Genomic subtraction for the identification of putative new virulence factors of an avian pathogenic Escherichia coli strain of O2 serogroup

Catherine Schouler, Frédérique Koffmann{dagger}, Cécile Amory, Sabine Leroy-Sétrin{ddagger} and Maryvonne Moulin-Schouleur

INRA–Centre de Tours, UR86, Pathologie bactérienne, 37380 Nouzilly, France

Correspondence
Maryvonne Moulin-Schouleur
dhomouli{at}tours.inra.fr


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
To identify putative new virulence factors of avian pathogenic Escherichia coli (APEC) strains, a genomic subtraction was performed between the APEC strain MT512 and the non-pathogenic E. coli strain of avian origin EC79. Seventeen DNA fragments were cloned that were specific for the APEC strain. Among them, nine were identified that were more frequent among pathogenic than non-pathogenic isolates in a collection of 67 avian E. coli. Chromosome or plasmid location, and the nucleotide sequence of these nine fragments were characterized. Four fragments were plasmid-located. The nucleotide sequence of two of them exhibited identity with the sequence of the RepF1B replicon of E. coli plasmids, and the amino-acid deduced sequences from the two other fragments exhibited similarity to the products of genes sitA of Salmonella Typhimurium and iroD of E. coli, which are involved in iron metabolism. Of the five chromosome-located fragments, three were predicted to encode parts of proteins that were significantly homologous to previously described proteins: TktA (transketolase) of Haemophilus influenzae, a FruA (fructokinase) homologue of Listeria innocua and Gp2 (large terminal subunit) of phage 21. The putative products of the two other chromosome-located fragments were homologous to proteins with unknown functions: Z0255 of E. coli strain EDL933 (EHEC) and RatA of Salmonella Typhimurium strain LT2. Both these chromosomal fragments, whose presence is correlated with serogroups O1 and O2 and to the virulence of APEC strains belonging to these serogroups, are good candidates for being part of novel virulence determinants of APEC. Moreover, several fragments were shown to be located close to tRNA selC, asnT or thrW, which suggests they could be part of pathogenicity islands. Six fragments that were shown to be part of whole ORFs present in the APEC strain MT 512 were also present in extra-intestinal pathogenic E. coli (ExPEC) strains of human and animal origin. Thus, the putative novel virulence factors identified in this study could be shared by ExPEC strains of different origins.


Abbreviations: APEC, avian pathogenic Escherichia coli; EHEC, enterohaemorrhagic E. coli; ExPEC, extra-intestinal pathogenic E. coli; UPEC, uropathogenic E. coli

The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AY187868AY187876.

{dagger}Present address: 3 rue Edouard Branly, 78390, Bois d'Arcy, France.

{ddagger}Present address: INRA–Centre de Clermont Ferrand/Theix, UR370, Microbiologie, 63122 St. Genès-Champanelle, France.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Escherichia coli is generally considered as a harmless bacterium in the normal intestinal microflora of humans and many animals. However, some strains are able to express virulence factors and to provoke intestinal or extra-intestinal diseases (Ambrozic et al., 1998). It is widely documented that the genome of pathogenic E. coli strains frequently includes 400–800 kbp more DNA as compared to the genome of non-pathogenic strains such as K-12/MG1655 (Bergthorsson & Ochman, 1995). This difference is mainly due to additional regions in the genome of pathogenic E. coli strains: temperate bacteriophages (Barondess & Beckwith, 1990), plasmids (Nataro & Kaper, 1998) or clusters of chromosomal genes called ‘pathogenicity islands' or ‘PAIs' (Dozois & Curtiss, 1999; Hacker et al., 1997). Several PAIs have been characterized on uropathogenic E. coli (UPEC) such as strains 536, J96 and CFT073 (Kaper & Hacker, 1999), on E. coli associated with neonatal meningitis (Bonacorsi et al., 2000), on Shiga-toxin-producing E. coli associated with haemolytic-uraemic syndrome (Pradel et al., 2002) and on avian pathogenic E. coli (APEC) strains (Brown & Curtiss, 1996; Parreira & Gyles, 2003).

APEC strains are responsible for extra-intestinal diseases in poultry. The most frequent form starts as a respiratory disease in chickens and turkeys of 4–9 weeks of age. Bacteria enter the respiratory tract of birds and colonize the airsacs. The aerosacculitis is then followed by a systemic infection: pericarditis, perihepatitis and septicaemia (Barnes & Gross, 1997; Dho-Moulin & Fairbrother, 1999). This infection causes important morbidity and mortality and significant economic losses in the poultry industry. APEC isolates commonly belong to serogroups O1, O2 and O78 (Dho-Moulin & Fairbrother, 1999). A study of genetic relationships between APEC strains has shown that they belong to a restricted number of clones and that most frequently encountered serotypes O2 : K1 and O78 are phylogenetically distant (White et al., 1993).

