Open Access

Genome sequence of adherent-invasive Escherichia coli and comparative genomic analysis with other E. coli pathotypes

  • John HE Nash1,
  • Andre Villegas1,
  • Andrew M Kropinski2,
  • Renan Aguilar-Valenzuela3,
  • Paulina Konczy2,
  • Mariola Mascarenhas2,
  • Kim Ziebell2,
  • Alfredo G Torres3,
  • Mohamed A Karmali1, 2 and
  • Brian K Coombes4Email author
BMC Genomics201011:667

DOI: 10.1186/1471-2164-11-667

Received: 1 July 2010

Accepted: 25 November 2010

Published: 25 November 2010

Abstract

Background

Adherent and invasive Escherichia coli (AIEC) are commonly found in ileal lesions of Crohn's Disease (CD) patients, where they adhere to intestinal epithelial cells and invade into and survive in epithelial cells and macrophages, thereby gaining access to a typically restricted host niche. Colonization leads to strong inflammatory responses in the gut suggesting that AIEC could play a role in CD immunopathology. Despite extensive investigation, the genetic determinants accounting for the AIEC phenotype remain poorly defined. To address this, we present the complete genome sequence of an AIEC, revealing the genetic blueprint for this disease-associated E. coli pathotype.

Results

We sequenced the complete genome of E. coli NRG857c (O83:H1), a clinical isolate of AIEC from the ileum of a Crohn's Disease patient. Our sequence data confirmed a phylogenetic linkage between AIEC and extraintestinal pathogenic E. coli causing urinary tract infections and neonatal meningitis. The comparison of the NRG857c AIEC genome with other pathogenic and commensal E. coli allowed for the identification of unique genetic features of the AIEC pathotype, including 41 genomic islands, and unique genes that are found only in strains exhibiting the adherent and invasive phenotype.

Conclusions

Up to now, the virulence-like features associated with AIEC are detectable only phenotypically. AIEC genome sequence data will facilitate the identification of genetic determinants implicated in invasion and intracellular growth, as well as enable functional genomic studies of AIEC gene expression during health and disease.

Background

Crohn's Disease (CD) is a chronic inflammatory bowel disease of the intestinal tract characterized by a strong activation of the intestinal immune system. A complex interaction of genetic, immunologic, and environmental factors contribute to the immunopathology of CD but despite intensive investigation over the last half-century, a unifying etiology of inflammatory bowel diseases (IBD) has not been uncovered [1, 2]. Abundant clinical and experimental data implicate luminal bacteria or bacterial products in both the initiation and perpetuation of chronic intestinal inflammation [24]. Some pathological manifestations observed in CD, including ulcers of the mucosa, mural abscesses and macrophage recruitment and activation, also occur in well-recognized infectious diseases caused by Shigella, Salmonella and Yersinia, in which invasion into mucosal epithelial cells is an important virulence trait [3]. However, a growing body of evidence indicates that the balance between host defence responses and the commensal microbiota plays a key role in the pathogenesis of IBD [2]. Patients with CD display an increased number of coliforms in their feces, particularly during periods of active disease [5] and E. coli antigens are found in most intestinal resection specimens from these patients [6]. Furthermore, it has been shown that early and chronic ileal lesions of CD patients harbour high levels of E. coli that might participate in disease pathogenesis [711]. E. coli strains isolated from the ileal lesions of CD patients can exhibit adherent and invasive capabilities in both gastrointestinal epithelial cells and macrophages [10, 12], a phenotype that was the basis for a new pathogenic group called adherent and invasive E. coli (AIEC) [12, 13]. AIEC are enriched in ileal lesions in human CD [7] and are associated with expression of proinflammatory cytokines and inflammation in mice expressing human carcinoembryonic antigen-related cell adhesion molecule (CEACAM) receptors [14]. The predominance of AIEC in human CD patients, in conjunction with a growing body of biological and animal model data [15] has generated intense interest into the possible role of AIEC in the initiation or maintenance of chronic inflammation associated with CD.

We previously reported on a clinical AIEC isolate with serotype O83:H1 (strain NRG857c) that was isolated from the terminal ileum of a patient with CD [16]. NRG857c belongs to the same serogroup as the historical AIEC isolate called LF82 first described over a decade ago [10] for which much of the experimental data on AIEC phenotypes have been documented. AIEC do not harbour common virulence factors found in various other pathogenic E. coli, and so the genetic basis for their invasive phenotype, proinflammatory nature and association with CD are not fully understood. Here, we report the complete genome sequence of AIEC NRG857c that includes a 150-kb plasmid. We found that AIEC are closely related to a group of extraintestinal pathogenic E. coli (ExPEC) associated with urinary tract infections and neonatal meningitis, a finding that confirms and extends previous work [17]. The comparison of this genome with other ExPEC, enteropathogenic E. coli, AIEC LF82, and commensal E. coli facilitated the identification of 41 high-confidence genomic islands and 66 genes unique to E. coli displaying the adherent and invasive phenotype.

Results and Discussion

Genome sequencing and gap closure

AIEC strain NRG857c was shotgun sequenced to 40-fold coverage using pyrosequencing. Assembly of the raw sequence data generated 48 contiguous regions (contigs) greater than 2-kb with a total size of 4.84-Mb. Contigs were assembled by aligning the larger contigs to an optical restriction map using MapSolver and by BLASTX analysis of contigs ends. The majority of gaps between contigs were identified because contigs ends were syntenic with single-copy genes in previously sequenced E. coli genomes. PCR primers were designed to amplify across these gaps followed by sequencing to generate "super-contigs" (see Additional File 1, Figure S1). Final gap closure was achieved after incorporation of sequence data for the seven ribosomal RNA operons. Plasmid contigs were identified by BLASTX analysis. Gap closure for the plasmid was done using BLASTN analysis of the terminal sequences from which PCR primers were designed. Amplification and sequencing of these regions resulted in the assembly, but not closure, of a single plasmid contig.

