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. 2010 Sep 9:10:236.
doi: 10.1186/1471-2180-10-236.

Suppression subtractive hybridization identifies an autotransporter adhesin gene of E. coli IMT5155 specifically associated with avian pathogenic Escherichia coli (APEC)

Jianjun Dai  1 , Shaohui Wang, Doreen Guerlebeck, Claudia Laturnus, Sebastian Guenther, Zhenyu Shi, Chengping Lu, Christa Ewers
Affiliations

Suppression subtractive hybridization identifies an autotransporter adhesin gene of E. coli IMT5155 specifically associated with avian pathogenic Escherichia coli (APEC)

Jianjun Dai et al. BMC Microbiol. .

Abstract

Background: Extraintestinal pathogenic E. coli (ExPEC) represent a phylogenetically diverse group of bacteria which are implicated in a large range of infections in humans and animals. Although subgroups of different ExPEC pathotypes, including uropathogenic, newborn meningitis causing, and avian pathogenic E. coli (APEC) share a number of virulence features, there still might be factors specifically contributing to the pathogenesis of a certain subset of strains or a distinct pathotype. Thus, we made use of suppression subtractive hybridization and compared APEC strain IMT5155 (O2:K1:H5; sequence type complex 95) with human uropathogenic E. coli strain CFT073 (O6:K2:H5; sequence type complex 73) to identify factors which may complete the currently existing model of APEC pathogenicity and further elucidate the position of this avian pathotype within the whole ExPEC group.

Results: Twenty-eight different genomic loci were identified, which are present in IMT5155 but not in CFT073. One of these loci contained a gene encoding a putative autotransporter adhesin. The open reading frame of the gene spans a 3,498 bp region leading to a putative 124-kDa adhesive protein. A specific antibody was raised against this protein and expression of the adhesin was shown under laboratory conditions. Adherence and adherence inhibition assays demonstrated a role for the corresponding protein in adhesion to DF-1 chicken fibroblasts. Sequence analyses revealed that the flanking regions of the chromosomally located gene contained sequences of mobile genetic elements, indicating a probable spread among different strains by horizontal gene transfer. In accordance with this hypothesis, the adhesin was found to be present not only in different phylogenetic groups of extraintestinal pathogenic but also of commensal E. coli strains, yielding a significant association with strains of avian origin.

Conclusions: We identified a chromosomally located autotransporter gene in a highly virulent APEC strain which confers increased adherence of a non-fimbriated E. coli K-12 strain to a chicken fibroblast cell line. Even though flanked by mobile genetic elements and three different genetic regions upstream of the gene, most probably indicating horizontal gene transfer events, the adhesin gene was significantly linked with strains of avian origin. Due to the nucleotide sequence similarity of 98% to a recently published adhesin-related gene, located on plasmid pAPEC-O1-ColBM, the name aatA (APEC autotransporter adhesin A) was adopted from that study.Our data substantiate that AatA might not only be of relevance in APEC pathogenicity but also in facilitating their reservoir life style in the chicken intestine, which might pave the way for future intestinal preventive strategies.