Various potential virulence factors are associated with APEC: they include F1 and P fimbrial adhesins, the aerobactin system of iron-uptake, the capsular K1 antigen, resistance to complement and several proteins such as the autotransporter Tsh (Dho-Moulin & Fairbrother, 1999). Nevertheless, not all steps of the infectious process can be explained by the presently known virulence factors and additional determinants remain to be characterized. By subtractive hybridization between an APEC strain and a K-12 E. coli strain, 12 specific chromosomal regions in the APEC O78 strain {chi}7122 were identified (Brown & Curtiss, 1996), out of which at least two might be involved in virulence. In this study we intended to detect and localize other DNA regions potentially related to virulence in another frequently encountered APEC, virulent serotype O2 : K1.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, plasmids and culture conditions.
Two avian E. coli strains, MT512 and EC79, were used for suppressive subtractive hybridization. The APEC strain MT512 (O2 : K1 : H7) was isolated from the trachea of a chicken suffering from respiratory disease [strain SI in Brée et al. (1989)]; it is lethal for 1-day-old chicks (LD50: 103 c.f.u.) and its virulence has been assessed by experimental reproduction of avian colibacillosis. The avirulent strain EC79 (O2 : K1– : H–) was isolated from the faeces of a healthy chicken; it is not lethal for 1-day-old chicks and is devoid of known virulence genes (Brée et al., 1989). These O2 strains are phylogenetically distant (White et al., 1993). The APEC strain BEN 2332 (O2 : K1 : H5) [former name MT78, Dho-Moulin et al. (1990)] or its nalR derivative BEN 2908 were used to extend the sequence of the identified fragments.

E. coli strains HB101 (Lacks & Greenberg, 1977) and MG1655 (Blattner et al., 1997) were used as non-pathogenic reference strains. E. coli strain JM109 was used for transformation with recombinant pGEM-T plasmid according to the manufacturer's instructions (pGEM-T Vector Systems kit; Promega).

In addition, a total of 304 avian E. coli strains originating from the laboratory collection (INRA–Tours, Bacterial Pathology Laboratory) were used to study the distribution of DNA fragments isolated by subtractive hybridization. These strains were epidemiologically unrelated and they included E. coli strains isolated from turkeys (175 isolates), chickens (115 isolates) and ducks (14 isolates). Serogroup was determined by slide agglutination with specific reagents against O1, O2 or O78 antigens (Biovac), and virulence was investigated using the lethality test for 1-day-old chicks (Dho & Lafont, 1984). The E. coli strains were routinely grown in brain heart infusion (BHI) (Difco) or Luria–Bertani broth (LB) at 37 °C with aeration. When necessary, LB medium was supplemented with 100 µg ampicillin ml–1 (Sigma-Aldrich).

Furthermore, eight E. coli strains isolated from human neonatal meningitis, five E. coli strains isolated from human urinary tract infections and seven extra-intestinal pathogenic E. coli (ExPEC) strains isolated from sheep, pig or calf were included to test for the presence of genes corresponding to the DNA sequences identified following subtractive hybridization.

DNA extraction.
Genomic DNA was prepared as described by Wilson (1987), or alternatively by the method described by Hull et al. (1981) for genomic subtraction experiments. Plasmid DNA extraction was performed as described by Birnboim & Doly (1979) for pGEM-T and as described by Takahashi & Nagano (1984) for native plasmids.

Genomic subtraction and cloning of fragments obtained.
Genomic DNA from the non-pathogenic E. coli strain EC79 was sheared by sonication to a maximum size of 1·8 kbp. The DNA fragments were then biotinylated with photobiotin acetate (Sigma-Aldrich) as described by Brown & Curtiss (1996). After photoactivation with an Ultra-Vitalux lamp (300 W; Osram), photobiotin acetate was extracted six times with 1-butanol, ethanol-precipitated and resuspended in 2·5x EE buffer (25 mM N-2-hydroxyethylpiperazine-N'-3-propane-sulfonic acid, 2·5 mM EDTA, pH 8). Genomic DNA from the APEC strain MT512 was digested to completion with Sau3AI (Roche Diagnostics), according to the supplier's instructions. The E. coli EC79 biotinylated DNA (10 µg) was hybridized with the E. coli MT512-digested DNA (0·4 µg), as described by Brown & Curtiss (1996). Biotinylated hybrid DNA fragments were eliminated using a streptavidin-coated magnetic beads solution (Dynal). The unbound DNA was then ethanol-precipitated and suspended in 1x EE buffer. An aliquot resulting from the first cycle of subtraction was saved and a second subtraction cycle was performed on the remaining fraction as described above. The DNA fragments in the two aliquots were then ligated to Sau3AI adaptors (Straus & Ausubel, 1990) using 5 U T4 DNA ligase (Rapid DNA ligation kit; Boehringer). Ligated DNA fragments were purified with the QIAquick PCR purification kit (Qiagen) then amplified by PCR using 100 pmoles of the phosphorylated adaptor strand as primer (primer 1, Table 1) (Straus & Ausubel, 1990). The temperature of the annealing step and duration of the extension were 65 °C and 1 min, respectively. The PCR products were finally cloned in pGEM-T according to the manufacturer's instructions.


