Flagellin and GroEL mediates in vitro binding of an atypical enteropathogenic Escherichia coli to cellular fibronectin
- Claudia T. P. Moraes†1,
- Juliana M. Polatto†1,
- Sarita S. Rossato1,
- Mariana Izquierdo2,
- Danielle D. Munhoz1,
- Fernando H. Martins1,
- Daniel C. Pimenta3,
- Mauricio J. Farfan2,
- Waldir P. Elias1,
- Ângela S. Barbosa1 and
- Roxane M. F. Piazza1Email author
© Moraes et al. 2015
Received: 2 June 2015
Accepted: 3 December 2015
Published: 18 December 2015
Enteropathogenic Escherichia coli (EPEC) is distinguished mainly by the presence of EPEC adherence factor plasmid (pEAF) in typical EPEC (tEPEC) and its absence in atypical EPEC (aEPEC). The initial adherence to the intestinal mucosa is complex and mediated by adhesins other than bundle-forming pilus, which is not produced by aEPEC. Extracellular matrix (ECM) proteins of eukaryotic cells are commonly recognized by bacterial adhesins. Therefore, binding to ECM proteins may facilitate colonization, invasion and/or signaling by intestinal pathogens. Previous studies from our group demonstrated that aEPEC O26:H11 (strain BA2103) showed high binding activity to fibronectin, not shared by its counterpart, aEPEC O26:HNM.
In the present study, using mass spectrometry after fibronectin-associated immunoprecipitation, two proteins, flagellin (50 kDa) and GroEL (52 kDa), were identified and BA2103 binding ability to fibronectin was inhibited in the presence of anti-H11 and anti-GroEL sera, but not by either naïve rabbit or other unrelated sera. It was also observed that the presence of purified flagellin inhibits adhesion of BA2103 to cellular fibronectin in a dose-dependent manner. Additionally, BA2103 GroEL is similar to the same protein of uropathogenic E. coli.
Our results suggest that flagellin may play a role in the in vitro interaction of BA2103 with cellular fibronectin, and GroEL can be an accessory protein in this process.
Adherence to intestinal cells is a critical step in enteropathogenic Escherichia coli (EPEC) pathogenesis. EPEC expresses intimin adhesin, an adherence factor chromosomally encoded by the eae gene , which is involved in the receptors recognition, located at the surface of target cells (translocated intimin receptor – Tir, β1-integrin and nucleolin) [2, 3]. The intimin-Tir interaction plays a role in attaching and effacement lesion followed by intestinal colonization. Moreover, EPEC is distinguished by the presence of EPEC adherence factor plasmid (pEAF) in typical EPEC (tEPEC) and its absence in atypical EPEC (aEPEC) . Also, lack of bundle-forming pilus (BFP) production [5, 6], presence of hemolysins  and autotransporter proteins [8, 9] existing in other diarrheagenic E. coli pathotypes characterize aEPEC [10, 11]. In tEPEC, the role of BFP either in initial contact or in bacteria-bacteria interaction is well established [12, 13]. However, in aEPEC this adhesion has been attributed to EspA and various accessory adhesins described in other pathogenic E. coli strains .
The ability to adhere to extracellular matrix (ECM) proteins has been shown to be essential for the virulence of several pathogens . The ECM proteins comprise a diverse group that function as a barrier, support for epithelial cells, and are responsible for development, growth, and maintenance of mammalian tissues . The composition of ECM differs in various organs, but fibronectin, collagen types I to XV, and laminin are common constituents . ECM proteins are commonly recognized by bacterial adhesins and have been shown to act as substrates for bacterial adherence to eukaryotic cells [15, 18–22]. Enteric bacterial pathogens can interact with ECM either during inflammation or in the tight junctions opening . Therefore, binding to ECM proteins may facilitate colonization, invasion, and/or signaling by intestinal pathogens . Fibronectin is an ECM molecule targeted by several pathogens and is formed by dimers covalently linked by a pair of disulfide bonds near their carboxyl termini . Fibronectin is responsible for connecting the collagen scaffold and other ECM components , and was the first eukaryotic cell receptor described for bacteria .
Recently, some conserved proteins, such as outer membrane protein A (OmpA), flagellin (FliC) and E. coli GroEL have been described as involved in adhesion, colonization, invasion and dissemination or as major antigens in many important pathogens [25–31]. FliC, the subunit of flagellum structure, contains highly conserved N- and C-termini, while its central region is significantly variable and provides antigenic differences . FliC is involved in motility and pathogenesis [33–36], and also can interact with cell surface polypeptide receptors on monocytes and activate Toll-like receptors 5 (TLR-5) . GroEL is a multitask protein, which function as a prototypical and indispensable molecular chaperone in stress survival. In addition, this protein presents moonlighting activities acting as a cell surface receptor for various pathogens ligands. Between 250–300 E. coli proteins bind to GroEL, 85 of them are obligate client to GroEL .
