factors led by the discovery of an active agfC gene. The conservation of agfC with agfBA throughout Salmonella, E. coli and even in Shigella, where the operon has been inactivated (Sakellaris et al., 2000), suggests that agfC is functionally important. The predicted amino acid sequence of AgfC has no motifs, domains or homologies in common with any known protein other than that encoded by csgC. In this study, we have shown agfC to be co-transcribed with agfBA, and that AgfC and AgfE are involved in extracellular Tafi assembly, thereby confirming the ENP model.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) was routinely grown at 37 °C for 24 h on T agar (Collinson et al., 1991), unless stated otherwise. E. coli BL21(DE3) (Stratagene), harbouring pET44(c) (Novagen) carrying an inserted sequence of agfC fused to the plasmid-encoded C-terminal hexahistidine tag (agfC-his), was grown at 37 °C for 18 h with agitation in 1.2 % (w/v) tryptone, 2.4 % (w/v) bacto-yeast extract and 0.4 % (v/v) glycerol broth supplemented with 100 µg ampicillin ml–1. S. Enteritidis 3b agfC, harbouring pARA-A5 (Mayer, 1995) carrying agfC-his, was grown at 37 °C for 18 h with agitation in 1 % (w/v) tryptone broth (pH 7.2) for cell localization, or T agar for transmission electron microscopy (TEM) experiments. Media were supplemented with 100 µg ampicillin ml–1 and 0.5 % (w/v) L-arabinose. E. coli XL-1 Blue (Stratagene) harbouring pBCKS (Stratagene), pHAG (Collinson et al., 1996), pGEM-T Easy (Promega) or pHSG415 (Hashimoto-Gotoh et al., 1981) was grown at 28 or 37 °C for 24 or 48 h with agitation in Luria–Bertani (LB) broth supplemented with 50 µg chloramphenicol ml–1 or 100 µg ampicillin ml–1 and 40 mg X-Gal ml–1, in addition to 1 mM IPTG, when required.
RT-PCR.
Cellular RNA from agar-grown cells (OD600 1.0) was stabilized with RNA Protect Bacteria Reagent (Qiagen). Cells were incubated with 0.4 mg lysozyme ml–1 in the presence of Superase (Invitrogen) and lysed using a QIAshredder (Qiagen). RNA was extracted using an RNA extraction kit (Qiagen), and contaminating DNA was digested with DNase I (Qiagen), according to the method of Wang et al. (2002). RT-PCR was carried out using Sensiscript reverse transcriptase (Qiagen), as recommended by the manufacturer, using the primers listed in Table 1.
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), and fused to a C-terminal hexahistidine tag by cloning into HindIII- and XhoI-digested pET44(c). E. coli BL21(DE3) cells harbouring pET44(c) : : agfC-his were grown to OD600 0.6 before induction with 0.1 mM IPTG at 30 °C for 18 h with agitation (soluble protein), or 1 mM IPTG at 37 °C for 4 h with agitation (insoluble protein). Protein was recovered from cells and purified using 50 % (w/v) Ni-nitrilotriacetic acid slurry (Qiagen), as recommended by the manufacturer.
Production of polyclonal serum against AgfC-his.
Affinity-purified AgfC-his was further SDS gel-purified and used to immunize a New Zealand white rabbit. Subcutaneous and intramuscular injections of 200 µg AgfC-his prepared in Emulsigen adjuvant (MPV Laboratories) were performed three times at 2-week intervals, with a final boost of 100 µg AgfC-his in adjuvant. Three weeks following the final booster injection, serum was collected, and the antibody titre was determined by ELISA using affinity-purified recombinant AgfC-his antigen. The specificity for AgfC was confirmed by immunoblotting and ELISA using affinity-purified recombinant AgfC-his antigen (data not shown).
Cellular localization.
S. Enteritidis 3b agfC harbouring pARA : : agfC-his was grown to OD600 0.6, and gene expression was induced with 0.5 % L-arabinose at 37 °C for 3 h with agitation. Cells were subjected to osmotic shock (Sweet et al., 1979), and cellular fractions were separated using standard methods (Sambrook & Russell, 2001). Samples were analysed by SDS-PAGE and Western blotting using AgfC-his rabbit serum or isoleucine tRNA synthase control rabbit serum.
N-terminal sequencing of AgfC-his.