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Table 1. Primers used for PCR amplifications and DNA probe synthesis

 
Pulsed-field gel electrophoresis (PFGE).
Bacterial cells grown in BHI broth to an OD600 of 1·0 were harvested by centrifugation. The cellular pellet was suspended in 1/2 volume of buffer TE100 (10 mM Tris/HCl, pH 9, 100 mM EDTA) and the suspension was incubated for 10 min at 37 °C. The bacterial suspension was then mixed with an equal volume of 1·6 % low-melting-point agarose (Sigma-Aldrich) and dispensed into plug moulds (Bio-Rad). Agarose plugs, first incubated for 2 h at 37 °C, without shaking, in a lysozyme solution (10 mM Tris/HCl, pH 9, 100 mM EDTA, 5 mg lysozyme ml–1, 0·05 % Sarkosyl), were then incubated overnight at 55 °C (without shaking) in a lysis solution (10 mM Tris/HCl, pH 9, 100 mM EDTA, 1 mg proteinase K ml–1, 1 % SDS). After lysis, agarose plugs were washed three times for 1 h each in a TE buffer (10 mM Tris/HCl, pH8, 1 mM EDTA); the first washing buffer was supplemented with 100 µM PMSF (Sigma-Aldrich). Plugs were then stored at 4 °C in TE buffer. For digestion, half-plugs were first equilibrated in the appropriate restriction enzyme incubation buffer, then 10–40 units of enzyme (BlnI, NotI, SfiI or XbaI) (Takara Bio Europe) were added and an overnight incubation was performed at the temperature prescribed. PFGE was conducted in a CHEF-DRIII apparatus (Bio-Rad). Gels (1 % agarose) were run at 14 °C for 24 h in TBE buffer (4 mM Tris, 4 mM borate, 1 mM EDTA, pH 8·3) at 6 V cm–1. Pulse times were increased from 10 to 30 s. Lambda ladder CHEF DNA size standard (Bio-Rad) was used as molecular size marker.

PCR amplification.
PCR amplification assays were carried out in a Perkin-Elmer temperature cycler 9600 (Applied Biosystems). The primers used are listed in Table 1. For the amplification of small fragments, DNA crude extracts prepared by a rapid boiling method, or purified DNA samples, were amplified in a 25 µl PCR using 1 U Taq DNA polymerase (Promega), 25 pmoles of the forward and reverse primers and 5 nmoles each dNTP (Promega) in 1x buffer. The PCR conditions were as follows: 94 °C for 3 min (1 cycle), 94 °C for 30 s, annealing temperature for 30 s and 72 °C for at least 1 min according to the size of the amplified fragment (30 cycles), then a final extension at 72 °C for 10 min. Whole genes were amplified using a similar protocol as described above, in a 50 µl PCR using 1 U Herculase (Stratagene), and an elongation time of 1 min kbp–1. In that case, sequences of primers were determined from the sequenced genome of E. coli CFT073 (Welch et al., 2002) for A12, D1, D7 and D11, and from the extended sequence of fragments A9 and D10 as determined from the APEC strain BEN 2332 (D10, GenBank accession no. AY307124), or its nalR derivative BEN 2908 (A9, GenBank accession no. AY395687); forward primers were designed from the start codon location and reverse primers from the stop codon location (primers 15–20, F and R, Table 1).

Dot- and Southern-blotting.
Dots were prepared from (i) crude extracts (rapid boiling method) of genomic DNA or (ii) amplified DNA fragments from subtractive hybridization in the case of reverse dot-blots (Kuhnert et al., 1997). They were hybridized with (i) labelled cloned fragments from subtractive hybridization or (ii) labelled genomic DNA in the case of reverse dot-blots. The Dig-High Prime labelling kit (Boehringer) was used for labelling of probes. Detection was performed with the DIG Luminescent detection kit (Boehringer), according to the supplier's instructions.

For Southern blotting, plasmids or digested chromosomal DNA separated by PFGE were passively transferred to positively charged membranes (Hybond-N, Amersham). Probes were labelled using the ECL Direct nucleic acid labelling system (Amersham). After hybridization, detection was performed using the ECL Gene detection system (Amersham), following the supplier's instructions.

DNA sequence analysis.
DNA sequencing was performed by the di-deoxy chain-termination method of Sanger et al. (1977) using the Dye Terminator cycle sequencing easy reaction kit (Perkin-Elmer) on an ABI PRISM 377 XL DNA Sequencer (Perkin-Elmer). Sequences were analysed with the program developed by the Genetics Computer Group (UWGCG) using GenBank and EMBL databases for homology research. The sequences of the isolated DNA fragments have been assigned GenBank accession numbers AY187868AY187876.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Recovery of genomic fragments of the APEC strain MT512 following subtractive hybridization
After two rounds of subtractive hybridization between the APEC strain MT512 and the non-pathogenic strain EC79, the remaining fragments were cloned into the pGEM-T vector. Sixty-two clones with inserts varying from 150 to 480 bp were obtained. Each insert was then amplified by PCR from the recombinant pGEM-T vector using primer Sau 11 (primer 2, Table 1) and hybridized with the labelled genomic DNA of strains MT512 and EC79 in a reverse dot-blot assay. Among the 62 clones, 17 contained inserts hybridizing only with labelled genomic DNA of strain MT512 and not with labelled genomic DNA of EC79. The specificity of these 17 DNA fragments for strain MT512 with respect to strain EC79 was confirmed by a direct dot-blot hybridization assay using each cloned fragment as a labelled probe.