Previous results from our group demonstrated that a subset of atypical EPEC presented ability to bind to immobilized ECM proteins. Among them, the O26:H11 (BA2103) strain consistently presented the highest binding ability to cellular fibronectin . Taking these results in consideration, we investigated in the current study putative candidates of this particular strain that might mediate binding to cellular fibronectin. Our results indicate that flagellin may play a role in the in vitro interaction with this ECM component, and GroEL can be an accessory protein in this process.
Atypical EPEC (aEPEC) strains BA2103 (serotype O26:H11) and 2271-1/85 (serotype O26:HNM, where NM indicates nonmotile) were previously characterized by the eae+/EAF-/bfpA- genetic profile, lack of production of BFP and production of the localized adherence-like (LAL) pattern on HEp-2 cells [5, 6, 39]. Molecular typing of fliC defined the H type of 2271-1/85 strain as H11 . Binding ability to ECM of supernatant proteins from BA2103 was described elsewhere . A non-pathogenic K12 E. coli DH5α was included as a control strain . Also, twenty O26:H11 aEPEC strains were employed in the fibronectin binding screening [5, 39]. This study does not involve humans, human data or animals, since all strains employed herein belong to the Bacteriology Laboratory bacterial collection of Butantan Institute, São Paulo, SP, Brazil.
Binding of E. coli strains to fibronectin
In order to confirm the binding ability of BA2103 strain to fibronectin and compare it to 2271-1/85 and DH5α strains, 40 μL of bacteria culture growth (1.5 × 108) were incubated in ELISA microplates previously coated with 1 μg of cellular fibronectin, or 1 μg of bovine serum albumin (BSA) (Sigma Chemical Co.) at 37 °C for 4 h . Also, 40 μL of serially diluted bacterial suspensions (from 108 to 105) were tested. An additional assay was done preincubating 107 bacterial cells with increasing concentrations of cellular fibronectin (from 0.2 to 25.6 μg) followed by incubation in ELISA microplates previously coated with 1 μg of cellular fibronectin. Quantification of adhered bacteria was determined by removing the bacteria adhered to fibronectin and BSA with PBS containing 0.05 % Triton X-100. Serial dilutions were plated onto Luria Bertani (LB) agar plates and the number of bacteria was determined by counting colony-forming units (CFU) . CFU values were obtained from triplicates of four independent experiments.
Additionally, the binding ability to cellular fibronectin of 20 strains belonging to the O26:H11 serotype was accessed employing the crystal violet staining measurement as described elsewhere . Experiments were performed in duplicate of two independent experiments and the average values were calculated.
Supernatant proteins were obtained by growing BA2103 in 50 mL of TSB at 37 °C for 18 h (150 rpm), followed by centrifugation at 10,000 g for 15 min. This supernatant was filtered through a 0.22 μm membrane and protein concentration was determined by a Bradford assay. Protein identification was achieved by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF) analyses. Prior to MALDI-TOF analyses, 100 μg of supernatant proteins from BA2103 were incubated with 25 μg/mL of fibronectin at 25 °C for 90 min. Fibronectin-associated proteins were obtained by immunoprecipitation analysis using A/G agarose columns (Pierce) conjugated with anti-fibronectin antibodies (Sigma). Proteins recovered from the column were visualized by silver-stained SDS-PAGE, and protein bands were excised from SDS gel for MALDI-TOF analyses (Mass Spectrometry Core Laboratory, University of Texas Medical Branch).
Sequence alignments of flagellin and GroEL from Escherichia coli O26:H11 strains
Amino acid sequences of flagellin (gi|260855903) and GroEL (gi|18028158) obtained by MALDI-TOF analyses were compared to other related sequences available in GenBank database. All sequence alignments and analyses were performed with Basic Local Alignment Search Tool (BLAST) program (http://blast.ncbi.nlm.nih.gov/BlastAlign.cgi), MUSCLE  and BioEdit Sequence Alignment Editor vs 126.96.36.199.
The fliC gene of BA2103 was amplified by PCR and sequenced using primers described elsewhere  in order to investigate minor nucleotide alterations. Three additional internal primers (sequence 1 fliC (R): GCCTGACCTGCTGCG; sequence 2 fliC (R): CACTGACTTACCATC; sequence 3 fliC (F): CATGTCTCGTGCG) were designed to obtain full-length fliC sequence.