Proteins in periplasmic osmotic shock samples were precipitated in a 10 % (w/v) TCA/acetone mixture overnight at 4 °C, washed in ice-cold acetone, centrifuged (16 000 g, 10 min), and allowed to air dry at room temperature for 1 h. The sample was solubilized in 90 % (v/v) formic acid (FA), lyophilized, resuspended in SDS-PAGE sample buffer, resolved by SDS-PAGE, transferred to a PVDF membrane (Bio-Rad), and stained with Sypro Ruby protein blot stain (Molecular Probes). Approximately 100 ng AgfC was excised, and eight cycles of Edman degradation were performed.
Generation of S. Enteritidis mutants.
Overlap-extension PCR (Horton et al., 1989) was used to generate deletion constructs for agfC (72 bp deletion; primers 17-A, agfC-C, agfC-B and 17-D), agfE (72 bp deletion; primers AgfE/F-A, AgfE-B, AgfE-C and AgfE/F-D) and agfF (101 bp deletion; primers AgfE/F-A, AgfF-B, AgfF-C and AgfE/F-D) containing six-frame translational stop codons at the beginning of each gene. All primers are listed in Table 1. Gene products were either cloned directly into EcoRI- and HindIII-digested pHSG415, or subcloned from pGEM-T Easy (Promega). Recombinant plasmids were transformed into S. Enteritidis 3b or S. Enteritidis 3b bcsA (White et al., 2003), and used to replace the native alleles in the chromosome (White et al., 2007). PCR was used to screen for deletion strains. Genotypes in final strains were confirmed by sequencing DNA regions of interest that were PCR-amplified from the chromosome. S. Enteritidis 3b fliC : : Tn10dCm was generated by random transposition (Gibson et al., 2006).
Recombinant DNA techniques.
Purified plasmids were electroporated into S. Enteritidis 3b or E. coli strains using standard techniques (Gene Pulser Electroprotocol; Bio-Rad). Recombinant plasmids were purified using QIAprep spin kits (Qiagen). Restriction enzyme digestions (New England Biolabs) and ligation reactions (Gibco-BRL) were carried out as recommended by the manufacturers. DNA fragments were purified from agarose gels using Qiaquick Gel Extraction kits (Qiagen). PCRs were carried out using Taq DNA polymerase (Boehringer Mannheim) or Proofstart DNA Polymerase (Qiagen) in buffer supplied by the manufacturer. Thermocycling was carried out in a PTC-100TM Programmable Thermal Controller (MJ Research). All reverse-transcription and PCR primers were purchased from Alpha DNA (www.alphadna.com).
TEM.
Cells were resuspended in 10 mM Tris/HCl (pH 8.0), placed onto 0.3 % (w/v) Formvar-coated 200 mesh copper grids, and negatively stained with 2 % uranyl acetate (pH 7) for 15 s. Samples were visualized with a Hitachi H7600 transmission electron microscope under HC-zoom mode at 100 kV.
Purification of fimbriae from S. Enteritidis bcsAagfC.
Fimbriae from S. Enteritidis bcsAagfC and S. Enteritidis bcsA were purified as described by Collinson et al. (1991). Briefly, cells were resuspended in 10 mM Tris/HCl (pH 7.2), vortexed (3x1 min) at medium speed, and harvested by centrifugation (6000 g, 10 min). The supernatant samples were lyophilized, resuspended in 750 µl SDS-PAGE sample buffer, and boiled for 10 min before electrophoresis (5 h at 150 V). After electrophoresis, SDS-insoluble material that did not enter the stacking gel was recovered from the well, washed in distilled H2O (dH2O), resuspended in 90 % (v/v) FA, and lyophilized. This material was resuspended in 750 µl SDS-PAGE sample buffer and electrophoresed a second time. The FA-insoluble material that did not enter the stacking gel was recovered from the well, resuspended in 6 M HCl, microwaved for 5 s at high power (900 W), resuspended in SDS-PAGE sample buffer, and electrophoresed a third time.
Hydrophobicity assay.
The protocol was carried out according to the method of Rosenberg et al. (1980). Cells were washed, resuspended and adjusted to OD600 0.6 in 13.6 mM sodium triphosphate buffer (pH 7.0). Aliquots (4 ml) of cells were overlaid with 1 ml n-octane in acid-washed test tubes. The suspension was mixed vigorously for 1 min by vortexing. The immiscible phases were allowed to separate for 15 min, the aqueous phase was removed, and the A400 was measured. The percentage hydrophobicity was calculated by dividing the decrease in A400 of the aqueous phase by the initial A400.