General features of the NRG857c AIEC genome

The chromosome of NRG857c is 4,747,819 bp (50.68% G + C content), encoding 4,431 genes (Figure 1, Table 1). The plasmid is 147,060 bp (50.92 G+C content) and encodes 155 genes (Table 1). The sequence of both the NRG857c chromosome and plasmid has been deposited in GenBank [GenBank: CP001855, GenBank: CP001856].
https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-11-667/MediaObjects/12864_2010_Article_3364_Fig1_HTML.jpg
Figure 1

Comparative genome atlas of NRG857c. The chromosome of NRG857c (two outermost rings are CDS on forward and reverse strand) was compared with those of selected E. coli strains, starting from the outer layer LF82 (AIEC; pale green), APEC-O1 (APEC; blue), CFT073 (UPEC; yellow), MG1655 (K12/commensal; purple) and enterohemorrhagic E. coli O157:H7 Sakai (EHEC, red). Genomic islands were plotted on the NRG857c chromosome (grey blocks). The G+C content and G/C skew are also plotted as indicated.

Table 1

General features of NRG857c genome and other E. coli strains

    

Chromosome

Plasmid(s)

Strain

Serotype

Pathotype

Phylogroup

Accession No.

Size (kb)

Total CDS

CDS density (%)

G+C (%)

Total tRNAs

Accession No.

Size (kb)

NRG857c

O83:H1

AIEC

B2

CP001855

4,748

4,431

88.2

50.7

84

CP001856

147

LF82

O83:H1

AIEC

B2

CU651637

4,773

4,312

87.7

50.7

84

CU638872

108

E2348/69

O127:H6

EPEC

B2

FM180568

4,965

4,703

88.2

50.6

92

FM180569 FM180570

97, 6

UTI89

 

UPEC

B2

CP000243

5,065

5,066

91.1

50.6

88

CP000244

114

CFT073

O6:K2:H1

UPEC

B2

AE014075

5,231

5,473

91.9

50.5

89

  

536

O6:K15:H31

UPEC

B2

CP000247

4,938

4685

88.7

50.5

81

  

APEC-O1

O1:K1:H7

APEC

B2

CP000468

5,082

4,467

87.5

50.6

94

DQ381420 DQ517526

241, 174, 105, 46

O157 Sakai

O157:H7

EHEC

E

BA000007

5,498

5,361

88.1

50.5

105

AB011548 AB011549

92, 3

MG1655 (K-12)

OR:H48:K-

Commensal

A

U00096

4,639

4,294

89.0

50.8

88

  

HS

O9

Commensal

A

CP000802

4,643

4,478

88.7

50.8

88

  

E24377A

O139:H28

ETEC

B1

CP000800

4,979

4,873

88.6

50.6

91

CP000795- CP000799 CP000801

79, 74, 70, 34, 6, 5

Phylogenetic position of NRG857c

The phylogeny of AIEC NRG857c was resolved in two ways. First, a phylogenetic tree based on the optical map data was constructed using the unweighted pair group method with arithmetic mean (UPGMA) along with the in silico derived NcoI fragments for other sequenced E. coli strains (Figure 2A). The second method involved multi-locus sequence typing (MLST) with seven housekeeping genes as described previously [18] (Figure 2B; Additional File 2, Table S1), followed by comparison to sequences from other strains [19]. In both analyses NRG857c clustered with avian pathogenic E. coli (APEC-O1), and the uropathogenic E. coli isolates 536 and CFT073. Also in this group was LF82, another AIEC strain of the same serotype as NRG857c (O83:H1) whose genome sequence was retrieved from Genoscope (http://www.genoscope.cns.fr see note added in revision). LF82 shows high sequence similarity to our strain as analyzed by MapSolver (Additional File 3, Figure S2), by BLASTN analysis (Figure 1), and by phylogenetic analysis (Figure 2).
https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-11-667/MediaObjects/12864_2010_Article_3364_Fig2_HTML.jpg
Figure 2

Phylogenetic analysis of NRG857c compared with representative strains of other enteric bacteria. (A) A phylogenetic tree based on the unweighted pair group method with arithmetic mean was constructed from the optimal map data and in silico NcoI restriction digests of other enteric bacterial chromosomes. (B) MLST-based analysis of NRG857c with other enteric bacteria was performed as described in the Methods and sequence data was used to construct a phylogenetic tree. Numbers on the tree branches represent bootstrap support from 1000 bootstrap replicates with a minimum cut-off of 65%. Accession numbers for gene sequences can be found in Additional File 2, Table S1.

A general comparison of the total genome content of NRG857c with several other E. coli pathotypes is shown in Table 1. The majority of human ExPEC belong to phylogenetic group B2 and are categorized based on their clinical spectrum of disease, including urinary tract infections (UPEC) and neonatal meningitis (NMEC) [2023]. AIEC strains cluster genetically with ExPEC and share some of their phenotypic traits including the ability to colonize mucosal epithelial cells, invade eukaryotic host cells, and to induce inflammatory responses in host animals [24, 25]. Although the prototype EPEC strain E2348/69 (serotype O127:H6) and other EPEC strains belong to the same phylogenetic group as the ExPEC strains [26], they are not generally considered to be invasive organisms. However, recent data suggests that at least two type III secreted proteins (EspT and EspF) can facilitate EPEC invasion into non-phagocytic cells and may define a new category of invasive EPEC [27, 28].

