Identification of Escherichia coli O157 : H7 genes influencing colonization of the bovine gastrointestinal tract using signature-tagged mutagenesis

Francis Dziva, Pauline M. van Diemen, Mark P. Stevens, Amanda J. Smith and Timothy S. Wallis

Mammalian Enteric Pathogens Group, Division of Microbiology, Institute for Animal Health, Compton, Newbury, Berkshire RG20 7NN, UK

Correspondence
Timothy S. Wallis
timothy.wallis{at}bbsrc.ac.uk


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Enterohaemorrhagic Escherichia coli (EHEC) cause acute gastroenteritis in humans that may be complicated by life-threatening systemic sequelae. The predominant EHEC serotype affecting humans in the UK and North America is O157 : H7 and infections are frequently associated with contact with ruminant faeces. Strategies to reduce the carriage of EHEC in ruminants are expected to lower the incidence of human EHEC infections; however, the molecular mechanisms underlying persistence of EHEC in ruminants are poorly understood. This paper reports the first comprehensive survey for EHEC factors mediating colonization of the bovine intestines by using signature-tagged transposon mutagenesis. Seventy-nine E. coli O157 : H7 mutants impaired in their ability to colonize calves were isolated and 59 different genes required for intestinal colonization were identified by cloning and sequencing of the transposon insertion sites. Thirteen transposon insertions were clustered in the locus of enterocyte effacement (LEE), which encodes a type III protein secretion system required for the formation of attaching and effacing lesions on intestinal epithelia. A putative structural component of the apparatus (EscN) is essential for intestinal colonization; however, the type III secreted effector protein Map plays only a minor role. Other Type III secretion-associated genes were implicated in colonization of calves by E. coli O157 : H7, including z0990 (ecs0850), which encodes the non-LEE-encoded type III secreted effector NleD and the closely related z3023 (ecs2672) and z3026 (ecs2674) genes which encode homologues of Shigella IpaH proteins. We also identified a novel fimbrial locus required for intestinal colonization in calves by E. coli O157 : H7 (z2199-z2206; ecs2114-ecs2107/locus 8) and demonstrated that a mutant harbouring a deletion of the putative major fimbrial subunit gene is rapidly out-competed by the parent strain in co-infection studies. Our data provide valuable new information for the development of intervention strategies.


Abbreviations: A/E, attaching and effacing; EHEC, enterohaemorrhagic Escherichia coli; EPEC, enteropathogenic Escherichia coli; LEE, locus of enterocyte effacement; STM, signature-tagged mutagenesis; Stx, Shiga toxin(s); Amp, ampicillin; Kan, kanamycin; Nal, nalidixic acid


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Enterohaemorrhagic Escherichia coli (EHEC) were first recognized as a cause of human disease in 1983 and are associated with diarrhoea and haemorrhagic colitis, which may be complicated by life-threatening renal and neurological sequelae, including haemolytic uraemic syndrome and thrombocytopaenic purpura (Karmali et al., 1983; Paton & Paton, 1998; Riley et al., 1983). EHEC, and in particular serotype O157 : H7, have since emerged as a major cause of severe diarrhoeal illness worldwide and EHEC infections are the leading antecedent to paediatric acute renal failure in many countries (Ammon, 1997; Mead et al., 1999; WHO, 1997). E. coli O157 : H7 strains are estimated to cause 73 480 cases and 60 deaths per annum in the United States, with serotypes other than O157 : H7 accounting for a further 36 740 illnesses (Mead et al., 1999). Non-O157 EHEC are an emerging problem, and indeed may be more prevalent than serogroup O157 in some countries (Bettelheim, 2000). EHEC are defined by their ability to produce one or more Shiga toxins (Stx), which mediate the systemic complications of EHEC infections, and to induce attaching and effacing (A/E) lesions on intestinal epithelia in which the bacteria adhere intimately to the apical surface of enterocytes on raised actin-rich pedestals and microvilli are locally destroyed (Nataro & Kaper, 1998).

Healthy ruminants are the principal reservoir of EHEC and human infections are frequently associated with the consumption of meat or dairy products contaminated with ruminant faeces (Borczyk et al., 1987; Orskov et al., 1987). Outbreaks have also been associated with direct contact with the farm environment and with the consumption of water, fruit and vegetables contaminated with ruminant manure (Locking et al., 2001; O'Brien et al., 2001). Indeed, the largest outbreak so far recorded involved over 6000 school children in Sakai City, Japan, and was associated with the consumption of contaminated white radish sprouts from a single farm (Michino et al., 1999). Recent surveys of cattle presented for slaughter in the United States and UK have revealed faecal prevalence rates of E. coli O157 : H7 of 28 and 4·7 %, respectively (Elder et al., 2000; Paiba et al., 2002), indicating that there is significant potential for contamination of the food chain and environment. By using a stochastic simulation model Jordan et al. (1999) predicted that a reduction in the carriage of EHEC in ruminants will lead to a decline in the incidence of human EHEC infections. The options for control of EHEC in ruminants were recently reviewed (Stevens et al., 2002a).

Natural and experimental infection of cattle with E. coli O157 : H7 results in efficient colonization of the intestinal tract with large numbers of bacteria being shed in the faeces for several weeks (Brown et al., 1997; Cornick et al., 2002; Cray & Moon, 1995; Dean-Nystrom et al., 1999; Grauke et al., 2002; Naylor et al., 2003; Wray et al., 2000). Clinical signs of EHEC-infection in calves may vary from subclinical to dysentery depending on serotype- and host-specific factors, with E. coli O157 : H7 infections tending to be asymptomatic in all but neonatal colostrum-deprived calves (Dean-Nystrom et al., 1997). It has been reported that a region of lymphoid follicle-dense epithelium in the terminal rectum is the principal site of E. coli O157 : H7 persistence in adult cattle (Naylor et al., 2003); however, persistence at other distal sites in the bovine intestines and the rumen has been described (Cray & Moon, 1995; Brown et al., 1997; Dean-Nystrom et al., 1999; Grauke et al., 2002).

