Enteroaggregative Escherichia coli expresses a novel flagellin that causes IL-8 release from intestinal epithelial cells (original) (raw)
EAEC 042 induces IL-8 release from intestinal cell lines. We previously reported that a prototype EAEC isolate, 042, causes Caco-2 cells to release IL-8 after as little as 3 hours of exposure to either live bacteria or to cell-free culture filtrates. We also demonstrated the heat stability and protease sensitivity of this IL-8–releasing activity, and its lack of inhibition by polymyxin B (8). Because proinflammatory cytokines are regulated differently in various immortalized cell lines, we tested T84 human colonic carcinoma cells and CCD-18Co human colonic fibroblasts. As shown in Figure 1, EAEC 042, the prototype strain that is pathogenic in volunteers, caused significantly more IL-8 release than did nonpathogenic E. coli or culture broth alone in all three cell types. Interestingly, T84 and CCD-18Co cells both express measurable IL-8 at baseline, whereas Caco-2 cells do not. For this reason, we used Caco-2 cells as the primary assay cells for the IL-8–releasing protein.
IL-8 release from intestinal cell lines in response to EAEC. Five-day-old monolayers of Caco-2 (black bars), T84 (gray bars), or CCD-18Co (white bars) cells were exposed for 3 hours to live EAEC 042, filtrates of saturated overnight cultures of EAEC 042, nonpathogenic (control) E. coli (HB101, K12, or GDI 20), or culture broth alone. The separate Y-axes reflect the tenfold greater amount of IL-8 release by CCD-18Co fibroblasts compared with Caco-2 or T84 colonic carcinoma cells. Error bars represent SEM.
Purification and identification of the IL-8–releasing protein from EAEC 042. We purified the IL-8–releasing protein from culture supernatants of EAEC 042. The appearance of the sequential purification products on 9% SDS-PAGE gels is shown in Figure 2.
Gel (9% SDS-PAGE) of sequentially purified, IL-8–releasing protein fractions from EAEC 042 culture supernatants. Samples were concentrated by membrane ultrafiltration to a volume of 25 μL and run simultaneously, with the exception of lane 7, which was run in a separate gel. Lane 1: Culture supernatant from overnight growth of 042. Lane 2: DEAE-cellulose batch adsorption and elution with NaCl. Lane 3: Sixty percent ammonium sulfate precipitation. Lane 4: Gel permeation chromatography in the presence of 6 M guanidine-HCl. Lane 5: Pooled, active gel permeation fractions, dialyzed to remove guanidine. Lane 6: Strong anion exchange and subsequent hydrophobic interaction chromatography. Lane 7: Hydroxyapatite chromatography.
The SDS-PAGE band from the hydroxyapatite chromatography step was sequenced by tandem mass spectroscopy. Eighteen trypsin peptides were identified that matched predicted sequences from a presumed S. dysenteriae flagellin, _fliC_SD (National Center for Biotechnology Information accession 442483). Eleven of these peptides were identical to the corresponding sequences predicted from _fliC_SD; the remainder had single amino-acid substitutions. None of the identified peptides matched predicted peptide sequences of the flagellin from E. coli K-12 (fliC gene product, accession 120319).
Flagella from EAEC cause IL-8 release from Caco-2 cells. To verify that the identified flagellin was indeed the IL-8–releasing protein from 042, we prepared flagella from EAEC 042 (Figure 3). We cut the higher- and lower-molecular-weight bands from a gel, eluted the proteins into water by diffusion, and dialyzed them thoroughly to remove SDS. The smaller proteins did not cause IL-8 release, whereas the protein of about 65 kDa (henceforth referred to as FliC-EAEC) was extremely potent, with a specific activity of approximately 333 U/μg, corresponding to half-maximal activity at approximately 10–10 M. Moreover, when EAEC flagella were treated at pH 3.0 and subsequently neutralized, they were still active and maintained their mobility and staining characteristics on SDS-PAGE (not shown). This suggests that depolymerized flagella (consisting largely of the flagellin protein) are as active as intact flagella, and hence that the flagellin itself is responsible for IL-8 release. As with EAEC culture supernatants, addition of polymyxin B (10 μg/mL) to Caco-2 cells did not reduce the IL-8–releasing effect of EAEC flagellin, suggesting that LPS did not contribute to this effect.
Gel (9% SDS-PAGE) of EAEC flagellar isolation. Samples were concentrated by acetone precipitation before loading. Lane 1: Flagella sheared from EAEC 042 and pelleted by ultracentrifugation. Lane 2: The approximately 65-kDa band, cut and eluted from a previous gel, dialyzed to remove SDS, and reVrun in SDS-containing loading buffer. Lane 3: The approximately 35-kDa band similarly eluted, dialyzed, and rerun. Specific activity was defined as the amount of sample producing half-maximal IL-8 release from Caco-2 cells at 3 hours of exposure.
