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. 2010 Sep 17;5(9):e12714.
doi: 10.1371/journal.pone.0012714.

Complete Genome Sequence of Crohn's Disease-Associated Adherent-Invasive E. Coli Strain LF82

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Free PMC article

Complete Genome Sequence of Crohn's Disease-Associated Adherent-Invasive E. Coli Strain LF82

Sylvie Miquel et al. PLoS One. .
Free PMC article

Abstract

Background: Ileal lesions of Crohn's disease (CD) patients are abnormally colonized by pathogenic adherent-invasive Escherichia coli (AIEC) able to invade and to replicate within intestinal epithelial cells and macrophages.

Principal findings: We report here the complete genome sequence of E. coli LF82, the reference strain of adherent-invasive E. coli associated with ileal Crohn's disease. The LF82 genome of 4,881,487 bp total size contains a circular chromosome with a size of 4,773,108 bp and a plasmid of 108,379 bp. The analysis of predicted coding sequences (CDSs) within the LF82 flexible genome indicated that this genome is close to the avian pathogenic strain APEC_01, meningitis-associated strain S88 and urinary-isolated strain UTI89 with regards to flexible genome and single nucleotide polymorphisms in various virulence factors. Interestingly, we observed that strains LF82 and UTI89 adhered at a similar level to Intestine-407 cells and that like LF82, APEC_01 and UTI89 were highly invasive. However, A1EC strain LF82 had an intermediate killer phenotype compared to APEC-01 and UTI89 and the LF82 genome does not harbour most of specific virulence genes from ExPEC. LF82 genome has evolved from those of ExPEC B2 strains by the acquisition of Salmonella and Yersinia isolated or clustered genes or CDSs located on pLF82 plasmid and at various loci on the chromosome.

Conclusion: LF82 genome analysis indicated that a number of genes, gene clusters and pathoadaptative mutations which have been acquired may play a role in virulence of AIEC strain LF82.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. AIEC strain LF82 chromosome circular map and synteny plots between pLF82 and pHCM2 and pMT1.
Circular representation of the E. coli LF82 genome (A). Circles display from the inside out: (1) GC skew (G+C/G−C using a 1 kbp sliding window). (2) Location of tRNAs (green), rRNAs (blue) and Insertion Sequences (grey). (3) GC deviation (mean GC content in a 1 kbp window – overall mean GC). Red areas indicate that deviation is higher than 2 Standard Deviation. (4) (5) and (6) Gene specificity of LF82 strain at strain level (K12, in blue), and at group level: E. coli B2 strains, in green, and E. coli commensal strains, in red. Genes sharing at least one homolog in an other E. coli of the same group and having more than 85 percent identity on at least 80% of its length were regarded as non specific. Synteny plots between the E. coli LF82 plasmid and the plasmid from Salmonella enterica serovar Typhi (upper comparison) and the plasmid from Yersinia pestis Pestoides (bottom comparison) (B). Synteny groups containing a minimum of five genes are shown in purple for colinear regions.
Figure 2
Figure 2. Recombination-insensitive phylogenetic analysis.
The analysis was based on the sequence of seven house-keeping genes (7497 nucleotides from genes arcA, aroE, icd, mdh, mtlD, pgi and rpoS) of 23 genomes reference strains including LF82. The major branches are labeled according to the major phylogroups A, B1, B2, D, E and F.
Figure 3
Figure 3. CRISPR regions of LF82 chromosome.
Genes are shown as boxes pointing towards the direction of transcription. CRISPR repeats are represented by “<” symbols.
Figure 4
Figure 4. Evolution of survival rate of mice challenged subcutaneously with various E. coli strains.
An inoculum of 2×108 bacteria was injected in OF1 mice and 10 mice were used for each bacterial strain tested. Strains were classified as non-killer (<2 of 10 mice killed), killer (>8 mice killed) or intermediate.
Figure 5
Figure 5. Genome organization of four putative pathogenic islands carrying virulence-related genes.
PAI I and PAI III present genes encoding type VI secretion system (t6ss), PAI II is similar to the Yersinia high pathogenicity island and PAI IV present a similar genetic organization of group 2 capsule gene clusters. Black arrows indicate genes associated to t6ss, grey arrows indicated genes with assigned functions. Depending of the inclination, hatched arrows represents hypothetical proteins or proteins absents in any sequenced E. coli strains. Characteristic features of type VI secretion system genes products are indicated in Table S4.
Figure 6
Figure 6. Phylogenic tree of sequenced E. coli strains.
Phylogenic analysis were performed with FimH (A), OmpA (C), OmpC (E) and YfgL (F) variants. Location of substitutions in crystal structures are shown for FimH (B) and OmpA (D). FimH and OmpA are presented as ribbon in yellow , . FimH structure is in complex with the chaperon FimC (blue ribbon) and mannose (stick). The substitutions are indicated in red. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches.
Figure 7
Figure 7. Phylogenic tree and studies on adhesion and invasion levels of E. coli strains.
Location of LF82 in the phylogenic tree based on analysis of concatenated FimH and OmpA amino acid sequences (A), adhesion (B) and invasion (C) abilities of LF82 and other sequenced B2 or non pathogenic strains to Intestine-407 cells. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches. Cell-associated bacteria were quantified after centrifugation and a 3-h infection period. Invasion was determined after gentamicin treatment for an additional 1h. Each value is the mean ± SEM of at least three separate experiments.

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