The molecular mechanisms underlying the carriage and virulence of EHEC in ruminants are poorly understood. A key locus mediating EHEC adherence in vitro and A/E lesion formation in vivo is the chromosomal locus of enterocyte effacement (LEE), which encodes a type III protein secretion system. One of the LEE-encoded type III secreted proteins (Tir) is translocated into the host cell plasma membrane where it acts as a receptor for the LEE-encoded outer-membrane protein intimin (DeVinney et al., 1999). Intimin is an important colonization factor for E. coli O157 : H7 in neonatal calves (Dean-Nystrom et al., 1998), lambs (Woodward et al., 2003), adult cattle and sheep (Cornick et al., 2002), gnotobiotic piglets (Donnenberg et al., 1993; McKee et al., 1995), rabbits (Ritchie et al., 2003) and streptomycin-treated mice (Judge et al., 2004). Intimin can also bind to {beta}1-integrins (Frankel et al., 1996) and cell surface-localized nucleolin (Sinclair & O'Brien, 2002); however, the importance of such interactions in colonization of the ruminant intestines is unknown. Mutation of Tir significantly impairs E. coli O157 : H7 colonization in calves (Stevens et al., 2004) and rabbits (Ritchie et al., 2003), indicating that the role of intimin in colonization can be explained, at least in part, by it binding to its translocated receptor. Translocation of Tir and other effector proteins into host cells requires the type III secreted EspA protein, which forms filaments connecting the bacteria to the host cell surface, as well as EspB and EspD, which are thought to form a membrane pore (reviewed by Frankel et al., 1998). EspA filaments are believed to act as a hollow conduit for the delivery of effectors; however, they also play a role in initial adherence (Cleary et al., 2004; Ebel et al., 1998), and an E. coli O157 : H7 non-polar espA deletion mutant exhibits reduced colonization in a murine model (Nagano et al., 2003). Immunization of cattle with E. coli O157 : H7 type III secreted proteins reduces faecal shedding of the bacteria following experimental infection (Potter et al., 2004); however, the contribution of the structural apparatus and individual type III secreted proteins in colonization and immunity in ruminants is unknown.

The only other EHEC factor reported to mediate intestinal colonization in calves is Efa1 (EHEC factor for adherence) (Stevens et al., 2002b). Efa1 was first identified as a factor influencing adherence of EHEC O111 : H to cultured epithelial cells (Nicholls et al., 2000) and is almost identical to an enteropathogenic E. coli (EPEC) factor (LifA, lymphostatin) that inhibits lymphocyte proliferation and proinflammatory cytokine synthesis (Klapproth et al., 2000). EHEC O5 and O111 efa1 mutants are markedly impaired in their ability to colonize calves and exhibit reduced adherence to intestinal mucosa; however, the mechanism of action of Efa1 remains obscure since efa1 mutations indirectly affect the expression and secretion of EspA and Tir (Stevens et al., 2002b). E. coli O157 : H7 lacks efa1, but encodes a full-length homologue on pO157 (toxB) and a truncated version of the gene (efa1'). Both genes have been implicated in adherence of E. coli O157 : H7 to cultured epithelial cells and are required for the expression and secretion of type III secreted proteins (Stevens et al., 2004; Tatsuno et al., 2001); however, recent studies have indicated that they do not influence intestinal colonization in calves or lambs by E. coli O157 : H7 (Stevens et al., 2004).

Adherence of EHEC to epithelial cells in vitro has been extensively studied and numerous putative adhesins have been identified by screening of EHEC transposon mutants, expression of EHEC gene libraries in E. coli K-12 or by blocking attachment with specific immune sera. These studies indicate that multiple elements contribute to EHEC adherence (Dytoc et al., 1993; Sherman et al., 1991; Tatsuno et al., 2000; Torres & Kaper, 2003). Roles have been identified for Iha, a 67 kDa outer-membrane protein in E. coli O157 : H7 similar to the product of Vibrio cholerae iron-regulated gene A (Tarr et al., 2000), the outer-membrane porin OmpA (Dytoc et al., 1993; Torres & Kaper, 2003), long polar fimbriae encoded by the E. coli O157 : H7 lpfABCC'DE operon (Torres et al., 2002), curli (Uhlich et al., 2002), an autoagglutinating adhesin (Saa) of LEE-negative Stx-producing O113 : H21 (Paton et al., 2001), Paa, which is required at an early stage of A/E lesion formation (Batisson et al., 2003), and certain flagella H-types (Girón et al., 2002).

The identification of putative EHEC virulence factors has been accelerated by determination of the complete genome sequence of strain EDL933, associated with an outbreak of haemorrhagic colitis in the United States in 1982 (Perna et al., 2001) and strain RIMD 0509952 isolated from the Sakai outbreak (Hayashi et al., 2001). Independent annotation of the genomes of the two strains has revealed the strains to be largely similar; however, comparison of the EDL933 genome with that of the non-pathogenic E. coli K-12 strain MG1655 has shown that it contains approximately 1·34 Mb of additional sequence distributed in 177 ‘O-islands' of greater than 50 bp in length (Perna et al., 2001). The function of only 40 % of O157-specific genes can be assigned at present. Acquisition of Stx-encoding bacteriophages by EPEC is believed to have contributed to the evolution of EHEC and therefore virulence genes may be conserved in both pathovars. Studies on the factors involved in EHEC intestinal colonization may therefore be relevant to EPEC, which are a major cause of infantile diarrhoea in the developing world.

To increase our knowledge of EHEC factors mediating colonization of the bovine intestines, we constructed a library of signature-tagged transposon mutants of E. coli O157 : H7 and screened the mutants by oral inoculation of calves. Signature-tagged mutagenesis (STM) is a DNA hybridization-based approach that allows the simultaneous screening of pools of mutants and identification of colonization-defective mutants by negative selection (Hensel et al., 1995). STM has proven to be extremely valuable in dissecting the molecular mechanisms underlying intestinal colonization by a variety of pathogens (West et al., 2003). Our data reveal key roles for the LEE-encoded type III secretion system and a novel fimbrial locus in colonization of the reservoir host by E. coli O157 : H7.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Bacterial strains, plasmids and growth conditions.
Bacterial strains and plasmids used in this study are listed in Table 1. Bacteria were cultured using Luria–Bertani (LB) broth or agar supplemented as appropriate with 20 µg nalidixic acid (Nal) ml–1, 50 µg kanamycin (Kan) ml–1 or 100 µg ampicillin (Amp) ml–1 and stored in medium supplemented with 15 % (v/v) glycerol at –70 °C. For oral inoculation studies bacteria were grown in brain heart infusion (BHI) broth (Oxoid). E. coli O157 : H7 was selected from faecal samples by growth on sorbitol MacConkey agar supplemented with 2·5 µg potassium tellurite ml–1 (T-SMAC) and antibiotics as appropriate. Minimal medium consisted of M9 salts plus 0·9 % (w/v) D-(+)-glucose. A spontaneous Nal-resistant derivative of E. coli strain EDL933 (Riley et al., 1983) was obtained by plating approximately 109 c.f.u. on T-SMAC containing 20 µg Nal ml–1. This strain, designated EDL933 NalR, exhibited identical growth kinetics and in vitro adhesion properties to the parent strain and was positive by PCR for the genes eae, stx1, stx2, espA, ehxA and toxB (data not shown).