Antibody capture of Tafi subunits in S. Enteritidis bcsA strains.
S. Enteritidis 3b bcsA strains were grown at 37 °C for 20 h with agitation in 1 % (w/v) tryptone broth (pH 7.2) supplemented with 30 % (v/v) polyclonal rabbit serum specific to AgfA (Collinson et al., 1991) or AgfB (White et al., 2001), with fetal calf serum as a non-specific negative control. For SDS-PAGE and immunoblotting, cells were adjusted to OD600 1 in binding buffer (20 mM Na2HPO4, 50 mM Tris/HCl, pH 7.2), centrifuged (6000 g, 10 min), resuspended in 250 µl 90 % (v/v) FA or dH2O, and lyophilized. The supernatant was incubated with 100 µl Protein A Sepharose 4 Fast Flow 50 % (w/v) slurry (Amersham Biosciences) at 4 °C for 1 h on a rotary shaker. Protein A beads were recovered by centrifugation (16 000 g, 10 min), and remaining supernatant proteins were precipitated by addition of acetone (Pohl, 1990). FA-treated samples were loaded directly onto SDS-PAGE columns, whereas all other samples were boiled for 5 min before loading. ELISA was performed according to the method of Engvall & Carlsson (1976) in high-binding, flat-bottomed, polystyrene 96 well plates (Costar). Cells were harvested (6000 g, 10 min), washed twice in 1 ml 100 mM glycine (pH 2.5), washed twice in 1 ml PBS (pH 7.4), adjusted to OD600 0.1 in each well, and dried at 90 °C for 4 h. AgfA or AgfB were detected using an AgfA-specific mAb ascites, or AgfB-specific polyclonal serum followed by goat anti-mouse or rabbit IgG–alkaline phosphatase conjugates (Cedarlane Laboratories).
SDS-PAGE, protein staining and immunoblotting.
Cells were resuspended in 1 ml 10 mM Tris/HCl (pH 8.0) and adjusted to OD600 0.1 (for protein staining) or OD600 1 (for immunoblotting), and 1 ml aliquots were vortexed (3x1 min) to shear off cell-surface material. The cells were harvested by centrifugation (6000 g, 10 min), resuspended in SDS-PAGE sample buffer, and boiled for 10 min before electrophoresis. Proteins in supernatant fractions were precipitated with acetone and resuspended in SDS-PAGE sample buffer. Tafi fimbrial protein samples were prepared as previously described (Collinson et al., (1991). SDS-PAGE was carried out according to the method of Laemmli (1970), with a 5 % (v/v) stacking gel and a 12 % (v/v) resolving gel. Proteins separated by SDS-PAGE were either protein-stained with Gelcode (Bio-Rad), or electrophoretically transferred to nitrocellulose using a Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad Laboratories), in buffer recommended by the manufacturer. Proteins were detected using immune serum specific to AgfA, AgfB, flagella (Salmonella H Antisera; Difco) or AgfC-his, followed by goat anti-rabbit immunoglobulin conjugated to IRDye800 (LI-COR Biosciences). Immunoreactive material was visualized using an Odyssey scanner (LI-COR Biosciences)
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The results indicated that agfC was co-transcribed with agfBA as a polycistronic agfBAC transcript, contrary to the results of previous attempts (Collinson et al., 1996; Hammar et al., 1995).
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agfC strain, AgfC-his was mostly localized to the periplasm (Fig. 2). N-terminal sequencing of the periplasmic form of AgfC-his revealed that it was cleaved between residues 8 and 9, generating a mature 100 aa protein. This was in contrast to the predicted cleavage site between residues 22 and 23 (SignalP 3.0, www.cbs.dtu.dk/services/SignalP).