Genomic islands and unique sequences associated with AIEC

Genomic islands (GI) comprise a horizontally acquired flexible gene pool that is a major driver in evolution and niche specialization of pathogenic bacteria [29]. Recent computational methods that take advantage of genetic signatures indicative of horizontal gene transfer enable the high-confidence prediction of GIs in annotated bacterial genomes [30]. To identify putative genomic islands in NRG857c, we used IslandViewer, which uses three independent methods for island prediction, IslandPick, IslandPath-DIMOB and SIGI-HMM. Using the methods and established thresholds described previously [31], we identified 35 genomic islands (GI-1 to GI-35) on the NRG857c chromosome ranging from 4 to 25-kb, with G+C content differing significantly from genome mean and with poor conservation among the other non-AIEC pathotypes shown in Figure 1 (see Additional File 4, Table S2 for full list of genomic islands and gene content analysis). We limited our comparative analysis here to the strains most related to NRG857c and to two well-described E. coli strains of commensal and pathogenic nature. The conservation of these 35 islands between NRG8578c and LF82 was high, suggesting that they may encode traits unique to the adherent and invasive phenotype. Five of the genomic islands (GI-6, -7, -8, -10 and -16) code for defective prophages, three (GI-14, -22, -29) are fimbrial islands, and three (GI-20, -26 and -30) appear to be involved in lipopolysaccharide or capsular polysaccharide biosynthesis. GI-23 is noteworthy because it encodes an EmrKY-TolC multidrug resistance efflux pump and the sensor kinase, EvgA, involved in acid resistance and multidrug resistance in E. coli[32]. GI-15 and GI-19 appear to be metabolic islands involved in the transport and metabolism of various sugars. An additional six genomic islands were identified on the large plasmid (PI-1 to PI-6 in Figure 3) (see Additional File 4, Table S2 for full list of plasmid islands and gene content analysis).
https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-11-667/MediaObjects/12864_2010_Article_3364_Fig3_HTML.jpg
Figure 3

Genomic islands in NRG857c. Genomic islands in the NRG857c chromosome (A) and plasmid (B) were predicted using stringent bioinformatics criteria as described in the Methods. Genomic islands are plotted to scale in blue and labelled clockwise on the genome maps. On the plasmid, genes involved in antimicrobial resistance are indicated in red.

To date, restriction profiles or other biased analyses such as pulse field gel electrophoresis (PFGE), MLST or typing for known virulence genes common to intestinal pathogenic E. coli have failed to uncover unique genetic determinants implicated in the AIEC phenotype [17]. To begin to identify single genetic determinants unique to AIEC, we carried out whole-genome comparisons between NRG857c, LF82, and 29 other non-AIEC genomes of E. coli. NRG857c and LF82 show considerable sequence similarity and synteny (Additional File 3, Figure S2) with 46 chromosomal genes unique to NRG857c and 10 chromosomal genes unique to LF82 (see Additional File 5, Table S3 for full list of genes unique to AIEC). The large plasmids from NRG857c and LF82 show almost no conservation between them (see below), suggesting that they have different ancestry.

Panseq, a Web-based tool designed to analyse the "pan-genome" of closely-related genome sequences, was used to identify genes common to AIEC strains NRG857c and LF82, but absent in other members of this phylogenetic cluster (i.e. APEC-O1, 536, and CFT073). We programmed Panseq to find unique sequences of at least 2-kb present in NRG857c and LF82 but absent in APEC-O1, 536 and CFT073. In this analysis, we found 21 sequences with a combined length of 155-kb that are unique to AIEC strains. Several of these sequences code for prophage elements including a 19.7-kb region encoding the morphogenesis and packaging modules of a P22-like prophage (NRG857_04720 - NRG857_04815). A second interesting region of 47.2-kb extends, with one interruption, from NRG857_09990 to NRG857_10240 and codes for several proteins involved in intermediary metabolism including transport of propanol/propanediol and galactitol. BLASTN analysis of this region revealed two sub-regions, one 20.3-kb and the other 4.4-kb, which are not found in the complete genome sequence of any other E. coli strain. The latter region shows 71% sequence coverage to a region from the complete genome of Citrobacter rodentium ICC168, while approximately half of the longer sequence is also found in an uncharacterized E. coli strain ATCC 8739. This 10.7-kb region has no nucleotide similarity with any other fully sequenced bacterium. BLASTX revealed similarity in this region to two hypothetical Vibrio coralliilyticus ATCC BAA-450 proteins [GenBank: ZP_05883689, GenBank: ZP_05883688] adjacent to orthologs in Burkholderia cenocepacia HI2424 [GenBank: YP_833853, GenBank: YP_833854], which are described as hypothetical proteins.