Distribution of MT512-isolated DNA fragments in a collection of avian E. coli strains
The presence of MT512-isolated DNA sequences was first assessed in a sample of 67 avian E. coli strains whose serotype and virulence for chicks had been previously determined (M. Moulin-Schouleur, unpublished results). E. coli strains MT512, EC79 and HB101 were used as controls. Dot-blot assays were performed on genomic DNA from the 67 avian E. coli strains using the 17 labelled DNA fragments as probes. Seven fragments (A5, A7, A8, A10, A11, B9 and C1) showed similar frequencies among pathogenic (APEC) and non-pathogenic avian E. coli strains (Table 2); furthermore, their presence was detected in the genome of E. coli HB101. The A2 fragment was as frequent in APEC strains as in non-pathogenic strains but it could not be detected in E. coli HB101. The nine other fragments (A9, A12, B3, B6, B7, D1, D7, D10 and D11) were significantly more frequently detected in APEC strains ({chi}2 test, P<=0·02) than in non-pathogenic strains (Table 2). Among them, fragment B7 was shown to be present in pathogenic strains only. Moreover, none of these nine fragments was detected in E. coli strain HB101. As these nine fragments more frequently encountered among APEC strains could be good candidates for the carriage of virulence determinants, they were retained for further characterization.


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Table 2. Incidence of E. coli MT512-isolated DNA fragments in a collection of avian E. coli strains

Pathogenicity was assessed by a lethality test on groups of five one-day-old chicks which were inoculated subcutaneously with an overnight broth culture of the E. coli strain (about 108 c.f.u.). Pathogenic strain, one to five chickens died of five inoculated; non-pathogenic strain, no chickens died of five inoculated. Results were expressed as a percentage of strains giving a positive reaction in the dot-blot assay, or detection (+) or absence of detection (–) of the fragment.

 
We noticed from this first investigation that the A9 and D10 sequences were absent from several APEC strains belonging to serogroup O78. We thus investigated their prevalence in another and larger sample of avian E. coli strains (237 isolates). Specific primers were designed for PCR amplification of internal regions of the A9 and D10 inserts (primers 13 and 14, F and R, Table 1) and PCR assays were conducted on 237 strains whose serogroups and virulence were determined. As previously shown in the initial study of strains (Table 2), the presence of the A9 sequence was significantly more frequent in pathogenic strains (35 %) than in non-pathogenic strains (5 %), ({chi}2 test, P=0·0061) (Table 3). Similarly, the presence of the D10 sequence was more frequent in pathogenic strains (32 %) than in non-pathogenic strains (0 %) ({chi}2 test, P=0·0027). Among pathogenic strains, the presence of A9 was significantly associated with serogroup O1 and O2 when compared with serogroup O78 ({chi}2 test, P=0), and the same result was observed for D10 ({chi}2 test, P=0) (Table 3).


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Table 3. Frequency of fragments A9 and D10 in relation to serogroup and pathogenicity

P, Pathogenic; NP, non-pathogenic. Pathogenicity was assessed by a lethality test on groups of five one-day-old chicks which were inoculated subcutaneously with an overnight broth culture of the E. coli strain (about 108 c.f.u.). Pathogenic strain, one to five chickens died of five inoculated; non-pathogenic strain, no chickens died of five inoculated. A9 and D10 sequences were detected in a PCR assay using internal primers 13F/13R and 14F/14R (Table 1). ND, Not determined (non-O1, -O2 or -O78).

 
Characterization of the genomic fragments carrying putative virulence determinants
The labelled MT512 DNA fragments were hybridized with a plasmid DNA preparation of E. coli MT512 in a Southern-blot assay. Four fragments (A12, B3, B6 and D11) hybridized with the large plasmid of MT512. The five remaining fragments (A9, B7, D1, D7, D10) which did not hybridize were thought to be located on the chromosome. The nucleotide sequences of these nine fragments were determined, as were their sizes, which were between 112 and 245 bp (Table 4). The nucleotide sequences showed that each insert was unique. BLAST searches in a non-redundant database at NCBI were conducted to find homologies with already described sequences (Table 4). The nine nucleotide sequences studied were not present in the genome of E. coli MG1655 (Blattner et al., 1997). Nucleotide sequences of the genomic fragments A12, D1, D7, D10 and D11 (but not A9), were shown to be present in the genome of the uropathogenic strain CFT073 (Welch et al., 2002).


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Table 4. Sequence analysis of MT512-isolated DNA fragments

 
Inserts B3 and B6 are homologous (96 and 99 % identity, respectively) to the RepFIB replicon of IncF plasmids, which is the main replicon of ColV plasmids (Ambrozic et al., 1998).

Insert B7 is homologous (99 % identity) to part of the gp2 gene of bacteriophage 21 encoding the terminase large subunit (Wu et al., 1988).