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Table 1. Bacterial strains and plasmids used in this study

 
Routine DNA manipulation techniques.
Standard techniques were used for DNA isolation, PCR, agarose gel electrophoresis, cloning, transformation, conjugation and hybridization (Sambrook et al., 1989). Restriction endonucleases, T4 DNA ligase and DNA polymerase were purchased from New England BioLabs.

Generation of a library of E. coli O157 : H7 signature-tagged transposon mutants.
A library of 2850 signature-tagged transposon mutants of E. coli O157 : H7 was constructed as described by Hensel et al. (1995), with minor modifications. Signature-tagged transposons (kindly supplied by Dr C. M. Tang, Imperial College London) were transferred into E. coli strain EDL933 NalR in 95 individual conjugative matings from E. coli S17-1{lambda}pir(pUTmini-Tn5Km2). Insertion mutants were selected on LB medium containing Nal and Kan. Mutants were examined for sensitivity to Amp to exclude bacteria that acquired Kan resistance by chromosomal integration of pUTmini-Tn5Km2. Auxotrophs were identified by absence of growth on M9 minimal salts medium and excluded from the library. Mutants were arrayed into 96-well plates such that all mutants in a given plate possessed a unique signature tag and the library stored at –70 °C in LB containing 15 % (v/v) glycerol.

Calf oral challenge model.
All animal experiments were performed in accordance with the Animal Scientific Procedures Act (1986) and approved by the local Ethical Review Committee. Friesian bull calves were used in this study and inoculated at 10–14 days of age. Calves were housed in containment level 3 accommodation in tanks on tenderfoot mats and fed 2 l milk replacer twice daily with free access to water. Prior to infection calves were confirmed to be culture-negative for EHEC and Salmonella by direct plating of rectal swabs on T-SMAC or Brilliant Green Agar (Oxoid), respectively. Presumptive EHEC colonies were screened for stx1 and stx2 genes by PCR using the primer pairs Stx1F (5'-ATAAATCGCCATTCGTTGACTAC-3')+Stx1R (5'-AGAACGCCCACTGAGATCATC-3'), and Stx2F (5'-GGCACTGTCTGAAACTGCTCC-3')+Stx2R (5'-TCGCCAGTTATCTGACATTCTG-3'). Animals excreting stx-positive E. coli or Salmonella were excluded from the study. All calves received colostrum from their respective dams for the first 24–48 h. Thereafter, no further colostrum was given. Total serum-Ig levels were measured at 1 or 2 days of age as a measure of colostrum intake by zinc sulphate turbidity assay. Only calves with a zinc sulphate turbidity measurement over 10 were used.

For calf oral challenge studies with single mutants, bacteria were amplified in BHI broth for 18 h at 37 °C and the OD600 was adjusted to approximately 1·2 (within 0·1 OD600 units). Approximately 1010 c.f.u. was given orally by syringe with the morning feed. Faeces were sampled twice daily by rectal palpation and animals were observed for intake of food and water, hydration status, diarrhoea severity and pyrexia. Viable bacteria excreted in the faeces were enumerated by plating triplicate tenfold dilution series onto T-SMAC containing appropriate antibiotics. Recovery of the EDL933 NalR map : : Tn mutant was confirmed by PCR analysis of isolated colonies using the primers map1 (5'-TATACCTGACAAGGTTATCCC-3') and map2 (5'-GTAGATAATGCCGGAGGATAG-3'), and recovery of EDL933 NalR escN : : Tn was confirmed using escN-for (5'-GATTGAAGGTTCGTTCC-3') and escN-rev (5'-CGAGTTTACCATCCACC-3').

Competition assays were performed essentially as described above, except that cultures of wild-type and mutant strains were adjusted to the same OD600 and mixed in a 1 : 1 ratio prior to inoculation. The ratio of wild-type to mutant bacteria was determined in the inoculum and in faecal samples collected twice daily thereafter by plating aliquots of the same tenfold dilutions (performed in triplicate) to both T-SMAC-Nal and T-SMAC-Nal-Kan agar. The number of viable wild-type bacteria was calculated by subtracting the viable count on T-SMAC-Nal-Kan from that obtained using T-SMAC-Nal. The competitive index for each day was determined by dividing the ratio of the mutant to wild-type bacteria in the faeces by the ratio of mutant to wild-type bacteria in the input. Animals were humanely killed at the end of the experiment or if pre-defined end points were reached by intravenous administration of pentobarbitone sodium.

Where appropriate faecal shedding data were statistically analysed after a log10 transformation for the effect of mutation by means of an F-test, with the data taken as repeated measurements (Proc Mixed, Statistical Analysis System; SAS Institute). P values <0·05 were taken to be significant.