Plasmid analysis

The 150-kb plasmid in NRG857c is different from the plasmid found in LF82. Whereas plasmid pNRG857c shows significant regions of identity to plasmids in other seropathotypes of E. coli, the 110-kb plasmid of strain LF82 (pLF82) has very little similarity to pNRG857c or pAPEC-O1 (APEC-O1), pColBM (APEC-O103), pUTI189 (UPEC UTI189) and pO157 Sakai (EHEC O157:H7) (Figure 4). The extrachromosomal plasmid in NRG857c is a antimicrobial resistance plasmid with a suite of genes encoding resistance to aminoglycosides, β-lactams, chloramphenicol, mercury, quaternary ammonium salts, sulfonamides, tetracycline, and trimethoprim, several of which appear to be enclosed as transposon blocks. The plasmid may be capable of conjugal transfer as it encodes several tra genes, although we have not experimentally tested this. In addition, there are genes for colicins M and V production and immunity. The antibiotic resistance genes are clustered in three regions of the plasmid in PI-2, PI-3 and PI-4 (Figure 3B). The mercury resistance cassette is identical to IS5075 found in IncA/C2 plasmids pRYC103T24 [GenBank: GQ293500.1], pLEW517 [GenBank: DQ390455.1], NR1 [GenBank: DQ364638.1] and R100 [GenBank: AP000342.1]. The β-lactam-macrolide region is identical to sequences present in plasmid pTZ3721 [GenBank: AB020531.1] and pTZ3723 [GenBank: AB038654.1]. Also of interest to us were several genes involved in siderophore production and iron metabolism. Plasmid pNRG857c has the sitABCD operon that encodes proteins involved in the periplasmic and inner membrane transport of iron and manganese. Two outer membrane proteins (IutA and FepA) are also encoded by the plasmid and are involved in translocation of iron across the membrane. IutA (NRG857_30235) is the ferric-aerobactin receptor, while FepA (NRG857_30015) is an iron-enterobactin outer membrane transporter, both of which are involved in the tonB-dependent transport pathway for iron and also the OM receptor for the colicins [33]. IutA and FepA are encoded on plasmids pAPEC-O103-ColBM, pAPEC-O1-ColBM, pCVM29188_146 (from Salmonella enterica serovar Kentucky, [34]), pVM01 (from the APEC strain E3, [35]), and pLVPK (from Klebsiella pneumoniae CG43, [36]). Interestingly, the chromosome contains a FepA paralog (NRG857_02640). The presence of several iron-acquisition genes suggests that Fur regulation of these plasmid-encoded genes occur [37, 38]. As predicted, the consensus DNA sequence for Fur binding (WAATDRNWNYNAWTW) is found in the upstream regulatory region, [39]) of the iroBCDE, sitABCD, iucABCD-iutA operons, and the shiF and fepA genes.
https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-11-667/MediaObjects/12864_2010_Article_3364_Fig4_HTML.jpg
Figure 4

Gene content analysis of plasmid pNRG857c and comparison to representative strains of other E. coli. BLASTN analysis was performed between each CDS in plasmid pNRG857c against each CDS in pLF82, pO157Sakai, pUTI89, and pAPEC-O1-ColBM. Genes in pNRG857c with orthologs in the other plasmids, defined as >85% identity over entire length of the gene, are connected with a coloured line.

Identification of other potential virulence determinants

The chromosome of AIEC strain NRG857c encodes a variety of potential virulence factors (Table 2). As mentioned above, the plasmid carries several potential virulence factors including genes for iron acquisition. This would suggest that the plasmid contributes to the overall virulence of this bacterium, however we have demonstrated previously that a plasmid-cured variant was still able to attach to and invade epithelial cells in vitro[16].
Table 2

Putative virulence factors in NRG857c genome

Locus Tag

Gene Name

Identity (%)

Function

Related Pathotype

Chromosome

Adhesins

NRG857_00540

hcpA

89

adherence

EHEC

NRG857_05010

csgE

100

assembly/transport component in curli production

Common

NRG857_17655

lpfA2

85

major fimbrial subunit of Long Polar Fimbriae (Lpf), named lpfA2

EPEC, EHEC

NRG857_21765

fimA

77

major fimbrial subunit of type 1 fimbriae

Common

NRG857_21795

fimH

99

adhesin of type 1 fimbriae

Common

Iron acquisition/Transport systems

NRG857_06120

fepC

79

ferric enterobactin transport ATP-binding protein

UPEC, EHEC

NRG857_09890

irp2

95

yersiniabactin biosynthetic protein

UPEC

NRG857_09895

irp1

91

yersiniabactin biosynthetic protein

UPEC

NRG857_09915

fyuA

95

pesticin/yersiniabactin receptor protein

UPEC

NRG857_17390

chuA

94

outer membrane receptor protein, heme utilization/transport protein

UPEC, EHEC

Capsular and somatic antigens

NRG857_14650

kpsM-II

94

involved in polysialic acid transport, group II (K1, K4, K5, K7, K12, K92...)

 

Haemolysins and haemagglutinins

NRG857_06035

clyA

95

cytolysin, cell lysis

ETEC

NRG857_01335

tsh

78

temperature-sensitive hemagglutinin of avian E. coli, autotransporter

APEC

Other

NRG857_00835

htrA/degP

97

stress protein, serine endoprotease

common

NRG857_02540

ompT

99

outer membrane protein 3b, other name: protease VII

common

NRG857_02570

ibeB

88

invasion gene locus (penetration of brain microvascular endothelial cells), putative resistance protein, putative outer membrane lipoprotein of copper ion antiporter

common

NRG857_04350

ompA

89

outer membrane protein (OMPA or OMPII)

common

NRG857_05660

iss2

100

gene for increased serum survival (similar to bacteriophage lambda Bor)

common

NRG857_07375

gadB

98

glutamate decarboxylase B, isozyme (amino acid catabolism and metabolism)

common

NRG857_11240

ompC

100

outer membrane protein

common

NRG857_13905

malX

94

maltose and glucose-specific IIABC component, pathogenicity island associated

UPEC

NRG857_15695

nlpI

100

lipoprotein

common

NRG857_17475

gadA

99

glutamate decarboxylase A, isozyme (amino acid catabolism and metabolism)

common

NRG857_19245

dsbA

100

oxidoreductase, thiol:disulfide interchange protein dsbA

common

NRG857_21885

ibeA

91

invasion protein, E. coli invasion of the blood-brain barrier, other name: ibe10

MENEC

Putative virulence associated genes

NRG857_00565

usp

93

uropathogenic specific protein (putative virulence island of UPEC)