Inserts D1 and D7 may be involved in various biosynthetic pathways. The translated D1 DNA sequence shares 80 % similarity with enzyme TktA of Haemophilus influenzae (Fleischmann et al., 1995), which is involved in the pentose-phosphate pathway. The translated D7 DNA sequence shares 79 % similarity with a fructose-like enzyme II component (FruA homologue) of a PTS system (Postma et al., 1993) from Listeria innocua (Glaser et al., 2001). However, the sequence of D1 and D7 inserts did not exhibit any similarity to the already described genes fruA and tktA, which are present in E. coli MG1655 (Blattner et al., 1997). Moreover, we demonstrated by PCR and by hybridization that the fruA and tktA genes of E. coli MG1655 are present in the pathogenic strain MT512 as well. The translated sequences of inserts of A12 and D11 are both homologous with proteins involved in iron uptake: 81 % similarity between the A12 polypeptide and SitA of Salmonella Typhimurium (Janakiraman & Slauch, 2000) and 98 % similarity between the D11 polypeptide and IroD of E. coli UPEC 536 (Dobrindt et al., 2001). Both corresponding genes sitA and iroD have been shown to be located on pathogenicity islands in Salmonella Typhimurium and E. coli UPEC 536, respectively.

The last two inserts (A9 and D10) possess ORFs whose putative products are homologous to proteins with unknown functions: with Z0255 of E. coli EDL933 (Perna et al., 2001) and RatA of Salmonella Typhimurium LT2 (McClelland et al., 2001), respectively. Moreover, the nucleotide sequence of insert A9 is also quasi-identical to clone TspeE4.A1, which has been identified in an E. coli strain responsible for neonatal meningitis (Bonacorsi et al., 2000).

Localization of the genomic fragments of strain MT512 carrying putative virulence determinants with respect to tRNA loci
Linkage of specific genomic fragments of pathogenic strains with tRNA loci may suggest the presence of a pathogenicity island (Hacker et al., 1997). To assess the potential presence of pathogenicity islands in the genome of strain MT512, we investigated the localization of the identified genomic fragments with regard to tRNA loci asnT, leuX, metV, pheU, pheV, selC and thrW. Genes of interest (iucA, fruA and tktA) were also used as probes in this study. PCR products from the inserts (primer 2, Table 1), amplifications of tRNA loci and genes of interest (primers 3–12, F and R, Table 1) were labelled and hybridized on Southern blots of genomic DNA from strain MT512 previously digested with the rare-cut enzymes BlnI, NotI, SfiI and XbaI (Fig. 1).



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Fig. 1. PFGE patterns of the APEC strain MT512. Probes that hybridized with restriction fragments are detailed for each restriction pattern. Probes that hybridized on the same BlnI restriction fragment: D11, B7, A12, fruA, metV, tktA and pheV on BlnI-1 (>600 kbp); leuX on BlnI-2 (350 kbp), selC and D7 on BlnI-3 (297 kbp); pheU on BlnI-4 (264 kbp); iucA, A12, B3 and D11 on the plasmidic fragment BlnI-5 (112 kbp); D1 on BlnI-6 (109 kbp); D10 on BlnI-7 (100 kbp); asnT on BlnI-8 (92 kbp); thrW and A9 on BlnI-9 (76 kbp). Probes that hybridized on the same NotI restriction fragment: iucA, A12, B3, D11 on the plasmidic fragment NotI-1 (>600 kbp); metV, tktA, pheV on NotI-2 (>600 kbp); pheU and leuX on NotI-3 (514 kbp); A12 on NotI-4 (385 kbp); thrW and A9 on NotI-5 (273 kbp); fruA on NotI-6 (232 kbp); selC and D7 on NotI-7 (169 kbp); D10 on NotI-8 (152 kbp); D11, B7 and A12 on NotI-9 (129 kbp); asnT and D11 on NotI-10 (93 kbp); D1 on NotI-11 (56 kbp). Probes that hybridized on the same SfiI restriction fragment: pheU and leuX on SfiI-1 (365 kbp); fruA and A12 on SfiI-2 (318 kbp); thrW and A9 on SfiI-3 (312 kbp); metV on SfiI-4 (222 kbp); B7 and A12 on SfiI-5 (216 kbp); D1 on SfiI-6 (167 kbp); D10 on SfiI-7 (153 kbp); asnT on SfiI-8 (109 kbp); D11 on SfiI-9 (82 kbp); tktA and pheV on SfiI-10 (73 kbp); iucA, A12 and B3 on the plasmidic fragment SfiI-11 (47 kbp); D11 on the plasmidic fragment SfiI-12 (36 kbp); selC and D7 on SfiI-13 (31 kbp). Probes that hybridized on the same XbaI fragment: pheU and leuX on XbaI-1 (>600 kbp); D11, B7 and A12 on XbaI-2 (456 kbp); thrW and A9 on XbaI-3 (425 kbp); asnT on XbaI-4 (345 kbp); D10 on XbaI-5 (279 kbp); selC and D7 on XbaI-6 (236 kbp); A12 on XbaI-7 (221 kbp); fruA on XbaI-8 (205 kbp); metV on XbaI-9 (197 kbp); D1 on XbaI-10 (168 kbp); tktA and pheV on XbaI-11 (153 kbp); iucA, A12, B3 and D11 on plasmidic fragment XbaI-12 (128 kbp).