H11 flagellin purification
Flagellin produced by BA2103 (O26:H11) was extracted from 50 mL cultures as described elsewhere with some modifications . Briefly, bacterial cells were collected by centrifugation, suspended in 2 mL of phosphate-buffered saline (pH 7.4) and sheared for 2 min in bench mixer (Genie 2) at maximal speed. Cells were kept on ice bath for 1 min and the procedure was repeated three times. The cell suspension was then centrifuged at 10,000 X g for 10 min to remove the bacterial cells. Culture supernatants containing the sheared flagellin were precipitated with acetone. The resulting pellet was suspended in PBS and, finally, submitted to heat treatment (65 °C for 30 min) to depolymerize filaments into flagellin monomers. Protein concentration was determined using BCA assay (Pierce) and purity was checked by SDS-PAGE and immunoblotting using anti-H11 serum. Also an ELISA was employed in order to check anti-H11 reactivity to purified flagellin.
Detection of flagellin and GroEL in E. coli strains by immunofluorescence
The presence of flagellin and GroEL was evaluated in three bacterial strains (BA2103, 2271-1/85 and E. coli DH5α) by immunofluorescence employing anti-H11 and anti-GroEL sera. E. coli strains were cultivated at 37 °C in LB broth for 16–18 h, and a smear from the bacterial pellets was fixed with 4 % paraformaldehyde. Slide glasses with fixed bacteria were washed with phosphate-buffered saline (PBS 0.01 M, pH 7.4) and blocked with 10 % goat serum in PBS (PBS-GS) at 25 °C for 1 h, followed by incubation at 25 °C for 2 h with rabbit anti-GroEL (1:500) or rabbit anti-H11 (1:200) antibodies diluted in PBS-GS and goat anti-rabbit IgG conjugated with FITC diluted 1:250 or 1:100, respectively at 25 °C for 1 h. Between incubations the reactions were washed twice with PBS, and then slide glasses were mounted with glass coverslips. Bacteria were visualized on Axioskop fluorescence microscope (Zeiss, Germany), with a 400X magnification.
Binding inhibition to fibronectin
In order to validate the interaction of flagella and GroEL with cellular fibronectin, an in vitro inhibition assay was performed. BA2103 was cultivated in LB broth and 1.5 x 107 of bacteria suspension was preincubated with serial dilutions of rabbit anti-H11 or rabbit anti-GroEL (1:500 to 1:8,000) (Sigma-Aldrich) antibodies at 37 °C for 2 h, at 50 rpm in a platform shaker. Bacteria were then incubated at 37 °C for 4 h in ELISA microplate wells previously coated with 1 μg of cellular fibronectin. Serial dilutions (1:500 to 1:8,000) of naïve rabbit serum, rabbit anti-H9, anti-human vitronectin (Sigma-Aldrich), anti-human C1q and anti-human C3 were employed as negative controls of binding inhibition assay. An additional assay was done by incubating increasing concentrations of purified flagellin (7.8 to 500 μg) in ELISA microplate wells previously coated with 1 μg of cellular fibronectin, followed by an incubation of bacteria culture growth (1.5 x 107). Quantification of adhered bacteria was achieved by removing them from microplate wells with PBS containing 0.05 % Triton X-100. Serial dilutions were plated onto LB agar plates and CFU was determined as described elsewhere . CFU values were obtained from triplicates of four independent experiments in the presence or absence of antisera or in the presence or absence of purified flagellin.
The colony-forming units (CFU) and absorbance values were analyzed by Graph Prism® 5.01, using unpaired Student’s t-test. The differences were considered statistically significant when p ≤ 0.05.
Binding of E. coli strains to cellular fibronectin
Furthermore, twenty aEPEC O26:H11 strains were screened by their binding ability to cellular fibronectin by crystal violet staining. Employing this method we were able to arbitrary separate three groups according to their ability to bind to cellular fibronectin: high-binding strains, including BA2103 and BA2459, and low-binding strains, including non-motile 2271-1/85. The statistical analyses showed that the absorbance’s means between both groups were statistically significant (p < 0.0001, R2 0.9025) and also the variances between both groups were significant (p < 0.0001). The third group presenting intermediate binding ability, comprised by three strains (0791-1/85; 4851-3/86 and IC50) (Fig. 1d).