UPEC

NRG857_00950

cadA

68

lysine decarboxylase

common

NRG857_03880

artJ

92

L-arginine periplasmic binding protein, supposed to be involved in virulence

common

NRG857_05150

mviM

93

putative virulence factor

common

NRG857_05155

mviN

86

putative virulence factor

common

NRG857_05410

b1121

90

hypothetical protein ycfZ, homologous to virulence factor

common

NRG857_19995

yjaA

100

hypothetical protein

common

NRG857_20725

cadA

99

Lysine decarboxylase

common

NRG857_20730

cadB

100

Lysine:cadaverine antiporter

common

NRG857_22200

nadAB

99

meningococcal adhesion, NAD biosynthesis

common

Plasmid

Colicins and microcins

NRG857_30019

cvaC

83

structural gene for microcin V

common

NRG857_30029

cma

99

structural gene for colicin M

common

Iron acquisition/Transport systems

NRG857_30008

iroB

93

siderophore

common

NRG857_30010

iroC

91

siderophore

common

NRG857_30012

iroD

99

siderophore

common

NRG857_30013

iroE

93

siderophore

common

NRG857_30015

iroN

97

siderophore

common

NRG857_30235

iutA

95

cloacin DF13/aerobactin outer membrane receptor protein

common

NRG857_30237

iucD

96

gene of the aerobactin operon, first product of the aerobactin biosynthesis pathway

common

Other

NRG857_30184

traT

84

complement resistance protein

common

NRG857_30283

ompT

76

outer membrane protein 3b, other name: protease VII

common

NRG857_30309

iss2

100

gene for increased serum survival (similar to Bacteriophage lambda Bor)

common

Antimicrobial resistance

NRG857_30085

blaTEM

94

ampicillin

common

NRG857_30067

tetC

71

tetracycline

common

NRG857_30068

tetA

91

tetracycline

common

NRG857_30075

catI

100

chloramphenicol

common

NRG857_30100

dhfrI

99

trimethoprim

common

NRG857_30095

sulII

93

sulfonamides

common

NRG857_30104

sulI

100

sulfonamides

common

(i) Type VI secretion system

We identified genes for a complete type VI secretion system (T6SS) that are associated with virulence in other invasive organisms (Table 3) [4042]. T6SS are phage-related secretion systems found in many Gram-negative pathogens and are thought to be involved in supporting an intracellular lifestyle, although their distribution is not restricted to pathogenic bacteria [43]. The T6SS in NRG857c is found in GI-2, a low GC region of the chromosome directly downstream from a tRNA which is a common integration site for mobile genetic elements. This T6SS island encodes the conserved core elements of the secretion apparatus, including the valine-glycine repeat protein G (VgrG/NRG857_01165), the ClpV ATPase (NRG_01105) and the hemolysin coregulated protein (Hcp/NRG857_01155) that is 100% identical to Hcp in APEC-O1 and the UPEC strains UT189 and 536. We also identified a second Hcp upstream of this conserved locus (NRG_01080) that is 100% identical to Hcp in E. coli S88 (O45:K1:H7) that causes neonatal meningitis [44], suggesting that this T6SS island is a mosaic with different ancestries. Other organisms, including Vibrio cholerae, have two hcp genes in different parts of the genome [45], which may impart different functionalities on the secretion apparatus. Whether the T6SS in AIEC facilitates intracellular survival and/or growth will require additional experimentation that we are currently pursuing.
Table 3

Type VI secretion system core proteins in NRG857c

Conserved domain(s)a

NRG857c ortholog

ImpA N-terminal related/COG3515

NRG857_01095 hypothetical protein

IcmF-related/DUF1215/COG3523

NRG857_01090 IcmF-related protein

DUF879/COG3519

NRG857_01135 hypothetical protein

DUF877/COG3517

NRG857_01145 hypothetical protein

DUF876/COG3522

NRG857_01115 hypothetical protein

DUF770/COG3516

NRG857_01150 hypothetical protein

DUF1305/COG3520

NRG857_01130 hypothetical protein

ClpV

NRG857_01105 putative ATP-dependent Clp proteinase

FHA domain/COG3456

NRG857_01125 hypothetical protein

COG3521

NRG857_01120 hypothetical protein

DotU (IcmH)-related/COG3455

NRG857_01110 hypothetical protein

Pfam04965/COG3518

NRG857_01140 hypothetical protein

Hcp/DUF796/COG3157

NRG857_01080 hemolysin co-regulated protein

 

NRG857_01155 hemolysin co-regulated protein

VgrG/DUF586/COG3501

NRG857_01165 Vgr-like protein

a as described in Reference 40

(ii) Adhesins

NRG857c contains genes that are important for adhesion and invasion of AIEC LF82, including nlp1, htrA, yfgL, and dsbA[4649]. The SPAAN program [50] as well as BLASTP with relaxed stringency was used to identify and extensive list of additional predicted adhesins (Table 4). The majority of the fimbrial operons in NRG857c are found in other E. coli strains, with the exception of the long polar fimbriae (Lpf; NRG857-17915-17923), which might be important for tissue tropism. A second Auf fimbrial system with a potential role as a colonization factor is encoded by genes NRG857_16960 through _17005. Other potential mediators of invasion include a hemagglutinin/invasin (NRG857_17920 to _17923) and an Ibe invasin (NRG857_21885 to _21890). In previous work, the invasion of brain endothelial cells was found to be mediated by the Ibe invasin, and was located on a genomic island called GimA [51]. The presence of GimA was almost exclusive to ExPEC strains of phylogroup B2, and we now show that ibe is also present in AIEC, suggesting it may be involved in invasive properties of certain strains.
Table 4