Screening of the mutant library and identification of non-colonizing mutants.
E. coli O157 : H7 transposon mutants defective in intestinal colonization in calves were identified essentially as described by Hensel et al. (1995), with minor modifications. Briefly, a set of 95 uniquely tagged mutants were individually amplified in LB overnight in 96-well plates at 37 °C with gentle agitation and then pooled. Approximately 1010 c.f.u. of pooled mutants were given orally to a single calf just before the morning feed. Bacterial cells from a 1 ml aliquot of the inoculum were collected by centrifugation and stored (‘input pool’). The 95 mutants were individually replicated onto duplicate LB-Nal-Kan agar plates and bacterial colonies transferred onto nylon membranes (Hybond-N, Amersham Pharmacia Biotech). Membranes were prepared for hybridization following alkaline lysis of bacteria and UV-cross-linking. An output pool was recovered from the faeces at 5 days post-inoculation. The number of viable bacteria being excreted per gram of faeces was estimated by plating of serial tenfold dilutions on T-SMAC-Nal-Kan agar on day 4 post-inoculation, then a suitable dilution plated on the fifth day post-inoculation to yield a representative output pool (in excess of 10 000 colonies). Colonies were harvested and pooled in 5 ml LB broth. Bacteria from 150 µl of this suspension were collected by centrifugation (‘output pool’). Chromosomal DNA was isolated from both input and output pools by cetyltrimethylammonium bromide/phenol extraction. Signature tags from both pools were then amplified by PCR in the presence of [32P]dCTP using the primers P2 (5'-TACCTACAACCTCAAGCT-3') and P4 (5'-TACCCATTCTAACCAAGC-3'). Primer sequences were removed from the amplified products by HindIII digestion and the labelled tags separately hybridized with identical colony blots.

The DNA flanking the site of transposon insertion from attenuated mutants was cloned by ligation of EcoRI- or EagI-restricted genomic DNA into similarly restricted pBluescript KS(+) and transformed into chemically competent E. coli TOP10F' cells (Invitrogen). Transformants were selected on LB-Kan-Amp agar. Plasmid DNA was purified using a QIAprep spin miniprep kit (Qiagen). The insert DNA sequence was obtained using a mini-Tn5Km2-specific primer, P6 (5'-CCTAGGCGGCCAGATCTG-3') for EagI clones or P10 (5'-TCCTCTAGAGTCGACCTGC-3') for EcoRI clones (Lark Technologies). Sequences were analysed using the BLASTN search algorithm at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/blast/) and coliBASE (http://colibase.bham.ac.uk).

Cell adherence assays.
HeLa cells (ATCC CCL2), grown to semi-confluency on coverslips in RPMI1640 buffered with 2 g sodium bicarbonate l–1 and supplemented with 10 % (v/v) fetal calf serum (PAA Laboratories), and 0·3 g L-glutamine l–1 were used to assess bacterial adherence and nucleation of F-actin at the site of attachment. An adaptation of the procedure described by Stevens et al. (2002b) was used to semi-quantitatively assess bacterial adherence. Briefly, overnight static LB cultures were standardized to an OD600 of 0·1 using Dulbecco Minimum Essential Medium (DMEM) plus 0·4 % (w/v) NaHCO3 and 1 % (w/v) D-(+)-mannose, diluted 1 : 2 with the same medium, then 1 ml was added onto the cells and incubated at 37 °C in a humidified 5 % CO2 atmosphere. Cells were washed twice at 3 and 6 h post-inoculation with fresh medium, then six times at 8 h post-inoculation before fixing overnight in 4 % (w/v) paraformaldehyde at 4 °C. Cells were stained for F-actin using FITC-conjugated phalloidin and for bacteria using rabbit anti-O157 typing serum, detected with anti-rabbit-Ig-Alexa568 (Molecular Probes), and viewed using a confocal laser scanning microscope as described by Stevens et al. (2002b). Adherence was quantified as the mean number of adherent bacteria per cell from three random fields each containing about 100 cells in triplicate assays of each mutant. The ability of each mutant to nucleate F-actin under the sites of bacterial attachment (FAS reactivity) was also scored.

Detection of EspD by Western blotting.
To measure secretion of the LEE-encoded type III secreted protein EspD, bacteria were grown to late-exponential phase (OD600 of about 1·0) in Minimal Essential Medium buffered with 25 mM HEPES. Bacteria were removed by centrifugation and secreted proteins were precipitated from the supernatant with 10 % (v/v) trichloroacetic acid overnight at 4 °C as described by McNally et al. (2001). Proteins were collected by centrifugation (about 10 000 g), washed once in acetone, resuspended in electrophoresis buffer and analysed by Western blotting using a mouse anti-EspD serum (kindly supplied by Dr D. Gally, University of Edinburgh) detected with a horseradish peroxidase-conjugated goat anti-mouse immunoglobulin secondary antibody. Blots were developed with 4-chloro-1-naphthol and 30 % (v/v) hydrogen peroxide (Sambrook et al., 1989), and EspD secretion levels were compared to internal positive (wild-type) and negative (escN mutant) controls.

Construction of a defined {Delta}z2200 : : KanR mutant of E. coli EDL933 NalR.
Replacement of gene z2200 with a Kan resistance cassette was achieved by integration of linear DNA following transient expression of bacteriophage {lambda} Red recombinase as described by Datsenko & Wanner (2000). Briefly, a Kan resistance cassette was amplified from plasmid pKD4 using primers MFS1P1 (5'-AATTGTTAACTCTCTTTATTCAGCTACTTAAATATAAATTTTGGAGAATTGTGTAGGCTGGAGCTGCTTC-3') and MFS2P2 (5'-CACACACCCTGTTTTAATTTCAGATGCCGTATAGCCACCTTAATATTGAACATATGAATATCCTCCTTAG-3'). The 5' termini of these primers comprise 50 nt identical to the sequence flanking the predicted start or stop codons of gene z2200 with the 3'-terminal 20 nt priming from pKD4. The amplified product was concentrated on Qiagen spin-columns, digested with DpnI for 1 h at 37 °C, then purified following agarose gel electrophoresis using a Qiagen gel purification kit. EDL933 NalR was transformed with the temperature-sensitive {lambda}Red-encoding plasmid pKD46 with selection for Amp resistance at 30 °C. The z2200 : : KanR PCR product was used to transform electrocompetent EDL933 NalR(pKD46) following induction of expression of {lambda}Red recombinase by growth in LB in the presence of 10 mM L-arabinose at 30 °C essentially as described by Datsenko & Wanner (2000). Mutants were selected on LB-Nal-Kan agar and confirmed to carry the insertion by PCR using the z2200 flanking primers MFS-for (5'-CTCTCTTAATTCAGCTACTT-3') and MFS-rev (5'-TTCAGATGCCGTATAGCCAC-3'). Plasmid pKD46 was cured from the mutant by overnight growth at 37 °C and the mutant was confirmed to be Amp-sensitive.


   RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Model for screening E. coli O157 : H7 signature-tagged transposon mutants
Our laboratory has studied intestinal colonization of conventional Friesian bull calves by EHEC strains of serotypes O5 : H, O111 : H, O157 : H7 (Stevens et al., 2002b, 2004) and O26 : H (P. M. van Diemen, F. Dziva, M. P. Stevens & T. S. Wallis, unpublished). Following oral inoculation of 4- to 14-day-old calves with about 1010 c.f.u., the EHEC strains can reliably be recovered in the faeces at >105 c.f.u. g–1 for the first 7 days post-inoculation. Furthermore, we have shown that E. coli O157 : H7 {Delta}tir, EHEC O5 {Delta}efa1 and EHEC O111 efa1 : : KanR mutants are shed in significantly lower numbers than the respective parent strains at just 5 days post-inoculation of calves (Stevens et al., 2002b, 2004), suggesting that the model can discriminate between colonizing and non-colonizing bacteria at this time. Indeed, a non-pathogenic prototrophic E. coli O43 : H28 isolate from healthy pigs could not be detected in the faeces just 6 days after inoculation of calves with about 1010 c.f.u. (P. M. van Diemen, F. Dziva, M. P. Stevens & T. S. Wallis, unpublished).

For STM studies we elected to use the sequenced E. coli O157 : H7 strain EDL933. A Nal-resistant derivative of EDL933 was constructed to enable counter-selection during transposon mutagenesis and to facilitate bacterial recovery from animals. No difference in the in vitro growth rate or cell adherence properties of the NalR mutant were observed relative to the parent strain (data not shown). In pilot experiments, about 1010 c.f.u. EDL933 NalR was given orally to four 10- to 14-day-old Friesian bull calves to determine the kinetics of bacterial excretion. In excess of 105 c.f.u. EDL933 NalR faeces g–1 were excreted for the first 7 days in the absence of clinical signs (data not shown). We then assessed if non-colonizing signature-tagged mutants of EDL933 NalR could be reliably identified by inoculating two calves with a single pool of 95 mutants, with recovery of output pools on days 5 and 10 days post-inoculation. Signature-tags were amplified from the input and output pools of EDL933 NalR mini-Tn5Km2 mutants in the presence of [32P]dCTP and hybridized to DNA from each of the individual mutants gridded on nylon membranes. Hybridization patterns were reproducible between output pools recovered from the two calves at 5 days post-inoculation (Fig. 1). Mutants exhibiting reduced colonization in calves were identified by the loss of hybridization signals on output blots compared to input blots. The hybridization patterns at 10 days post-inoculation accurately reproduced those at 5 days post-inoculation and were similar from both calves (data not shown). The number of non-colonizing mutants identified as a proportion of the input pool was comparable to that observed in STM screens with other pathogens.



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Fig. 1. Representative hybridization results from the initial screen of E. coli O157 : H7 EDL933 NalR signature-tagged transposon mutants in calves. Colony blots were probed with 32P-labelled tags amplified from bacteria in the inoculum (a) and from bacteria recovered from the faeces of two separate calves at 5 days post-inoculation (b, c).

 
For reasons of practicality and cost calves rather than adult ruminants were used in the present study. Recent studies have indicated that a region of follicle-associated epithelium in the terminal rectum may be the principal site of E. coli O157 : H7 colonization in adult cattle (Naylor et al., 2003). It is not known if E. coli O157 : H7 exhibits a specific tropism for the terminal rectum in calves or if lymphoid follicles have matured at this site in animals of the age used; therefore, output pools were recovered from faeces rather than intestinal mucosa. Follicle-associated epithelium is not restricted to the terminal rectum in calves and indeed is abundant in the proximal colon and distal ileum (Liebler et al., 1988; Parsons et al., 1991). Further studies will be required to determine the principal site(s) of E. coli O157 : H7 persistence in calves before an attempt can be made to identify bacterial factors mediating mucosal adherence at the appropriate location.

Identification of E. coli O157 : H7 genes required for intestinal colonization in calves
A total of 1900 EDL933 NalR mini-Tn5Km2 mutants were screened by oral inoculation of 10- to 14-day-old calves with recovery of output pools at 5 days post-inoculation. One hundred and one mutants were identified as absent or poorly represented in output pools relative to the inocula. The site of transposon insertion was determined in 79 mutants by subcloning of EcoRI or EagI fragments from genomic DNA with selection for mini-Tn5Km2-encoded resistance to Kan. Sequences were aligned to the genome of EDL933 and other pathogens using the BLASTN algorithm. Fifty-nine different genes were implicated in colonization of the bovine gastrointestinal tract by E. coli O157 : H7. Some genes required for intestinal colonization were independently identified on more than one occasion. E. coli O157 : H7 genes required for intestinal colonization of calves were grouped into six arbitrary categories: (i) type III secretion-associated genes, (ii) genes encoding surface structures, (iii) other O-island genes, (iv) regulatory genes, (v) genes involved in central intermediary metabolism and (vi) hypothetical genes (Table 2). Selected findings are highlighted below.


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Table 2. E. coli O157 : H7 genes required for intestinal colonization in calves

The site of transposon insertion relative to the predicted start codon, the gene number (EDL933 annotation) and results of in vitro assays are shown.

 
(i) Type III secretion-associated genes.
Thirteen transposon insertions mapped to the LEE, indicating that it plays a key role in intestinal colonization of calves by E. coli O157 : H7. The LEE-encoded intimin and Tir proteins are known to be required for colonization of the bovine intestines by E. coli O157 : H7 (Dean-Nystrom et al., 1998; Cornick et al., 2002; Stevens et al., 2004), but were not disrupted in any of the mutants isolated. Since Tir is delivered into host cells via the type III secretion system it could be inferred that structural components of the apparatus would also be required for colonization; however, this has previously not been established in calves. We isolated five escC mutants, two escV mutants and single escN, sepQ and rorf3 mutants (Table 2). Mutations in escC, escV and sepQ genes have previously been shown to prevent secretion of EspA and Tir in E. coli O157 : H7 and to impair adherence to Caco-2 cells (Tatsuno et al., 2000). EscN is a putative inner membrane ATPase but the function of rOrf3 is unknown. Both EscN and rOrf3 are required for secretion of EspB and Tir in Citrobacter rodentium (Deng et al., 2004). Consistent with these data, all mutants lacking putative structural components of the apparatus were observed to show reduced secretion of EspD, weak adherence to HeLa cells and an absence of F-actin nucleation under sites of attachment (Table 2).

