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Infection and Immunity, April 2007, p. 1661-1666, Vol. 75, No. 4
0019-9567/07/$08.00+0     doi:10.1128/IAI.01342-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Enterohemorrhagic Escherichia coli O157:H7 gal Mutants Are Sensitive to Bacteriophage P1 and Defective in Intestinal Colonization

Department of Molecular Biology and Microbiology, Tufts University School of Medicine and Howard Hughes Medical Institute, 136 Harrison Avenue, Boston, Massachusetts 02111

Received 21 August 2006/ Returned for modification 20 October 2006/ Accepted 9 November 2006

	ABSTRACT

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Enterohemorrhagic Escherichia coli (EHEC), especially E. coli O157:H7, is an emerging cause of food-borne illness. Unfortunately, E. coli O157 cannot be genetically manipulated using the generalized transducing phage P1, presumably because its extensive O antigen obscures the P1 receptor, the lipopolysaccharide (LPS) core subunit. The GalE, GalT, GalK, and GalU proteins are necessary for modifying galactose before it can be assembled into the repeating subunit of the O antigen. Here, we constructed E. coli O157:H7 gal mutants which presumably have little or no O antigen. These strains were able to adsorb P1. P1 lysates grown on the gal mutant strains could be used to move chromosomal markers between EHEC strains, thereby facilitating genetic manipulation of E. coli O157:H7. The gal mutants could easily be reverted to a wild-type Gal⁺ strain using P1 transduction. We found that the O157:H7 galETKM::aad-7 deletion strain was 500-fold less able to colonize the infant rabbit intestine than the isogenic Gal⁺ parent, although it displayed no growth defect in vitro. Furthermore, in vivo a Gal⁺ revertant of this mutant outcompeted the galETKM deletion strain to an extent similar to that of the wild type. This suggests that the O157 O antigen is an important intestinal colonization factor. Compared to the wild type, EHEC gal mutants were 100-fold more sensitive to a peptide derived from bactericidal permeability-increasing protein, a bactericidal protein found on the surface of intestinal epithelial cells. Thus, one way in which the O157 O antigen may contribute to EHEC intestinal colonization is to promote resistance to host-derived antimicrobial polypeptides.

	INTRODUCTION

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Enterohemorrhagic Escherichia coli (EHEC) is an emerging cause of food-borne illness. After ingestion of contaminated food, humans can develop symptoms ranging from mild diarrhea to the severe, and at times life-threatening, condition hemolytic-uremic syndrome. Currently, EHEC is the most common cause of pediatric renal failure in the United States (15). Besides causing significant human disease, EHEC is a major economic burden due to the high cost associated with the recall of contaminated beef (www.fsis.usda.gov/FSIS_Recalls/index.asp). Several EHEC serotypes cause disease, but the O157 serotype is by far the most common cause of EHEC-related disease in North America, Europe, and Japan (6).

Lipopolysaccharide (LPS), found in the outer membrane of gram-negative bacteria, is composed of lipid A, core oligosaccharide, and repeating O-antigen subunits. The O antigen is covalently linked to the outer region of the core oligosaccharide, and it appears to act as a barrier that can protect enteric pathogens against toxic agents encountered in host gastrointestinal tracts (23). For example, galU and galE mutants of Vibrio cholerae, which lacked O antigen, were defective in intestinal colonization, although they had no growth defect in rich medium. These mutants were more sensitive than O-antigen-producing strains to killing by complement and cationic antimicrobial peptides, suggesting that their defect in colonization was attributable to their sensitivity to bactericidal substances elaborated by the host gastrointestinal tract (17).

Like many enteric pathogens, E. coli O157 produces LPS that contains an extensive O antigen. The O157 O-antigen subunit consists of N-acetyl-D-perosamine, L-fucose, D-glucose, and N-acetyl-D-galactose (22). Production of N-acetyl-D-galactose requires that its precursor, galactose, be modified by the enzymes GalE, GalT, GalK, and GalU. Salmonella enterica serovar Typhimurium and E. coli gal mutants do not make O antigen (8, 18, 25).

The inner region of the LPS core oligosaccharide, which is conserved in many enteric bacteria, serves as the receptor for bacteriophage P1. Phage P1 has been a workhorse for genetic manipulation of E. coli K-12 for many decades. P1-mediated generalized transduction enables movement of mutations for generation of isogenic bacterial strains, which is often required for proving the linkage between particular genotypes and phenotypes. In S. enterica serovar Typhimurium, which has an LPS core oligosaccharide similar to that of E. coli K-12, the long O antigen obscures the core oligosaccharide and prevents P1 from adsorbing to the bacteria. O-antigen mutants (gal, galE, and galU) of S. enterica serovar Typhimurium have been shown to be P1 sensitive (18).