Predicted invasion and adhesion factors in NRG857c

Locus Tag

Protein

Ortholog in:

Function

Identity (%)

SPAAN Pad-Value

Invasion

NRG857_06210

putative transcriptional regulator

SMS-3-5

putative invasion gene expression up-regulator SirB

99

NA

NRG857_06250

hypothetical protein

SMS-3-5

putative invasin

93

0.44

NRG857_12485

putative intimin or invasin protein (SivH-like)

UMN026

putative intimin attaching and effacing protein or invasin protein (sivH-like)

96

NA

NRG857_13980

dinucleoside polyphosphate hydrolase

O157:H7 EDL933

putative invasion protein

100

0.12

NRG857_21885

invasion protein IbeA

SMS-3-5

invasion protein IbeA

93

0.35

Adhesion

NRG857_00700

putative fimbrial-like adhesin protein

UTI89

putative fimbrial-like adhesin protein

90

0.83

NRG857_00705

protein YadK

ED1a

protein yadK, putative fimbrial-like adhesin

95

0.79

NRG857_00710

putative fimbrial-like adhesin protein YadL

S88

putative fimbrial-like adhesin protein YadL

80

0.87

NRG857_00715

putative fimbrial-like adhesin protein YadM

ED1a

putative fimbrial-like adhesin protein YadM

100

0.86

NRG857_00730

predicted fimbrial-like protein

S88

putative fimbrial-like adhesin exported protein

95

0.87

NRG857_00985

lipoprotein involved with copper homeostasis and adhesion

UTI89

lipoprotein involved with copper homeostasis and adhesion

99

0.62

NRG857_01440

putative adhesin

S88

putative adhesin

95

0.91

NRG857_01490

putative autotransporter

S88

Putative adhesin; putative outer membrane autotransporter barrel

82

0.91

NRG857_03200

hypothetical protein

K-12 substr. W3110

predicted fimbrial-like adhesin protein

95

0.26

NRG857_04950

PgaD putative PGA biosynthesis protein

K-12 substr. MG1655

required for biofilm adhesin polysaccharide PGA synthesis

88

NA

NRG857_04960

PgaB outer membrane N-deacetylase

K-12 substr. MG1655

biofilm adhesin polysaccharide PGA export lipoprotein with a polysaccharide deacetylase activity needed for export

94

NA

NRG857_04965

outer membrane protein PgaA

K-12 substr. MG1655

biofilm adhesin polysaccharide PGA export, predicted OM protein

94

NA

NRG857_05015

DNA-binding transcriptional regulator CsgD

K-12 substr. DH10B

DNA-binding transcriptional regulator of adhesion determinants

87

0.25

NRG857_05965

hypothetical protein

536

Putative adhesin

100

0.59

NRG857_06960

putative autotransported outer membrane protein involved in cell adhesion

S88

putative autotransported outer membrane protein involved in cell adhesion

78

0.96

NRG857_07415

predicted fimbrial protein-like protein

UTI89

putative fimbrial adhesin FmlD precursor

93

0.90

NRG857_07420

predicted fimbrial protein-like protein

IAI1

putative fimbrial-like adhesin exported protein

89

0.74

NRG857_07425

fimbrial-like adhesin protein

APEC O1

fimbrial-like adhesin protein

98

0.74

NRG857_08345

Hypothetical protein

SE11

putative adhesin

98

0.66

NRG857_09925

hypothetical protein

SE11

putative adhesin

95

NA

NRG857_10700

putative exported fimbrial-like adhesin protein

UTI89

putative Yeh fimbiral adhesin YehA precursor

91

0.81

NRG857_10715

putative fimbrial-like adhesin protein

ED1a

putative fimbrial-like adhesin protein

89

0.82

NRG857_10720

hypothetical protein

APEC O1

putative fimbrial-like adhesin protein

100

0.33

NRG857_11325

adhesin

O157:H7 str. EC4115

putative outer membrane autotransporter adhesin

78

0.95

NRG857_11815

putative exported fimbrial-like adhesin protein

S88

putative exported fimbrial-like adhesin protein

92

0.76

NRG857_11820

fimbrial-like protein YfcQ precursor

S88

putative fimbrial-like adhesin exported protein

99

0.83

NRG857_11825

hypothetical protein

S88

putative fimbrial-like adhesin exported protein

96

0.57

NRG857_11840

putative fimbrial-like adhesin protein

S88

fimbrial-like adhesin protein

76

0.78

NRG857_15155

putative fimbrial protein

S88

putative fimbrial-like adhesin protein

100

0.75

NRG857_15170

putative fimbrial adhesin

UTI89

putative Yqi fimbrial adhesin

95

0.84

NRG857_16975

putative fimbrial-like adhesin protein AufG

ED1a

putative fimbrial-like adhesin protein AufG

94

0.62

NRG857_17635

LpfE protein precursor

O26:H11 str. 11368

putative fimbrial adhesin protein

86

0.68

NRG857_17920

putative haemagglutinin/Invasin

CFT073

putative adhesin

87

0.98

NRG857_21795

type 1 fimbiral adhesin FimH

APEC O1

type 1 fimbrial adhesin FimH

100

0.95

In mouse models of AIEC-induced colitis, inflammation requires type I pili expression by the bacterial cells, as no colitis is induced by ΔfimH mutant bacteria [14]. Colitis in this model requires the expression of human CEACAM receptors by transgenic mice, suggesting that the type I pili of AIEC can induce a proinflammatory response via CEACAM receptors in the gut mucosa. In support of this, FimH, the adhesin tip protein, is necessary but not sufficient for adhesion of AIEC strain LF82 to Intestine-407 cells [52]. Polymorphisms in the FimH sequence have been identified in E. coli isolated from IBD patients and healthy individuals. In particular, 7 amino acid variants are associated with E. coli from IBD tissue and 2 variants are associated with E. coli from healthy individuals [53]. Interestingly, FimH in NRG857c contains two disease-associated amino acid variants (N91S, S99N, and none of the SNPs associated with healthy tissue (A48V, A140V). Whether or not these variants are associated with different inflammatory responses or subtle differences in adherence in vivo will be important areas for future work.