 
Two probes regularly co-localized with tRNA: A9 with thrW (smallest restriction fragment of 76 kbp, BlnI-9) and D7 with selC (smallest restriction fragment of 31 kbp: SfiI-13) (Fig. 1).

The iucA-amplified probe was used as a marker for ColV plasmid DNA fragments. A12, B3, D11 and the iucA probe hybridized on the same 112 kbp fragment BlnI-5 and on the same 128 kbp fragment XbaI-12 of strain MT512, confirming their localization on the ColV plasmid of MT512 (Fig. 1). The restriction pattern and the probe localization indicated that this plasmid was not cut with the restriction enzyme NotI (fragment NotI-1), and possessed at least two SfiI cleavage sites (fragments SfiI-11 and SfiI-12).

However, the A12 and D11 probes were also shown to hybridize on chromosomal DNA fragments as assessed by their co-localization with probes from chromosomal genes: tktA, metV, fruA and pheV (BlnI-1 fragment). Furthermore, the D11 probe co-localized with the asnT probe on a 93 kbp fragment (NotI-10), and the A12 probe co-localized with the fruA probe on a 318 kbp fragment (SfiI-2) (Fig. 1). These results indicated that duplicates of A12 and D11 could exist at both chromosome and plasmid locations.

The B7 probe hybridized with several restriction fragments of strain MT512. Using the complete lambda phage genome as a probe we could then demonstrate the presence of lambdoid phage sequences at different positions on the chromosome of strain MT512, confirming the results obtained with the B7 probe. Moreover, the B7 and the lambda phage probes co-localized with A12 on a 129 kbp chromosomal fragment (NotI-9), and with A12 and D11 on a 456 kbp fragment (XbaI-2).

The D1 and D10 probes did not co-localize with any of the other tested probes, whatever the restriction enzyme used (Fig. 1).

The observed co-localization of some of the tRNAs was in agreement with the genetic map of E. coli MG1655. tRNA tktA and pheV co-localized on a 153 kbp fragment (XbaI-11), and pheU and leuX co-localized on a 365 kbp fragment (SfiI-1).

Using the genetic map of E. coli MG1655 as reference, a hypothetical scheme of MT512 chromosome was then drawn, based on the hybridization results (Fig. 2).



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Fig. 2. Hypothetical location of additional sequences in the genome of the APEC strain MT512. The genome of E. coli MG1655 was used as a reference to locate tRNA loci and genes on the genome of E. coli MT512. Additional sequences in the genome of E. coli MT512 were approximately located according to the vicinity of a tRNA locus: *, <93 kb away from asnT; **, <76 kb away from thrW; ***, <31 kb away from selC. D1 and D10 could not be located.

 
Presence of genes corresponding to the isolated genomic fragments in ExPEC strains from humans and animals
As the sizes of the isolated fragments from MT512 were only about 200 kbp, we determined if these fragments represented a whole gene or part of a gene. Fragments B3 and B6 were not investigated as they could not correspond to a putative virulence gene; nor fragment B7 that corresponds to the large subunit of lambdoid phage terminase. The whole genes corresponding to genomic fragments A9, A12, D1, D7, D10 and D11 were detected by amplification in the APEC strain MT512 (primers 15–20, F and R, Table 1) and they were then searched for in ExPEC strains of human and animal origin.

The six genes were detected in all eight E. coli strains from human neonatal meningitis (Table 5), but only some of them were present in E. coli strains from urinary tract infection, with the exception of strain 536 and strain J96. The sequence corresponding to fragment A9 was not detected in E. coli EDL933, confirming that the observed homology between the putative product of A9 and Z0255 (Table 4) was restricted to the protein level. Among the seven ExPEC strains of animal origin, none possessed all of the six genes (Table 5).


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Table 5. Presence of genes from APEC in ExPEC strains of various origins

Hu, Human origin; Av, avian origin; ds, different size – the size of the amplified fragment was lower than expected.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
APEC strains are major pathogens for poultry, however, our knowledge of virulence factors is still incomplete. A genomic approach, such as subtractive hybridization, is well-adapted to screen for genomic regions that differentiate pathogenic from non-pathogenic strains and that could represent good candidates involved in virulence. Using the subtractive hybridization method on the APEC strain MT512 (O2 : K1 : H7) we isolated 62 fragments among which 17 (27·4 %) were specific for strain MT512, and were absent from the genome of the non-pathogenic strain EC79, used as the subtractive strain. None of the fragments that differentiate E. coli strain MT512 from strain EC79 were recovered in this experiment, as shown by the absence of sequences belonging to the aerobactin operon or to the kps gene cluster, for instance. Using the same method on an APEC strain of O78 serogroup, Brown & Curtiss (1996) identified 12 unique regions as compared to E. coli K-12. More recently, Stocki et al. (2002) identified 62 fragments specific for two avian strains (48 fragments specific for one strain and 14 specific for the other strain) following suppression subtractive hybridizations against an E. coli K-12 strain. No common sequences were identified between our results and those of Brown & Curtiss (1996), or Stocki et al. (2002). This confirms that numerous fragments differentiate pathogenic from non-pathogenic E. coli strains, and indicates the limits of the subtractive hybridization method that mainly results in an enrichment of specific sequences.