Mutants were also identified that have defects in LEE-encoded secreted proteins/chaperones. Mutant 19F5 harboured an insertion in cesF (rorf10) which encodes a chaperone for the secreted effector protein EspF. In EPEC, CesF is required for the ability of EspF to cause a loss of transepithelial resistance and increased paracellular permeability in infected monolayers (Elliott et al., 2002). In contrast, mutation of cesF in E. coli O157 : H7 reduces the expression of EspF but does not dramatically alter the reduced transepithelial resistance phenotype, possibly owing to the presence of two other EspF-like homologues encoded by cryptic prophages CP-933U (U-EspF) and CP-933M (M-EspF) (Viswanathan et al., 2004). It is presently unknown whether CesF is required for the secretion of the EspF-like proteins or indeed other effectors. Mutants were also isolated with insertions in espD and map. EspD is required for the formation of EspA filaments and is translocated into the host cell plasma membrane where it is believed to mediate pore formation in concert with EspB (Kresse et al., 1999; Wachter et al., 1999; Ide et al., 2001). Map (mitochondrial associated protein, Orf19) is translocated into host cells, localizes to mitochondria and collapses their membrane potential (Kenny & Jepson, 2000). Map also induces filopodia formation following initial contact between EPEC and host cells (Kenny et al., 2002; Jepson et al., 2003). Screening of signature-tagged transposon mutants of C. rodentium by oral inoculation of mice revealed escD, orf4, espD, eae and tir to be required for intestinal colonization (Mundy et al., 2003), confirming that the LEE is a key conserved colonization factor of A/E pathogens. Mundy et al. (2003) also observed that a transposon mutant with an insertion 24 bp upstream of map was attenuated following oral inoculation of mice.

Type III secretion-associated genes unlinked to the LEE were also implicated in colonization of calves by E. coli O157 : H7. An insertion was mapped to z0990 (ecs0850), which was recently reported to encode a homologue of the C. rodentium non-LEE-encoded type III secreted effector NleD (Deng et al., 2004). NleD is encoded proximal to another type III secreted effector (NleC/Z0986) in cryptic prophage CP-933K (O-island 36); however, it is unlikely that the transposon insertion would be polar since nleD is predicted to be the last gene in an operon. We also isolated mutants with insertions in z3026 (ecs2674), 5' of z3023 (ecs2672) and in z3022 (ecs2671/yedL). The hypothetical z3022 (yedL) gene is conserved in E. coli K-12 but separates two O157-specific genes (z3023 and z3026), the products of which are 92 % identical to each other and about 50 % similar in a 100 aa overlap to Shigella IpaH proteins. Five copies of the ipaH gene exist on the large plasmid of Shigella flexneri, each encoding a conserved C-terminal portion containing leucine-rich repeats, but differing in the sequence encoding the N termini. IpaH9·8, IpaH7·8 and IpaH4·5 are known to be type-III-secreted by S. flexneri (Buchrieser et al., 2000). IpaH7·8 facilitates bacterial escape from endosomes (Fernandez-Prada et al., 2000) and IpaH9·8 is translocated to the nucleus (Toyotome et al., 2001).

(ii) Genes encoding surface structures.
A mutant was isolated with an insertion in z2203 which encodes a putative fimbrial usher protein and forms part of a fimbrial locus part conserved in E. coli K-12 and shared by EDL933 (z2199z2206) and the Sakai outbreak strain RIMD 0509952 (ecs2114ecs2107; referred to as locus 8 in the supplementary material in Hayashi et al., 2001). EDL933 contains 10 putative fimbrial loci, three of which are conserved in E. coli K-12, and little is known about their relative contribution to virulence.

One mutant was isolated (9B3) with an insertion 1729 bp upstream of the known E. coli O157 : H7 adhesin Iha, in the intergenic region separating two small hypothetical ORFs of unknown function (z1182 and z1181). The 9B3 mutant showed a slight reduction in adherence in vitro; however, we were unable to reproducibly demonstrate a marked effect of the 9B3 mutation on the expression of Iha by EDL933 NalR by Western blotting using an Iha-specific antibody (kindly supplied by Dr Phil Tarr, University of Washington, Seattle, USA) (F. Dziva & R. Rashid, unpublished observations). EDL933 is unusual in having a duplication of the O-island that encodes Iha, urease and tellurite resistance (O-islands #43 and #48); therefore, effects of the 9B3 mutation may be masked by expression of Iha from the distal locus.

Mutant 3D8 contains an insertion in a z0390 (ecs0350), which encodes a putative adhesin 24·16 % similar over 342 aa to the C-terminal domain of the high-molecular-mass adhesin HmwA of Haemophilus influenzae. HmwA plays a key role in mediating bacterial adherence to human epithelial cells (St Geme et al., 1993). The C-terminal domain of HmwA is thought to tether the adhesin to the bacterial surface and is required for adherence (Grass & St Geme, 2000).

Five insertions were clustered in O-island #84 in a locus required for biosynthesis of the E. coli O157 : H7 lipopolysaccharide (LPS) O-antigen (manC, mannose-1-phosphoguanosyltransferase; fcl, fucose synthetase; wbdP, glycosyltransferase; per, perosamine synthetase; and 3' of manB). A mutation affecting the putative perosamine synthetase (per/rfbE) has previously been reported to increase adherence of E. coli O157 : H7 to cultured HeLa cells (Bilge et al., 1996). LPS also plays a role in colonization of the intestines of infant rats by neonatal meningitis-associated E. coli K1 (Martindale et al., 2000) and other enteric pathogens (reviewed by West et al., 2003). Antibodies against LPS inhibit EHEC adherence to Henle 407 cells in vitro; however, pre-incubation of cells with LPS does not competitively inhibit adherence, suggesting that LPS is unlikely to be an adhesin per se (Paton et al., 1998; Sherman & Soni, 1988). E. coli O157 : H7 mutants deficient in O-antigen biosynthesis still form A/E lesions (Cockerill et al., 1996), and it remains unclear why LPS mutations impair intestinal colonization. LPS mutations have pleiotropic effects on cell envelope stability, cell surface hydrophilicity and the correct insertion and folding of membrane proteins and may also unmask underlying antigens.