(iii) Transcriptional regulators of virulence genes

NRG857c contains global transcriptional regulators including phoP-phoQ, envZ-ompR, slyA and the negative regulators hns, hha, and fis involved in genome architecture and transcriptional regulation [54]. Although these transcriptional factors are common to many bacterial species, in most Gram-negative pathogens they coordinate transcription of virulence genes including secretion system, toxins, adhesins and flagellar biosynthesis machinery [55, 56]. With this completed genome sequence, functional genomics approaches are now possible to understand the regulons of these transcription factors and their roles in intracellular survival and growth of AIEC. Indeed, Fis levels in the cell have already been associated with regulating the adhesive properties of AIEC strain LF82 [57].

(iv) Iron acquisition

Iron acquisition is an essential virulence trait in other ExPEC and these systems are expressed during urinary tract infections in vivo[58, 59]. Since NRG875c had an abundance of iron uptake systems, we designed experiments to test the role of iron acquisition during infection. We made an aerobactin transport mutant by deletion of iutA and tested whether this iron transport system was important for intracellular survival and the ability to colonize animals. We found that the iutA mutant was able to synthesize but not transport aerobactin (Additional file 6, Table S4). To investigate the invasive properties of ΔiutA, we conducted standard gentamicin protection assays in J774.1 macrophage cells, which did not reveal a significant difference in the uptake at 2 h of the wild type and the iutA mutant (Figure 5A). However, by 4 h after infection and thereafter, the iutA mutant had a significant defect in intracellular survival and/or replication compared to wild type cells. To determine whether the transport of aerobactin was important for bacterial infection in vivo, streptomycin pre-treated mice were infected with wild type NRG857c and the isogenic iutA mutant as described previously for a Salmonella infection model [60]. Wild type NRG857c was recovered in ~50-fold more abundance in the intestinal tissue compared to ΔiutA (Figure 5B).
https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-11-667/MediaObjects/12864_2010_Article_3364_Fig5_HTML.jpg
Figure 5

Iron uptake by the aerobactin system is important for intracellular survival and for mouse colonization. (A) J774.A1 macrophage cells were infected with wild type NRG857c or iutA mutant cells. The survival of intracellular bacteria was determined at various times after infection. Data are the mean survival of intracellular bacteria with standard deviation. (*, P < 0.05, Mann Whitney) (B) The aerobactin iron transport system improves colonization in vivo. Groups of mice were infected orally with wild type NRG857c or iutA mutants. Colonization of the small intestine by NRG857c AIEC was determined three days after infection by enumerating the number of cfu in tissue homogenates. Data are the means with standard errors. (**, P < 0.005, Mann Whitney).

Conclusions

The two broad hypotheses accounting for the immunopathology of IBD, including deregulation of the intestinal immune system, and dysbiosis of the commensal microbiota [61], are likely not mutually exclusive. Both pathways could be operationalized at the same time and in response to known genetic and environmental triggers. Regarding the genetic correlates of the AIEC phenotype, our genome sequence and comparative analyses provide many testable hypotheses to uncover the adhesive, invasive, and proinflammatory nature of AIEC. The fact that the 35 genomic islands in NRG857c are, in many cases, highly orthologous in LF82 but weakly conserved or absent in other E. coli pathotypes and commensal organisms is suggestive that these genomic islands may have an influential role in the expression of the AIEC phenotype. It is also likely that evolved differences in gene expression, or regulatory evolution, has played a pivotal role in generating phenotypic diversity involved in pathogen-like behaviour of AIEC, as we have shown previously for another intracellular pathogen [62, 63]. Functional genomics studies enabled by this work will be forthcoming.

Methods

AIEC strain and genome sequencing

Escherichia coli AIEC strain NRC857c was isolated from a biopsy of a Crohn's disease patient at the Charite Hospital, Germany [16]. A mutant in aerobactin transport (designated RAA002) was created by disruption of the iutA gene using allelic exchange from a suicide plasmid as described previously [64]. For preparation of genomic DNA, wild type NRC857c cells were grown on solid Luria-Bertani (LB) agar at 37°C. Genomic DNA was extracted from 10 mg of bacteria scraped from a plate using the BioRobot EZ1 with the EZ1 DNA kit (Qiagen, Hilden, Germany). For plasmid purification, bacteria were grown in 4 L of LB broth and plasmid was isolated using a Maxi-prep kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Total genomic DNA was sequenced using a Genome Sequencer FLX System (454 Life Sciences, Branford, CT, USA) at the McGill University and Genome Quebec Innovation Centre (Montreal, QC, Canada).

Phylotype grouping, optical mapping, and in silico similarity clustering

Phylogenetic determinations were performed by in silico MLST using seven housekeeping genes (aspC, clpX, fadD, icdA, lysP, mdh and uidA). Analysis was performed using the software package MEGA4 [65, 66] and the Neighbour-Joining method under the Tajima-Nei model. An optical map of NRG857c was generated using the restriction enzyme NcoI (OpGen Inc., Madison, WI) and used for contig ordering. Unweighted Pair-Group Method using Arithmetic averages (UPGMA) similarity clustering of the restriction fragments generated in the whole genome optical map of NRG857c with in silico maps of publicly available E. coli isolates was performed using MapSolver version 2.1.1 (OpGen Inc., Madison, WI).