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agfC mutant produces aberrant AgfA fimbriaeagfC strains were compared by TEM, fibres 20 nm in diameter were abundant on the cell surface of the agfC mutant (Fig. 3b), in comparison to the wt where fewer 20 nm fibres were observed (Fig. 3a). In contrast to normal Tafi (5–7 nm), these 20 nm fibres were not stained with an AgfA-specific mAb by immunogold-TEM (data not shown). To confirm that the increase in the number of 20 nm fibres was due to the agfC mutation, agfC was expressed in trans in the agfC strain; TEM of the complemented strain (Fig. 3c) showed it to be similar to wt, with infrequent occurrences of something that resembled the 20 nm fibres on the cell surface. Moreover, 20 nm fibres that had been sheared off cells were enriched in the supernatant from the agfC mutant, but not from the wt or agfC+agfC expressed in trans (data not shown).
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agfC mutant were compared by SDS-PAGE and immunoblotting (Fig. 4a). There were no discernible differences in the quantities of FliC flagellin detected. Additionally, comparison of SDS-PAGE protein profiles from whole-cell lysates of the wt and the agfC mutant did not reveal any differences between the strains (data not shown). The morphological appearance of these 20 nm fibres, as observed by TEM, was similar in both cellulose+ (agfC) and cellulose– (bcsAagfC) strains (data not shown), indicating that they did not consist of cellulose-containing complexes.
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agfC mutant, according to the previously described Tafi purification method (Collinson et al., 1991). After electrophoresis, the gel-impermeable SDS-insoluble material from agfC was recovered from the wells, and found to be highly enriched with large curved fibres (Fig. 3d). This was in contrast to that of the wt strain, which consisted primarily of Tafi fibres. When treated with 90 % FA, AgfA subunits were detected in both strains (Fig. 4b, +FA). However, significant amounts of FA-insoluble material remained in the agfC mutant. This material was recovered, washed again with 90 % FA, and solublilized in 6 M HCl by microwave treatment for 5 s. Subsequent SDS-PAGE and immunoblotting revealed a 16.5 kDa protein band and some higher molecular oligomers that were recognized by Tafi polyclonal antiserum (Fig. 4c, +HCl). No corresponding bands were detected in samples from the wt strain (Fig. 4c). Analysis of the 16.5 kDa SDS-PAGE band (Fig. 4c, +HCl) by MALDI-TOF MS yielded only peptides that matched the AgfA amino acid sequence (data not shown). Although the AgfA monomers reacted with AgfA-specific polyclonal antisera, they were not strongly recognized using an AgfA mAb that recognizes a conformational epitope (data not shown), suggesting that they were structurally altered. Unlike Tafi fibres, no AgfB subunits were detected in these large fibres by MALDI-TOF MS or Western blotting with AgfB-specific polyclonal serum (data not shown), which readily detects both AgfB and AgfB–AgfA dimers derived from Tafi (White et al., 2003). Consequently, we called these 20 nm fibres aberrant Tafi.
Changes in S. Enteritidis agfC cell-surface properties
To determine if any surface-related alterations occurred as a consequence of the proliferation of aberrant Tafi, the overall cell hydrophobicity of the agfC mutant was compared to that of wt, using the bacterial adherence to hydrocarbons (BATH) test (Rosenberg et al., 1980), which measures the relative hydrophobicity according to partitioning into n-octane. The mean±SEM generated from three independent experiments for the agfC mutant was 65.3±2.3 %, whereas the value for the wt was 46.2±4.3 %, representing an approximately 40 % increase for the agfC mutant.
AgfC and AgfE mediate Tafi assembly by the ENP pathway
Since aberrant Tafi were present in the agfC mutant, it suggested that the normal assembly process was altered, which implies that AgfC plays a role in correct assembly of native Tafi fibres. We therefore wanted to investigate Tafi assembly in more detail. Strains with isogenic deletions in agfE and agfF, two proposed Tafi assembly factors (Chapman et al., 2002), were generated to facilitate comparison with the agfC mutant. agfB and agfA mutants have been described previously (White et al., 2001). To determine whether subunits were accessible to specific antibodies, wt and mutant strains were grown in liquid phase in the presence of AgfA- or AgfB-specific immune serum. If the subunits are secreted or surface-accessible as soluble monomers, as proposed in the ENP pathway, we reasoned that specific antibodies would capture the subunits before Tafi polymerization occurred (Fig. 5a). After growth, antibody–subunit complexes were recovered from culture supernatants with Protein A–Sepharose using a pull-down assay, and analysed by immunoblotting. In addition, AgfA or AgfB subunits not captured by antibodies, but incorporated into polymerized Tafi at the cell surface, were measured by ELISA and quantified.