Several additional strains of bacteria were tested for the presence of IL-8–releasing flagella (Table 1). No detectable IL-8 release was induced in Caco-2 cells by the addition of 50 μL of flagellar solution (0.5–5.0 μg/well) from either GDI 20 (a nonpathogenic human isolate from Brazil), Vibrio cholerae, C1845 (a diffusely adherent E. coli), or BL21. In contrast, proinflammatory flagella were detected in eight of ten EAEC isolates from diverse sources, as well as enterohemorrhagic E. coli O157:H7 and enteropathogenic E. coli E2348. Interestingly, there was a dissociation between reactivity with H18 antiserum and IL-8 release, suggesting that the H-antigenic epitopes on FliC-EAEC may be separate from the regions of the molecule responsible for proinflammatory activity.
Aflagellar EAEC 042 does not cause IL-8 release. To determine if flagellar expression is necessary for 042 to cause IL-8 release, we used a 588-bp internal fragment of fliC to direct insertion of the R6K suicide vector pJP5603 into the EAEC 042 flagellin gene (_fliC_EAEC). This strategy produced a partial merodiploid, 042:_fliC_–, harboring the suicide vector (Figure 4a). The identity of 042:_fliC_– as a derivative of 042 was confirmed by antibiotic susceptibility pattern (resistant to chloramphenicol and tetracycline), by aggregative adherence to HEp-2 cells, by biofilm formation, by the presence of a plasmid with the same size and restriction patterns as the 042 AA plasmid, and by agglutination with O44 antiserum (Denka Seiken Co., Tokyo, Japan). Southern hybridization confirmed the site of insertion of the suicide plasmid (Figure 4b). Moreover, plasmid rescue liberated a novel plasmid of approximately 15 kb harboring the R6K origin and kanamycin resistance cassette. Sequencing of the portions of this plasmid flanking the pJP5603/fliC junction revealed the predicted duplicated copy of the 588-bp region of the fliC gene, confirming the partial merodiploid (not shown).
Construction of 042:_fliC_–. (a) Restriction maps of pJP5603 (3.1 kb) with the cloned 588-bp PCR fragment of _fliC_EAEC (gray bar), wild-type _fliC_EAEC (dashed line), and the map of the desired transconjugant. Restriction sites and locations are shown (Bam, _Bam_HI; Ssp, _Ssp_I; Stu, _Stu_I; Spe, _Spe_I; Xba, _Xba_I; Eco, _Eco_RI). (b) Southern hybridization of 042 and 042:_fliC_– using the 588-bp PCR fragment from the 042 genome as a probe. Lane 1: Probe, 1 ng. Lane 2: 042, _Spe_I/_Stu_I cut. Lane 3: 042, _Spe_I/_Ssp_I cut. Lane 4: 042:_fliC_–, _Spe_I/_Stu_I cut. Lane 5: 042:_fliC_–, _Spe_I/_Ssp_I cut. Kmr, kanamycin resistance cassette; TTA, stop codon; ori R6K, origin of replication.
IL-8–releasing activities of 042 and 042:_fliC_– were compared using both live bacteria and culture supernatants. As shown in Figure 5c, neither live 042:_fliC_– (n = 3) nor its culture supernatants (n = 5) caused detectable IL-8 release from Caco-2 cells at similar bacterial density to that from active cultures of 042 (as determined by dilutional colony counts of the bacterial cultures).
Motility and IL-8–releasing activity of 042 and 042:_fliC_–. (a) Growth at 72 hours on swarm plates without kanamycin. Bacteria picked from the periphery (swarming area) of the lower left plate had lost kanamycin resistance. (b) Electron microscopy with negative staining for flagella. Bar = 1 μm. (c) IL-8 release from Caco-2 cells incubated for 3 hours with culture supernatants of EAEC 042 grown in 1% tryptone (n = 5), 1% tryptone with 3% NaCl (n = 2), culture supernatants (sup) (n = 5), or live cultures (n = 3) of 042:_fliC_– grown in 1% tryptone. P < 0.05 by Kruskal-Wallis test.
042:_fliC_– demonstrated reduced motility compared with wild-type 042 when grown on swarm plates (Figure 5a). Other phenotypic characteristics of 042 (growth, adherence, biofilm formation) were not altered in 042:_fliC_–. Reversion to a motile state was able to occur spontaneously in the absence of kanamycin selection, presumably by a recombination event facilitated by the repeated portion of the fliC gene. Reversion to wild-type motility also restored IL-8–releasing activity (not shown).