Gap closure

Outward facing primers annealing to adjacent contigs were designed using Primer3Plus, synthesized by SigmaGenosys (Oakville, ON, Canada) and used to amplify DNA of NRG857c using the Expand Long Template PCR system (Roche, Mannheim, Germany). PCR products were analysed on agarose gels, purified with a Montage PCR purification kit (Millipore, Billerica, MA, USA) and sequenced using Sanger sequencing (University of Guelph, ON, Canada). Finished sequence was assembled using SeqManPro (DNASTAR Inc., Madison, WI). For ribosomal RNA (rRNA) operons, primers were designed using the syntenic flanking sequences of each rRNA operon in the E. coli strain CFT073 [67]. These seven rDNA amplicons were sequenced using the flanking primers and specifically designed 16S (rrs) and 23S (rrl) primers based on sequence alignment with CFT073 rDNAs.

Genome annotation and in silico identification of genes unique to AIEC strains, NRG957c and LF82

The genome sequence was subjected to automated annotation using the NCBI Prokaryotic Genomes Automatic Annotation Pipeline with the resulting GenBank data incorporated into Kodon (Applied Maths Inc., Austin, TX) for manual curation. A protein database was constructed from 22 Escherichia coli genomes available in GenBank. All of the open reading frames of NRG857c predicted by Glimmer 3 [68] were searched against the protein database using BLASTX running locally [69]. The same comparison was performed using the LF82 nucleotide sequences. A script written with the BioPerl toolkit [70] was used to parse the BLAST output files for sequences that did not have any matches, or sequences with only weak matches using the criteria: (E-value ≥ 0.01), or (Percent Identity < 50%), or (<50% of the query length was used in the BLAST alignment). The predicted ORFs of NRG857c were compared against those of strain LF82 to identify those unique to each strain. Additional comparative genomics analyses were carried out using Panseq [71] and 29 publicly-available E. coli genome sequences (see Additional File 7, Table S5 for list of E. coli genomes and accession numbers used for comparative analyses). The functions of identified sequences were predicted using the annotation engine AutoFACT [72]. Circular genome atlases were generated using CGView [73, 74] or Circos [75].

Gentamicin protection assays

J774A.1 macrophage cells were seeded at 5 × 105 cells/well in DMEM with L-glutamine and 10% FBS for 16 h prior to infection. Cells were infected at a multiplicity of infection of 10 with wild type NRG857c or the iutA mutant. Infected cells were incubated at 37°C for 2 h, then washed and treated for 2 h with 100 μg/ml gentamicin. At various times post-infection, cells were washed and lysed with 0.1% Triton X-100 in PBS, followed by serial plating on LB agar. Gentamicin protection experiments were performed in triplicate and reported as the percent survival with standard error with statistical significance determined by Student's t test.

Mouse infections

All animal experiments were performed in accordance with protocols approved by the local animal ethics committee at the University of Texas Medical Branch, Galveston, Texas. Female ICR mice of 20-25-g (Charles River Laboratories) were used after 72 h of quarantine as described previously [76]. Briefly, food-restricted animals received streptomycin (5 g/L in drinking water supplemented with 7% fructose) for 48 h prior to oral inoculation with NRG857c or the iutA mutant. Groups of mice (n = 6) were orally inoculated with a suspension of NRG857c bacteria in a final volume of 0.4 mL delivered by gavage (20-gauge needle). The animals were maintained for 72 h, after which the animals were killed and the small intestines removed for homogenization and enumeration of the bacterial load. Groups were compared using the Mann Whitney non-parametric test.

Siderophore utilization and iron uptake bioassays

The synthesis of siderophores by AIEC O83:H1 was analyzed by the colorimetric Arnow assay to detect catechol siderophores [77] and the ferric perchlorate assay for hydroxamates [78]. To restrict the iron availability in liquid or solid medium, the iron chelator 2,2'-dipyridil was used. To examine the ability to use various siderophores or iron compounds as iron sources, overnight cultures of AIEC O83:H1 were diluted to 1 × 105 bacteria per ml and seeded into L agar containing 2,2'-dipyridil. Plates were spotted with 5 μl of 8 μM hemin or 5 μl of an overnight culture of a siderophore-producing strain. A sterile disk containing 20 μl of 10 mM FeSO4 was placed on each plate. Growth was monitored around the spots or disk after 18 to 24 hours at 37°C.

Declarations

Acknowledgements

We thank Dr. Paul Stothard at the Department of Agricultural, Food and Nutritional Science at the University of Alberta for help in generating Figure 1, and Dr. Alexander Swidsinski for providing the original NRG857c isolate. This work was supported by an Innovation in IBD grant to BKC from the Crohn's and Colitis Foundation of Canada and by an operating grant to BKC from the Canadian Institutes of Health Research (MOP-82704). BKC is a CIHR New Investigator (MSH-83721) and the recipient of the Early Researcher Award from the Ontario Ministry of Research and Innovation and the Young Investigator Award in the Biological Sciences from Boehringer Ingelheim (Canada) Ltd.

Note added in revision

While this paper was being revised, Miquel and colleagues reported the genome sequence of LF82, a prototype AIEC isolate [79].

Authors’ Affiliations

(1)
Office of Biotechnology, Genomics and Population Health, Public Health Agency of Canada
(2)
Laboratory for Foodborne Zoonoses, Public Health Agency of Canada
(3)
Department of Microbiology and Immunology, University of Texas Medical Branch
(4)
Michael G. DeGroote Institute for Infectious Disease Research and the Department of Biochemistry and Biomedical Sciences, McMaster University

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