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, Cell Pellet+FA). In the agfB and agfA control strains, AgfA and AgfB, respectively, were detected as soluble proteins in the supernatants (Fig. 5, Supernantant–FA). When wt cells were grown in the presence of AgfA- or AgfB-specific serum, a proportion of soluble AgfA or AgfB was captured (Fig. 5, Beads–FA). This indicated that during normal Tafi assembly, AgfA and AgfB were in a form accessible to antibodies. The presence of AgfA-specific immune serum caused a 69 % reduction in the amount of polymerized Tafi on the wt cell surface (Fig. 5b), indicating that the antibodies were competing for polymerization-competent subunits. In contrast, the levels of polymerized Tafi on the cell surface of the agfC mutant did not change significantly, and soluble AgfA was not captured with AgfA-specific antibodies. This could be complemented back to the wt phenotype with agfC expressed in trans (data not shown). The agfE mutant was similar to the agfC mutant, in which basal levels of polymerized Tafi were not altered and soluble AgfA was not captured. This suggested that, in the absence of AgfC and AgfE, AgfA assembly was different and was not proceeding through accessible intermediates. The agfF mutant did not produce polymerized Tafi on the cell surface, and soluble AgfA was detected outside of the cell. These results were similar to those for the agfB control mutant.
The results of parallel experiments using AgfB-specific antibodies were in contrast to those found using AgfA-specific antibodies: soluble AgfB was captured in all five strains, with a corresponding decrease in the amount of polymerized AgfB detected by ELISA (Fig. 5c). These results indicate that AgfC and AgfE affect the antibody accessibility of AgfA but not of AgfB. These results reveal that AgfC and AgfE facilitate the extracellular assembly of AgfA.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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agfC mRNA was detected by RT-PCR using Sensiscript (Qiagen). Although this technique is not quantitative, we believe that agfC is transcribed at low levels, since previous transcription studies using less sensitive techniques did not detect agfC (csgC) transcripts (Collinson et al., 1996; Hammar et al., 1995). AgfC protein was also produced in minute amounts, as it was not detected in cell extracts. Similarly, other fimbrial systems have minor proteins that are present in minute quantities, making clear definitions hard to assign because of the difficulty of detection (Craig et al., 2003). Although this is artificial, mature AgfC was found primarily in the periplasm when overexpressed in trans. Therefore, we hypothesize that the periplasm is the normal cellular compartment for native AgfC.
Deletion of AgfC caused changes on the cell surface of S. Enteritidis 3b. The agfC mutant cells showed increased cell-surface hydrophobicity and produced 20 nm fibres. We hypothesize that the increase in hydrophobicity is the result of increased production of 20 nm fibres. Although the fibres are morphologically similar to flagella, the evidence that supports the hypothesis that they are not composed of flagellin subunits but rather of Tafi subunits is: (1) flagella monomer levels were the same in both the wt and the agfC mutant, despite an increase in the abundance of 20 nm fibres; (2) regular flagella fibres are soluble in SDS buffer and would have disappeared in the first step of the purification procedure; in contrast, the aberrant Tafi fibres were not soluble in SDS buffer; (3) once the fibres were solubilized, AgfA was detected by immunoblotting and MS, whereas FliC was not detected by either technique.
Aberrant Tafi did not depolymerize upon treatment with FA, but required the presence of HCl and heat treatment. After depolymerization, AgfB was not detected, either as protein fragments or immunologically, indicating that the aberrant Tafi were composed solely of AgfA. This is unlike normal Tafi, which are composed of both AgfA and AgfB at a ratio of approximately 20 : 1 (White et al., 2001). We therefore hypothesize that the AgfA subunits, as present in aberrant Tafi, have adopted an alternative conformation leading to polymerization of thicker filaments, and that this conformation has not conscripted AgfB subunits as in normal Tafi. Formation of this alternative conformation is somehow prevented by AgfC, perhaps by complexing (chaperoning) with pre-assembly AgfA, although we have no direct evidence to show this. Consequently, AgfC biases AgfA assembly toward normal Tafi structure. Fibres morphologically similar to those in the agfC mutant have been observed in E. coli, in which overexpression of CsgB (AgfB) fused to a maltose-binding protein results in the production of morphologically altered, highly ordered CsgA (AgfA) assemblages that, although uncharacterized, appear curved, loosely aggregated, 10–15 nm in diameter and do not incorporate CsgB (AgfB) (Bian & Normark, 1997). Additionally, others have observed that in the absence of CsgE (AgfE), curli (Tafi) fibres are morphologically altered (Chapman et al., 2002). This indicates that AgfA has the ability to form alternative tertiary, and hence quaternary structures, when normal assembly factors are disturbed.