Identification of proteins involved in the recognition of cellular fibronectin by BA2103
Identification of fibronectin-associated proteins by MALDI-TOF analyses (**p value = 0,05 and Protein Significance Score = 71)
Average Mass (kDa)
flagellin (Escherichia coli O26:H11 str.11368)
GroEL (Escherichia coli)
lysine-tRNA ligase (Escherichia coli)
In silico analysis were performed in order to investigate which features of BA2103 flagellin or GroEL would confer their binding activities to cellular fibronectin. Multiple alignments of flagellin sequences from O26:H11 strains showed that these proteins are highly conserved, and no sequence unique to aEPEC BA2103 flagellin (FliCBA2103) was observed (Additional file 1: Figure S1). Considering that hypervariable domains of flagellin are involved in bacterial adhesion , we hypothesize that fibronectin-binding sites could be located on this region of the FliCBA2103. To test this, we performed an alignment by BLASTp of this region (residues 177 to 394) and fibronectin-binding domain of the Staphylococcus aureus FnBPA, a well-characterized fibronectin-binding protein . We found a 14-amino acid sequence (YDVGGDAYTVNVDS) showing 64 % similarity to two motifs of FnBPA, which could be a putative Fn-binding site within FliCBA2103 (Additional file 2: Figure S2).
GroEL Sequence Identity Matrix values generated after alignment by ClustalW among E. coli J96 O4:K6, Shigella sonnei 3233-85 and E. coli 11368 O26:H11
The role of flagellin and GroEL in the in vitro interaction of BA2103 to cellular fibronectin
A competitive inhibition assay between BA2103 and flagellin was done by incubating a fixed concentration of cellular fibronectin with increasing concentrations of purified flagellin (Additional file 4: Figure S4) followed by incubation with BA2103 bacterial cells. These results corroborate that flagellin may mediate in vitro binding of BA2103 to cellular fibronectin; since flagellin was able to block in a dose-dependent manner the subsequent interaction of BA2103 bacterial cells to cellular fibronectin (Fig. 4b).
Bacteria can produce proteins that interact with ECM components. These proteins are qualified as MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) . Thus, ECM proteins can play a bridging role between bacteria and host cells, contributing to activation of signal transduction pathways and control of later steps of pathogenesis . In addition, bacterial colonization and invasion is related to laminin and fibronectin adherence in epithelial cells .
The high adhesiveness ability of the O26:H11 strain to ECM components, mainly fibronectin and laminin , prompted us to characterize in vitro binding ability of BA2103 to fibronectin with the aim of characterizing putative bacterial proteins involved in this interaction. Our results confirmed the binding of BA2103 bacterial cells to fibronectin. This kind of interaction has been described for enteroaggregative and enterohemorrhagic E. coli fimbrial adhesins [20, 21]. In order to identify which proteins could be involved in this process, fibronectin-associated immunoprecipitation was performed.
Our initial thought was that hemolysins could be partially involved in these interactions, since high levels of binding were observed with bacterial supernatant from the strains harboring ehly, ehx and sheA genes . Therefore, we employed bacterial supernatants for fibronectin-immunoprecipitation in the present study. Surprisingly, no hemolysins were identified; on the other hand, after MALDI-TOF analyses four proteins were identified, three of them (lysine tRNA ligase, GroEL and H11 flagellin) present in E. coli. Despite the fact that, one cannot exclude the functions of tRNA ligase as a moonlighting protein as previously showed for glycil-tRNA . It is worth to mention that flagellin is abundant in E. coli and is certainly present both in the bacterial membrane and in the supernatant due to physical rupture during cultivation. The same can be applicable for GroEL, considered a moonlighting protein and present not only in the cytoplasm but also on the bacterial membrane and extracellularly . Thus, we decided to focus on the role of flagellin and GroEL.
It has been described that flagella not only contribute to bacterial motility but are also involved in biofilm formation, binding to host proteins, adherence, invasion, and colonization of host cells [25, 33, 36, 49–51]. Concerning EPEC, the detected binding of H6 flagella, purified from the prototype tEPEC E2348/69, to ECM components was in a dose-dependent manner to collagen and with less affinity to laminin and fibronectin, but not to vitronectin. Furthermore, the prototype tEPEC E2348/69, but not fliC mutant bound to ECM proteins . Also, FliC in aEPEC serotype O51:H40 plays a role in adhesion, invasion and IL-8 production .
In our study, both aEPEC strains belong to the serotype O26:H11 but only the motile BA2103 produce flagellin and interact with fibronectin, leading us to attribute a role for the H11 flagellin in mediating bacterial binding to fibronectin. Thus, the interaction between flagellin and fibronectin may contribute to aEPEC BA2103 efficiency in tissue colonization, indirectly demonstrated herein by a significant inhibition of the binding of aEPEC binding to fibronectin in presence of different dilutions of rabbit anti-H11 serum, but not to naïve rabbit serum. Furthermore, purified flagellin (H11) was able to block in a dose-dependent manner the subsequent interaction of BA2103 bacterial cells to cellular fibronectin.