An insertion was mapped to 30 bp upstream of a gene encoding a putative flagellin structural protein (z0469/ecs0464). This gene is conserved in E. coli K-12 (yaiU), is encoded distal to the flagellar biosynthesis locus and plays an unknown role. The role played by flagella and motility in EHEC persistence is unknown. Previous studies have shown that in contrast to the H6 flagellin of EPEC O127 : H6, the E. coli O157 : H7 flagellin does not influence adherence to cultured cells (Girón et al., 2002). Furthermore, non-motile sorbitol-fermenting E. coli O157 : H are a major cause of haemolytic uraemic syndrome in Germany (Karch & Bielaszewska, 2001), and O5 : H and O111 : H EHEC strains cause transient enteritis in calves with extensive mucosal adherence (Stevens et al., 2002b), indicating that flagella are not essential for carriage and virulence of EHEC.

(iii) Other O-islands.
An insertion was mapped to gene z3341 (ecs2970), which is encoded within CP-933V between the stx1AB genes encoding Stx1 and bacteriophage genes involved in host cell lysis. Stx-receptors exist on bovine crypt epithelial cells (Hoey et al., 2002) and recent studies have shown that Stx1 depletes a subset of intraepithelial lymphocytes in a bovine ligated ileal loop model of infection (Menge et al., 2004). It is possible that modulation of mucosal immune responses by Stx1 may facilitate persistence of EHEC in the intestines. However, by Western blotting using Stx1-specific rabbit polyclonal antiserum we did not detect any obvious differences in expression or secretion of Stx1 by the 11G2 mutant during growth in LB medium (data not shown). Further studies are required to dissect the role of Stx in colonization of the bovine intestines by EHEC, but are beyond the scope of the present study.

(iv) Regulatory genes.
An insertion was mapped to sdiA, which encodes a quorum sensing regulator thought to sense an acyl homoserine lactone autoinducer (AI-1) owing to its similarity to Vibrio fisheri LuxR. SdiA negatively regulates the expression of EspD and intimin in E. coli O157 : H7 (Kanamaru et al., 2000), and one might have anticipated that upregulation of LEE expression caused by mutation of sdiA would enhance the carriage and virulence of EHEC. Mutation of sdiA has been reported to increase the virulence of Salmonella enterica serovar Typhimurium following oral inoculation of mice (Volf et al., 2002). However, the observed defect in E. coli O157 : H7 carriage caused by mutation of sdiA may be explained by positive regulation of other colonization factors.

HrpA is a putative ATP-dependent RNA helicase that is involved in endonucleolytic cleavage of mRNA from the daa locus encoding F1845 fimbriae of diffusely adhering diarrhoeagenic E. coli (Koo et al., 2004). E. coli O157 : H7 lacks the daa fimbrial locus; however, it is possible that HrpA has a wider role in mRNA processing in E. coli. A mutant was also isolated with a transposon insertion in z2318 (ecs2011), which is encoded upstream of hrpA on the same strand. The effect of this mutation on hrpA expression is unknown.

The ymcC gene is conserved in other Gram-negative pathogens and E. coli K-12, and is encoded in a locus putatively involved in exopolysaccharide synthesis. Two independent insertions were mapped to arp, which encodes an ankyrin-like regulatory protein involved in fatty acid and phosphatidic acid biosynthesis. Two mutants were independently isolated that lack the putative regulator yibD, although the site of transposon insertion was identical in both cases. The targets of the YibD are unknown.

No plasmid-encoded genes were found to be required for intestinal colonization in the present study. This is consistent with an earlier study in which plasmid-cured strains of E. coli O157 : H7 colonized gnotobiotic piglets in a manner similar to the parent strain (Tzipori et al., 1987).

Characterization of E. coli O157 : H7 mutants defective in intestinal colonization
Since our data indicated that the LEE plays a key role in colonization of the bovine intestines by E. coli O157 : H7, we sought to determine if any of the transposon insertions unlinked to the LEE impaired intestinal colonization by indirectly affecting LEE function. Adherence was semi-quantitatively assessed using a standard HeLa cell assay. The secretion of EspD was assessed by Western blotting and nucleation of F-actin under sites of adherence scored as an indicator of LEE function. All mutants with transposon insertions located outside the LEE retained the ability to secrete EspD and were FAS-positive (Table 2). Only mutants lacking structural components of the LEE-encoded type III secretion apparatus exhibited a complete absence of adherence to HeLa cells; however, slight variations in the number of adherent bacteria per cell were seen in non-LEE mutants. Consistent with previous observations, mutations affecting map and cesF did not affect the secretion of EspD or development of FAS lesions.

To assess the degree of attenuation caused by mutation of a structural component of the type III secretion system and the secreted effector protein Map, we separately inoculated calves with EDL933 NalR or the transposon mutants EDL933 NalR escN : : Tn (9B5) or EDL933 NalR map : : Tn (11D4). Approximately 1010 c.f.u. of each mutant was separately administered to 10- to 14-day-old calves (3 calves per mutant strain and 2 calves for the wild-type) and the course of faecal excretion monitored twice daily for 7 days. Whilst the map transposon mutant was shed in comparable numbers to the parent strain for the duration of the experiment, the escN mutant was shed in significantly lower numbers than the parent from just 2 days post-inoculation (P<0·05) and was excreted at a level six orders of magnitude lower than the parent and map mutant by day 7 post-inoculation (Fig. 2). These data suggest that whilst type III secretion is crucial for intestinal colonization of calves by E. coli O157 : H7, the effector protein Map plays a subtle role that may only be evident in competition (e.g. during initial screening of the mutant bank). Consistent with these findings, a C. rodentium map mutant was found to be attenuated in competition following oral inoculation of mice (Mundy et al., 2003); however, in single infection studies a map mutant showed initial slight attenuation and induced less colonic hyperplasia, but was shed in higher numbers than the wild-type strain later in infection (Deng et al., 2004; Mundy et al., 2004). The data obtained by infection of calves with pure cultures of mutants identified in the primary screen validate the sensitivity of the STM approach for identifying EHEC colonization factors. We did not attempt to complement the escN mutant, since the independent isolation of 11 E. coli O157 : H7 mutants lacking structural components of the type III secretion apparatus indicates that it is extremely unlikely that the observed defect in intestinal colonization is the result of second-site mutations or polar effects.