Several strains were examined for flagella by electron microscopy. As shown in Figure 5b, 042 is highly flagellated, whereas 042:_fliC_– has no visible flagella (over many fields). In addition, 042 grown in LB with 3% NaCl had greatly reduced flagellar expression and did not release IL-8 from Caco-2 cells; adding 3% NaCl after overnight growth had no effect on IL-8 release.
EAEC flagellin expressed in BL21 causes IL-8 release. As described in Methods, the fliC gene was cloned into an expression vector with an NH2-terminal 6-His tag. Maximal expression occurred 2 hours after addition of IPTG to mid–log phase cultures, although some expression was present in unstimulated cultures, reflecting basal transcription at the T7 promoter. As shown in Figure 6, FliC-EAEC, but not the expressed control protein, immunoreacted with anti-flagellar antibody, whereas both proteins reacted with Ni2+-HRP. Crude lysates of BL21-pLysS:p_fliC_, but not BL21-pLysS:pCRT7/NT-E3, caused IL-8 release from Caco-2 cells. The IL-8–releasing specific activity of lysates was increased by purification over a Ni2+ column (not shown).
Expression and purification of FliC-EAEC. (a) BL21(DE3)pLysS transformed with p_fliC_ or pCRT7/NT-E3 (expression control) were harvested from mid–log phase cultures at the indicated times after addition of IPTG. Ten microliters of bacterial lysate was loaded per well for 9% SDS-PAGE, and transferred to PVDF membrane after gel was run. The membrane was probed with Ni2+-HRP to detect polyhistidine-containing proteins, stripped with 0.2 N NaOH, and reprobed with polyclonal anti-flagellar antiserum. A lane containing 2.5 μg of EAEC 042 flagella (A) was included in the gel as a control. For IL-8 release, purified flagella (72 ng) or bacterial lysate (1 μg) was added to 500-μL wells of Caco-2 cells in the same experiment, and supernatant was assayed after 3 hours of incubation. (b) BL21(DE3)pLysS:p_fliC_ was harvested 2 hours after addition of IPTG to mid–log phase culture. Lysates were purified by nickel affinity chromatography, visualized on 9% SDS-PAGE gels, and transferred to a PVDF membrane that was probed with Ni2+-HRP. Lane 1: EAEC 042 flagella, 1.4 μg. Lane 2: Crude lysate, 15 μg. Lane 3: Flow-through from nickel column, 12.5 μg. Lane 4: Eluate from column, 10.8 μg. For IL-8 release, 350 ng of flagella, 1 μg of bacterial lysate, and 1 μg of eluted flagellin were tested.
H18 antiserum inhibits IL-8 release by the 042 flagellin. As mentioned earlier, EAEC 042 has a serotype of O44:H18. We used commercially available antiserum to the H18 flagellum (Denka Seiken Co.) to attempt to adsorb FliC-EAEC and inhibit IL-8 release from Caco-2 cells. This antiserum immunoreacts by Western blot with flagellin purified from 042 or FliC-EAEC expressed in BL21 (not shown). Addition of 1–10 μL of H18 antiserum to Caco-2 cells inhibited IL-8 release from Caco-2 cells stimulated with 042 culture supernatants, purified flagella, or His-tagged FliC-EAEC to 51.2 ± 11.2% of control levels (mean ± SD, n = 4, P = 0.025). No inhibition of IL-8 release was seen with H7 antiserum or polyclonal anti-Salmonella a-z antiserum.
Sequencing of fliCEAEC and comparison to other bacterial flagella. The nucleotide sequence of fliC_EAEC was obtained from p_fliC (submitted to GenBank; accession AF 194946). The predicted amino acid sequence of this gene is shown in comparison to flagellins from S. dysenteriae, S. flexneri, and E. coli K12 in Figure 7. The four proteins are nearly identical over the first 150 and last 90 amino acids. FliC-EAEC and FliC-SD are completely identical over the first 200 residues, and more than 98% identical over the last 420, but only 42% identical from amino acid 220 to 270. A database search of this region of FliC-EAEC using the FASTA program from Genetics Computer Group (Madison, Wisconsin, USA) found no significant homology to any known protein.
Comparison of deduced amino acid sequences of flagellins from EAEC 042 (EAEC), S. dysenteriae (SD), S. flexneri (SF), and E. coli K12 (K12). Trypsin peptides identified by mass spectrometry from the initial purification product of EAEC 042 supernatants are underlined. Amino acids in the EAEC flagellin differing from those in S. dysenteriae are shown above the line. The arrows represent the locations of the forward and reverse primers used to generate the 588-bp fliC probe. FliC-EAEC accession AF 194946.