Even though the number of recovered specific sequences of MT512 was low, the subtractive hybridization method used here gave good results, as it resulted in the identification of nine DNA fragments which were found to be associated with the pathogenicity of APEC strains. These fragments are not present in the genome of E. coli strains HB101 or MG1655. The efficacy of the subtractive procedure used here was confirmed by the recovery of clones B3 and B6, which harboured inserts with highly homologous sequences to the main replicon of ColV plasmids (Ambrozic et al., 1998). They are probably part of the large plasmid of strain MT512 (130 kb) which belongs to the ColV plasmid family, whereas strain EC79 possesses no plasmid (M. Moulin-Schouleur, unpublished data).

The other DNA fragments identified are very probably of interest for virulence. Fragment B7 corresponded to the gp2 gene encoding the major subunit of the lambdoid bacteriophage 21. Wang et al. (1997) have demonstrated the presence of a specific fragment of bacteriophage 21 in strains belonging to the ECOR collection. Although phage 21 was globally distributed throughout the major groups of the ECOR collection (39 %), it was relatively infrequent among strains of the A and B1 groups. In contrast, it was detected in 13 out of the 15 strains of group B2 which corresponds to a group of highly pathogenic strains frequently implicated in extra-intestinal infections (Bingen et al., 1998; Boyd & Hartl, 1998; Goullet & Picard, 1986). It thus seems that a correlation exists between the presence of bacteriophage 21 and the pathogenicity of E. coli strains. The observation that fragment B7 was only identified in APEC strains, not in non-pathogenic avian strains, supports such a correlation. More generally, some phages are known to be implicated in bacterial virulence (Cheetham & Katz, 1995), such as phages encoding Shiga-like toxins SLTI and SLTII or enterohaemolysin (Mühldorfer & Hacker, 1994). Phages are also implicated in the transfer of virulence genes. The presence of bacteriophage 21 in APEC strains will be investigated further in relation to the pathogenicity of these strains.

Despite the homology of translated sequences of D1 and D7 fragments with TktA from H. influenzae (Fleischmann et al., 1995) and a FruA homologue from Listeria innocua and Listeria monocytogenes (Glaser et al., 2001), respectively, D1 and D7 sequences did not show any homology at the nucleotide level with tktA and fruA genes of E. coli MG1655 (Blattner et al., 1997). Moreover, we showed that strain MT512 harbours both genes tktA and fruA in addition to D1 and D7 sequences. D1 and D7 fragments are parts of whole genes of the APEC strain MT512 that are absent in E. coli MG1655 and in the EHEC strain EDL933. However, these genes are present in the UPEC strain CFT073 and in other human ExPEC strains isolated from meningitis or urinary tract infections as shown in our study. In E. coli CFT073, whose genome has been sequenced, fragment D7 is part of gene c4485 that is included in a 7 kbp genomic island comprising eight genes encoding putative PTS components and located between yicH and yicI (Welch et al., 2002). Fragment D1 is 100 % identical to an internal part of gene c4759 in E. coli CFT073 that encodes a putative transketolase. This gene is located in a 16 kbp genomic island inserted between metE and ysgA that includes 19 putative ORFs (Welch et al., 2002). Only some of these ORFs have been assigned hypothetical functional products: putative permease, putative glucose-specific IIBC component of a PTS system and a carbonate kinase-like protein. No direct correlation between the presence of these islands and virulence properties is actually established. However, genes tktA and fruA are involved in sugar metabolism and one hypothesis is that the products of genes corresponding to fragments D1 and D7 might be involved in different physiological roles to that of tktA and fruA products, or could play a similar role but in particular conditions, such as bacterial infection. The presence of several genes encoding homologous proteins can also reflect the metabolic flexibility of pathogenic bacteria. Genes involved in sugar metabolism have already been described in pathogenic bacteria (Spiroplasma citri and Legionella pneumophila) and have been implicated in virulence (Gaurivaud et al., 2000; Cianciotto, 2001).