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Fig. 2. Course of faecal excretion of E. coli O157 : H7 strain EDL933 NalR wild-type (diamonds, n=2), EDL933 NalR escN : : Tn (squares, n=3) and EDL933 NalR map : : Tn (triangles, n=3), following oral inoculation of 10- to 14-day-old calves. The data represent the least square mean±SEM.

 
We identified an E. coli O157 : H7 mutant with an insertion in z2203, which encodes a putative fimbrial usher protein. In a separate screen of EHEC O26 : H signature-tagged transposon mutants in calves an insertion in a downstream gene (z2205/ecs2108) was also found to impair intestinal colonization (P. M. van Diemen, F. Dziva, M. P. Stevens & T. S. Wallis, unpublished results), indicating that genes encoded by this fimbrial locus may play an important role in the carriage of EHEC. To address the role played by the putative fimbriae encoded by this locus we constructed a defined E. coli O157 : H7 mutant lacking z2200 (ecs2113) which encodes the putative major fimbrial subunit. This was accomplished by integration of a linear DNA fragment comprising a Kan resistance gene flanked by the sequences immediately 5' and 3' of the z2200 ORF following transient expression of phage {lambda} Red recombinase. The ability of the mutant strain to colonize the bovine intestines was then tested by co-infection of four 10- to 14-day-old calves with the EDL933 NalR parent strain and EDL933 NalR {Delta}z2200 : : KanR mutant, with recovery of bacteria twice a day over 12 days. The mean competitive index of the mutant strain at 6 days post-inoculation was just 0·05, at which point the reduction in the amount of the mutant strain being excreted compared to the wild-type was highly significant (P=0·005; Fig. 3). These data suggest that the attenuation observed in the original E. coli O157 : H7 z2203 transposon mutant was unlikely to be the result of a second-site mutation and further validate the STM method for screening EHEC mutants in calves.



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Fig. 3. Course of faecal excretion of E. coli O157 : H7 strain EDL933 NalR wild-type (diamonds) and EDL933 NalR {Delta}z2200 : : KanR (squares), following co-infection of four 10- to 14-day-old calves. The number of viable wild-type bacteria was calculated by subtracting the viable count on T-SMAC-Nal-Kan from that obtained using T-SMAC-Nal. The data represent the least square mean±SEM.

 
The sequence of the z2199z2206 region in E. coli O157 : H7 strain EDL933 is highly conserved in the Sakai outbreak strain RIMD 0509952; however, the annotation of the latter strain does not predict the existence of an ORF (z2203) overlapping with z2202 (Fig. 4). It is not clear whether this reflects a sequencing or annotation error or a genuine polymorphism between the strains. The locus is largely conserved in the genomes of other pathogenic E. coli; however, polymorphisms exist proximal to the z2200 gene (Fig. 4). The locus is partially conserved in E. coli K-12 strains MG1655 and W3110; however, the z2200, z2201 and z2203 genes are absent.



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Fig. 4. Genetic organization of a putative fimbrial locus of E. coli O157 : H7 EDL933 required for intestinal colonization in calves. ORFs are represented by arrows and are aligned to the homologous regions from E. coli O157 : H7 strain RIMD 0509952, EPEC strain E2348/69, enteroaggregative E. coli (EAEC) strain 042, uropathogenic E. coli (UPEC) strain CFT073 and E. coli K-12 strain MG1655. The gene names from E. coli O157 : H7 strain EDL933 are shown at the top. The genes in E. coli O157 : H7 are designated ecs2114ecs2107 and have been referred to collectively as locus 8 (see supplementary material in Hayashi et al., 2001). HipA is a putative DNA-binding protein regulating peptidoglycan biosynthesis. z2207 is predicted to encode the major subunit of an oxidoreductase.

 
Conclusions
We report the first comprehensive survey for E. coli O157 : H7 genes required for colonization of the bovine intestines. A library of 1900 signature-tagged transposon mutants was successfully screened by oral inoculation of calves and the site of transposon insertion mapped in 79 non-colonizing mutants. Our data provide valuable new information on the molecular mechanisms underlying EHEC persistence in calves that will facilitate the development of strategies to control EHEC in the ruminant reservoir.

The LEE-encoded type III secretion system was found to play a key role in intestinal colonization of calves by E. coli O157 : H7 and several known or putative type III secreted proteins were implicated in the colonization process. This is the first direct evidence for the involvement of the type III secretion apparatus in the persistence of EHEC in the bovine gut. Previous studies have focussed on the role of intimin and its translocated receptor (Dean-Nystrom et al., 1998; Cornick et al., 2002; Stevens et al., 2004) and the efficacy of vaccines based on type III secreted proteins (Potter et al., 2004). Mutation of a structural component of the type III secretion apparatus (EscN) was highly attenuating following oral inoculation of calves. By implication, the EHEC proteins delivered into host cells via the type III secretion system must play important roles in determining the outcome of infection. The repertoire and function of EHEC type III secreted proteins in the carriage and virulence of EHEC in calves therefore requires further study.

Colonization of the bovine intestine by EHEC also requires multiple elements encoded outside the LEE. We provide evidence that a novel fimbrial locus conserved among pathogenic E. coli is required for intestinal colonization of calves by E. coli O157 : H7. It remains unclear if genes encoded by this locus encode fimbriae per se, or if they contribute to the surface expression or composition of fimbriae encoded at distal loci. Further studies are required to address the role of genes encoded by this fimbrial locus in EHEC adherence and intestinal colonization. It is possible that the fimbrial subunit(s) and other colonization factors identified in this study may be useful as subunit vaccines for the control of EHEC infection.


   ACKNOWLEDGEMENTS
 
The authors gratefully acknowledge the financial support of the Department for the Environment Food & Rural Affairs, UK (project number OZ0707), Biotechnology & Biological Sciences Research Council, UK (project number 201/D10261) and the European Union (project number QLK2-CT-2000-00600).


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
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Received 30 June 2004; revised 30 July 2004; accepted 10 August 2004.



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