The role of AgfC in maintaining the conformational integrity of AgfA prompted a further investigation into Tafi assembly factors. The ENP pathway for E. coli K-12 curli is based on growth complementation of E. coli K-12 CsgA donor (csgB– or csgF–) and acceptor (csgB+) strains, and Congo red detection of polymerizing curli (Bian & Normark, 1997). Since this type of analysis is not feasible with wt Salmonella spp. (White et al., 2003), we devised a quantitative analytical test for this system. When S. Enteritidis 3b was grown in the presence of AgfA- or AgfB-specific antiserum, soluble subunits were captured and Tafi polymerization was partially inhibited. From these results, it is evident that both AgfA and AgfB monomers are transiently soluble outside the cell before polymerization on the cell surface. This supports the unique ENP pathway for Salmonella Tafi, as originally proposed for E. coli curli (Hammar et al., 1996). In contrast, soluble AgfA subunits could not be captured in the agfC or agfE mutants, even though Tafi polymerization was still occurring. This corroborates previous studies in which a csgE (agfE) mutant was unable to donate CsgA (AgfA) subunits when cross-streaked against the CsgB+ recipient, indicating that, in the absence of CsgE, AgfA is no longer secreted, even though Tafi is still polymerized on the cell surface (Chapman et al., 2002). Together, these results reveal that Tafi assembly can still occur without proceeding through the extracellular-antibody-accessible pathway, demonstrating that extracellular Tafi assembly is not obligatory in these mutants. In the wt strain, the ENP pathway accounted for approximately two-thirds of Tafi polymerization when observed this way. One way to interpret these results is that a proportion of Tafi can assemble through an intracellular pathway in the wt strain, and in the absence of AgfC or AgfE, the extracellular pathway is lost. Alternatively, in the absence of AgfC or AgfE, the antibody is no longer able to recognize and capture conformationally altered AgfA subunits.
Unlike AgfA, AgfB could be captured by AgfB-specific antibody in the wt and the individual deletion strains before polymerization. Therefore, AgfC and AgfE specifically affect AgfA but not AgfB. Thus, the roles of AgfC and AgfE may involve maintaining an assembly-competent AgfA for extracellular polymerization into Tafi. Evidence indicates that the soluble form of AgfA (CsgA) is primarily 
-helical and is not folded into the parallel 
-sheet predicted for AgfA in Tafi (Collinson et al., 1999), but it will slowly assemble spontaneously when purified (Chapman et al., 2002). Presumably, the bacterial assembly apparatus (AgfBCEFG) provides the nucleation/assembly platform to prevent premature subunit polymerization while crossing the periplasm, facilitating folding, and accelerating the process. The results here suggest that AgfC and AgfE may be involved in facilitating Tafi assembly by influencing AgfA export and/or conformation, thus facilitating extracellular polymerization. This role is similar to that of chaperone proteins.
This study reports experimental observations of AgfC in Tafi assembly. Experimental observations have been made for CsgE (AgfE) and CsgF (AgfF) (Chapman et al., 2002), but due to the insoluble nature of these fimbriae and their unique assembly pathway, mechanisms involving the Tafi assembly machinery have still not been experimentally defined. AgfC, E and F are all small with no known homologies in the databases. It is at least known that CsgE (AgfE) and CsgF (AgfF) bind CsgG, forming an outer-membrane complex (Robinson et al., 2006).
The detection of the agfC transcript in S. Enteritidis 3b, and the finding that AgfC and AgfE direct AgfA assembly traffic may be instructive for further studies on Tafi (curli) assembly. These studies may also provide insight into other pathways of aggregative fibre assembly, such as in prion-related diseases.
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ACKNOWLEDGEMENTS |
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Edited by: D. L. Gally
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 25 August 2006;
revised 24 October 2006;
accepted 15 November 2006.
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20 nm diameter) fibres and small arrows indicate thin (5–7 nm) Tafi fibres. Bars, 100 nm. At least 10 images of each strain were generated from at least five independent experiments.