Most APEC strains possess and express the aerobactin iron-acquisition system (Dho-Moulin & Fairbrother, 1999), like MT512. As shown by the presence of fragments A12 and D11, the APEC strain MT512 should possess at least two additional systems involved in iron acquisition that might contribute to the fitness of the strain. Several iron-acquisition systems were similarly demonstrated in the APEC strain {chi}7122 (Dozois et al., 2003). The nucleotide sequence of fragment D11 exhibits identity to a part of gene iroD encoding a ferric enterochelin esterase, and we have shown that the whole gene is present in the APEC strain MT512, such as in human ExPEC strains studied, whereas it is absent in E. coli MG1655 and EDL933 strains. The iro operon may be plasmid- or chromosome-located and is part of a pathogenicity island (PAI) in several E. coli strains: in E. coli CFT073 the iro operon is contained in a 115 kbp PAI located at serX (Welch et al., 2002), and in E. coli 536 it is found in PAI III (68 kbp) which is located at thrW (Dobrindt et al., 2001). The iro operon can also be located on plasmids, for example plasmid p300 in an UPEC strain (Sorsa et al., 2003), and plasmids pTJ100 (GenBank accession no. AY567838), pColV (GenBank accession no. AF449500) and pAPEC-1 (GenBank accession no. AF449498, Dozois et al., 2003) of APEC strains. Fragment A12 is also part of a whole gene that is present in MT512 and human ExPEC strains in this study. Its sequence is 100 % identical to a part of gene sitA that is found in a genomic island in strain CFT073, inserted between ycfD and ycgX (Welch et al., 2002). Gene sitA is also present in plasmids pAPEC-1 (GenBank accession no. AY598030) and pJT100 (GenBank accession no. AY567838) of APEC strains, and in Shigella flexneri (Runyen-Janecky et al., 2003). The presence of gene sitA has also been demonstrated within the centisome 63 pathogenicity island of Salmonella Typhimurium strain LT2 where it encodes a putative iron transport system (Zhou et al., 1999). Acquisition of iron is essential for bacteria to survive in an host which produces high-affinity iron-binding proteins. The capacity for bacterial pathogens to possess and use different iron-acquisition systems seems to be very frequent, as multiple systems have been identified in numerous bacterial pathogens such as Vibrio cholerae, Shigella flexneri and H. influenzae (Weinberg, 1995). This emphasizes the importance of iron in pathogenicity. Moreover, the localization of fragments A12 and D11 on the ColV plasmid of MT512, like the aerobactin operon, strengthens the hypothesis of the role of this large plasmid in APEC virulence (Ike et al., 1992).

As with other fragments, D10 is part of a whole gene in strain MT512, which is also present in E. coli CFT073 (gene c3029) and in other human ExPEC strains, whereas it is absent in the genome of E. coli MG1655 and EDL933 strains. In E. coli CFT073, gene c3029 is part of a 14 kbp genomic island inserted between xseA and engA; its putative product shares homology with RatA of Salmonella Typhimurium that is located on CS54 island involved in colonization of the caecum of mice (Kingsley et al., 2003). On the other hand, no homology was found in the databases with the nucleotide sequence of fragment A9. However, the putative product of the gene containing A9 in strain MT512 (determined according to the extended sequence of A9, GenBank accession no. AY395687), shares homology with putative protein Z0255 of E. coli EDL933, whose function is unknown.

With the exception of A9, all the identified fragments isolated from APEC strain MT512 in this study show homology with parts of genomic islands described in pathogenic E. coli strains, and their localization has noticeable importance considering their putative role in pathogenicity. Fragments A9 and D7 are located near the tRNA loci thrW and selC, respectively, and could be part of PAIs, as defined by Hacker et al. (1997). Other fragments, such as A12 and D11 might also be located in PAIs as deduced from similarities of their putative products with SitA and IroD proteins which have been described as encoded by PAIs (Dobrindt et al., 2001; Janakiraman & Slauch, 2000). Fragments A12 and D11 also demonstrated both a chromosomal and a plasmid location that could indicate duplication or multiple acquisitions of similar genes by APEC. Further studies are needed to demonstrate the role of these genomic regions in the pathogenicity of APEC for chickens.

The presence of some fragments, such as A9 and D10 seemed to be related to the serogroup of the APEC strains. They were more frequently encountered in APEC strains of the same serogroup (O2) as strain MT512 than in strains of the O78 serogroup. This could be related to the observation that the genetic relationships between O2 and O78 strains are low (White et al., 1993). This result also strengthens the hypothesis of different virulence mechanisms expressed by APEC strains of O2 and O78 serogroups. The identified sequences could constitute preliminary results for the characterization of virulence determinants that differentiate O2 and O78 APEC strains.

Of nine genomic sequences identified in this study, at least six can be considered as potential new virulence factors and are parts of whole genes of APEC strain MT512. All these genes are also present in human ExPEC strains isolated from meningitis, and furthermore, these genes can also be found in strains isolated from urinary tract infections in humans or from septicaemia in animals. These observations strongly support the idea of close relationships between avian and human E. coli strains responsible for extra-intestinal infections. However, ExPEC strains from human and animal origins also share virulence factors such as P fimbriae, resistance to complement or iron-acquisition systems. Therefore, it is difficult to define at present if avian E. coli are specific for avian species or if they could be also responsible for human infections.


   ACKNOWLEDGEMENTS
 
We thank P. Gilot and P. Germon for critical reading of the manuscript, and Annie Brée for her precious help in animal experiments. We are grateful to M. Peloille for her participation in sequencing. F. K. was financially co-supported during her PhD by the INRA (National Institute for Agronomic Research) and by Sanders–Aliments (France). This work was also supported by a grant from the Commission of the European Communities (Contract FAIR6-CT-98-4093).


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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 20 April 2004; revised 16 June 2004; accepted 18 June 2004.



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