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Figures

Figure 1
Figure 1
APEC IMT5155 aatA: genomic locus and predicted protein structure. A: Scheme of the genomic locus of aatA in IMT5155. The open reading frame (ORF) of aatA is indicated as grey arrow. Black bars indicate PCR fragments amplified using oligonucleotides marked as black arrows. The size of each fragment and the ORF are given in brackets. B: Scheme of the predicted protein AatA. The 3,498 bp ORF results in the 124-kDa APEC autotransporter adhesin A. Sequence analyses revealed the given domain structure. At the N-terminus a signal peptide (SP) is predicted which probably enables the sec machinery to secrete AatA across the cytoplasmic membrane. The autotransporter repeat (ATr) is found in many AT adhesins and proteins, which are predicted as AT adhesins. The alignment below the protein structure shows conserved amino acid (aa) residues within one AT repeat. C-terminal of the AT repeat lies the predicted functional passenger domain found in AT adhesins (PD). The AT-adhesin-typical translocation domain (TD) resides at the C-terminus of the protein. C: Scheme of fusion protein AatAF. Using oligonucleotides B11-for and B11-rev the 1,222 bp fragment aatA_1222, comprising the region for the AT repeat and the functional PD was amplified by PCR and cloned into pET32a(+) for expression. The 64-kDa fusion protein AatAF contains an enterokinase recognition site (EK), an S tag, a thrombin site (T), a His6 tag and a thioredoxin tag (Trx) fused to the N-terminus of the adhesin peptide to enhance protein solubility and to simplify protein purification.
Figure 2
Figure 2
Comparison of the genome regions surrounding aatA of IMT5155, APEC_O1, B_REL606 and BL21. In total we sequenced 6,154 bp of the strain IMT5155 including the aatA gene, 1,072 bp upstream and 1,584 bp downstream of aatA. Our sequence was compared with the comparable 6,154 bp genome regions of the sequenced strains harbouring aatA homologs: APEC_O1, B_REL606 and BL21. Open reading frames (ORFs) are shown as arrows. White arrows represent known genes, predicted ORFs are shown in grey and insertion sequences or an ORF encoding a putative transposase are indicated in black. IS2i: interrupted insertion sequence.
Figure 3
Figure 3
Phylogenetic tree of autotransporter adhesins including AatA. The phylogenetic trees were calculated with the Neighbor-Joining-Algorithm on the basis of a ClustalW multiple alignment of 24 protein sequences from known adhesins of the autotransporter family including AatA. The percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. Protein sequences were obtained from the NCBI database. A: Phylogenetic tree (NJ-tree) obtained using the complete 24 protein sequences. B: NJ-tree obtained using only the last 256 amino acid residues according to the smallest protein HadA in ClustalW analyses. Here, only proteins clustering in one phylogenetic branch with AatA are shown. C: The amino acid residue alignment of the C-termini of AIDA-I and AatA are shown highlighting identical residues (*indicates fully conserved residues, :indicates fully conserved strong groups, .indicates fully conserved weaker groups). Symbols indicate the species: *Escherichia coli, #Neisseria meningitidis, °Haemophilus influenzae, +Yersinia enterocolitica, 'Moraxella catarrhalis, ´´Helicobacter pylori, $Xylella fastidiosa, **Salmonella Typhimurium, and &Bordetella pertussis.
Figure 4
Figure 4
Comparison of the AatA proteins of IMT5155, APEC_O1, BL21, and B_REL606. AatA amino acid sequences were compared using MegAlign (Lasergene 6, DNASTAR, WI, USA). Proteins are depicted as schemes indicating specific protein domains as predicted (SP: signal peptide; ATr: autotransporter repeat region; PD: passenger domain; TD: transmembrane domain). Amino acid differences are shown as lines. Red lines indicate differences to the IMT5155-AatA amino acid sequence. The total number of amino acid substitutions is given for each protein domain below the protein schemes.
Figure 5
Figure 5
Purification of AatAF after expression in E. coli BL21. The internal part of aatA encoding the passenger domain of AatA was cloned into pET32a(+) leading to the expression of the 64-kDa fusion protein AatAF. BL21 cells were incubated in LB at 37°C without (lane 1) or with (lane 3) addition of IPTG. Proteins of total extracts (lane 1 and 3) and of eluates of the purified AatAF (lane 4) were separated on an SDS-PAGE and stained with coomassie. TE: total extract, -: without IPTG, +: with IPTG; M: protein marker.
Figure 6
Figure 6
Expression of AatA in different E. coli strains. The purified fusion protein (lane 1) and total protein extract of BL21(pET32a:aatAF) (lanes 2 and 3), expressing AatAF under the control of the IPTG-inducible promoter, AAEC189(pUC18:aatA+P) expressing aatA under the control of the native promoter and AAEC189(pUC18) (lanes 4 and 5), APEC_O1 (lane 6), IMT5155 (lane7), CFT073 (lane 8) and MG1655 (lane 9) were separated on an SDS gel and blotted to polyvinylidene fluoride membrane. The membrane was then incubated with anti-AatA antibody.
Figure 7
Figure 7
AatA plays a role in adhesion to chicken fibroblast DF-1 cells. A: Adhesion of AAEC189(pUC18) and AAEC189(pUC18:aatA+P), expressing aatA under its native promoter, to DF-1 cells. Monolayers of DF-1 cells were incubated with E. coli strains for 3 h at 37°C. Adherent bacterial cells were harvested and the number was determined. B: The anti-AatA antibody inhibits binding capacity of IMT5155 to DF-1 cells. IMT5155 was incubated with preimmune serum (control) and with anti-AatA antibody, respectively. After washing, bacteria of each experiment were added to monolayers of DF-1 cells and incubated for 3 hours. Adherent bacterial cells were harvested and the number was determined. C: Pre-incubation of DF-1 cells with AatAF protein reduces adhesion capacity of IMT5155 to these cells. Confluent monolayers of DF-1 cells were incubated with BSA (control, 50 μg/well) or purified and refolded AatAF protein (50 μg/well) for 1 h at 37°C prior to the addition of IMT5155 cells. After 3 h of incubation, adherent bacterial cells were harvested and the number was determined. A-C: Columns represent the mean value of three independent wells per strain. Standard errors of the mean values are indicated as error bars. The experiment was repeated three times showing comparable results.
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References

    1. Kaper JB. Pathogenic Escherichia coli. Int J Med Microbiol. 2005;295(6-7):355–356. doi: 10.1016/j.ijmm.2005.06.008. - DOI - PubMed
    1. Kim KS. In: Escherichia coli: Virulence Mechanisms of a Versatile Pathogen. Orlando MSD, editor. Florida, USA: Academic Press; 2002. Meningitis-Associated Escherichia coli; pp. 269–286.
    1. Barnes HJ, Gross WB. In: Diseases of poultry. 10. Gross WB, editor. Ames: Iowa State University Press; 1999. Colibacillosis; pp. 131–141.
    1. Dobrindt U. (Patho-)Genomics of Escherichia coli. Int J Med Microbiol. 2005;295(6-7):357–371. doi: 10.1016/j.ijmm.2005.07.009. - DOI - PubMed
    1. Blondeau JM. Current issues in the management of urinary tract infections: extended-release ciprofloxacin as a novel treatment option. Drugs. 2004;64(6):611–628. doi: 10.2165/00003495-200464060-00004. - DOI - PubMed

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