In an attempt to characterize BA2103 flagellin features that confer its binding to cellular fibronectin, in silico analysis was performed. No fibronectin-binding domain has been described in flagellin so far. Despite that, our data demonstrate a specific interaction between FliCBA2103 and fibronectin, suggesting the presence of binding sites for this ECM molecule. As hypervariable domains of flagellin bear adhesive properties , we selected this specific region of FliCBA2103 to search for Fn-binding sites, identifying a 14-amino acid residue similar to two motifs of S. aureus FnBPA involved in interactions with fibronectin . The YDVGGDAYTVNVDS identified sequence could be a putative Fn-binding site, although specific experiments such as peptide array and site-directed mutagenesis need to be performed for mapping the Fn-binding sites within FliCBA2103.
GroEL was described as a conserved and immunodominant protein in Brucella and avian pathogenic E. coli [52, 53]. Besides its classical function as a chaperone, a role as adhesin- or invasin-mediating factor has been described for different pathogens such as Mollicutes, Salmonella Typhimurium, Mycobacterium bovis and E. coli [54–56]. Moreover, it has been shown that GroEL homolog of E. coli, the 60-kDa-heat shock protein, is not only intracellularly located but also associated with cell surface . Our immunofluorescence data indicate that GroEL was also found to be associated with cell surface in all three tested strains. In aEPEC BA2103 anti-GroEL serum significantly reduced its binding capacity to fibronectin, which further supports a function for this protein as one adhesin, since no reduction was observed when unrelated sera were employed. The folding activity of GroEL on substrate proteins is dependent of its ATP induced conformational change . There are three important domains described for this protein: apical, intermediate and equatorial 1 and 2 . The hydrophobic binding is found on apical domain  and the flexibility of this site of protein facilities the binding with substrates, GroES (co-chaperonin) and non-natives polypeptides . In our results, the MALD-TOF analysis identified a GroEL of BA2103 O26:H11 similar to GroEL to uropathogenic E. coli J96 . When compared to the majority of the H11 FliC E. coli subtype sequences in GenBank, a difference of 65 amino acids was verify between them at Equatorial domain 1. Although no changing has been observed at the conserved binding site, a significant size alteration can affect conformation of GroEL. As the function of GroEL is folding related, conformational changes could improve exposition of binding site and higher the binding to fibronectin, consequently. This interaction would facilitate the interaction between this ECM and others E. coli proteins . Nevertheless, structural analyses are necessary to elucidate this mechanism.
The role of flagellin and GroEL in the in vitro interaction of aEPEC BA2103 to cellular fibronectin herein demonstrate cannot be extended to all O26:H11 aEPEC strains. In fact, among the collection of twenty O26:H11 strains tested in this study, only BA2459 presented the same binding ability, as previously described . The non-motile strains were classified in the low-binding group and it is worth to mention that among these strains, three harbor the ehxA gene (O26 TR EPM; 3451-3/86 and 2012–1) .
A frequent attribute among aEPEC strains is the finding of specific phenotypes or putative virulence factors produced by subgroups, sometimes one strain belonging to the same serotype [9, 36, 61, 62]. Adhesion, invasion, and IL-8 production in cultured intestinal epithelial cells mediated by flagella , induction of mucus production , production of hemolysis and binding to ECM molecules , as well as toxin production  are examples of such characteristic of aEPEC.
Taken together our results suggest that flagellin may play a role in the in vitro interaction of BA2103 with cellular fibronectin, and GroEL can be an accessory protein, in this process as an additional strategy acquired by this strain. Besides, considering ours and previous data, it is clear that these data cannot be extended to all O26:H11 aEPEC strains. The significance of these proteins in host cell interaction of other aEPEC serotypes is under investigation by our group.
Availability of supporting data
All the supporting data are included as additional files.
This work was supported by grants 04/12136-5 and 06/05145-0 from São Paulo Research Foundation (FAPESP) and from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq – 470648/2008-2). SSR was recipient of FAPESP fellowship (06/58303-5). Work in the MJF laboratory was supported by grant 1120809 from Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT). The rabbit anti-H11 and rabbit anti-H9 sera were kindly provided by Prof. Tânia A.T. Gomes and Prof. Beatriz E. C. Guth (Federal University of São Paulo, Brazil).
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