P1-mediated generalized transduction of EHEC has not been described previously. Here, we adopted the strategy that was used to facilitate P1 transduction in Salmonella. Wild-type EHEC O157:H7 strains are resistant to P1, but O157:H7 gal mutants were found to be P1 sensitive and permitted P1-mediated movement of genetic markers between EHEC strains. Furthermore, we developed a method that allows a simple reversion to convert P1-sensitive strains to the wild type. Interestingly, we found that the P1-sensitive galETKM O157:H7 mutant was extremely attenuated in the ability to colonize the infant rabbit intestine and had increased sensitivity to bactericidal permeability-increasing protein (BPI).

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Bacterial strains and growth. The strains used in this study are shown in Table 1. Unless indicated otherwise, strains were grown in LB broth or on LB agar plates. For antibiotic selection, agar plates were supplemented with ampicillin (80 µg/ml), spectinomycin (100 µg/ml), or tetracycline (6 µg/ml). MacConkey agar plates with 1% galactose or M63 [22 mM KH₂PO₄, 40 mM K₂HPO₄, 15 mM (NH₄)₂SO₄, 0.5 mg/liter FeSO₄] agar plates supplemented with 0.2% galactose and 0.1% Casamino Acids were used to test whether a strain could metabolize galactose.

We constructed the pTHE001 plasmid to generate an insertion mutation in galE. First, a 460-bp internal fragment of the galE gene was amplified by PCR using primers TE013 (5'-GCAAGGATCCGACGTTTGTTGAAGGCGATA) and TE014 (5'-GGCATAAGGGAATTCGGAATGCCTTGCGGA). The PCR product was digested with BamHI and then cloned into the BglII site of the conditional plasmid pGP704 (16). The resulting plasmid, pTHE001, was mobilized using the RP4⁺ helper strain WM3064 (provided by W. Metcalf, University of Illinois, Urbana-Champaign) into EDL933.

To generate the Gal⁺ revertant TEA040, we first moved the galE::pTHE001 mutation from TEA007 into the galETKM::aad-7 strain (TEA026) by P1 transduction, selecting for ampicillin resistance. The resulting strain was then used as a recipient for P1 transduction of the galETKM⁺ allele from TEA023. Gal⁺ transductants were selected on M63 agar plates supplemented with 0.2% galactose and 0.1% Casamino Acids.

P1 adsorption and sensitivity assays. P1 adsorption assays were performed using a P1 lysate grown on E. coli K-12 strain MC4100. Approximately 100 µl of an overnight culture was pelleted by centrifugation and resuspended in 100 µl of LB broth. Then 100 µl of the P1 lysate was added to the cells. After 15 min of incubation at 37°C, cells were pelleted by centrifugation at 6,000 rpm in an Eppendorf centrifuge at 4°C for 2 min. The P1 titers of the supernatants were then determined by plaquing, using MC4100 as an indicator strain. To plaque P1, we spotted phage lysates on top agar (LB broth containing 2 mM MgSO₄, 10 mM CaCl₂, and 0.7% agar) lawns of MC4100.

To test for sensitivity of strains to P1 lysis, each strain was cross-streaked against P1. A single line of P1 (100 µl; 10⁹ PFU/ml) was allowed to dry on an LB agar plate. For each bacterial strain, a single streak was then drawn perpendicular to a line of phage P1. Strains resistant to P1 grew on both sides of the line of P1; susceptible strains were partially lysed following an encounter with P1.

P1 lysate production. P1 lysates of various EHEC strains were generated by growing 1:100 dilutions of overnight cultures of each strain in 2.5 ml of LB broth containing 2 mM MgSO₄ and 10 mM CaCl₂ and incubating the preparations for 1 h at 37°C with agitation. Then 100 µl of a P1 lysate grown on MC4100 was added to each culture. After 2 to 3 h of incubation at 37°C with agitation, lysis of the cultures was observed. Tubes were transferred to ice, and any remaining intact bacteria were lysed with 0.5 ml chloroform. Lysates were then centrifuged at 13,000 rpm for 1 min, diluted in phosphate-buffered saline (PBS), and spotted on top agar lawns of MC4100 for titration. Lysates were stored at 4°C in the dark with 0.5 ml chloroform.

P1 transduction. Overnight cultures (0.5 ml) of recipient bacteria grown in LB broth were pelleted and resuspended in 100 µl MC (5 mM MgSO₄, 50 mM CaCl₂). About 50 µl of P1 lysate was added to the cells, which were then incubated at 37°C for 15 to 30 min. LB broth with 10 mM sodium citrate (0.5 to 1 ml) was added to each tube and incubated for 1 h at room temperature. Each tube was centrifuged at 6,000 rpm for 2 min, and the pellets were resuspended in 100 µl 1 M sodium citrate and plated on selective media.

In vitro competition assays. A 1:1 mixture of a mutant (TEA026) and wild-type strain EDL933 or TEA040 initially containing 5 x 10⁷ bacteria/ml was incubated in LB broth at 37°C with agitation overnight. Each assay mixture was then diluted and plated on LB agar. After overnight growth, bacteria were replica plated on selective media to determine the numbers of mutant and wild-type bacteria. Each assay was performed at least three times.

Competition assays with infant rabbits. We used the infant rabbit model to test the colonization ability of EHEC strains (27). Three-day-old New Zealand White rabbits were orally inoculated with a 1:1 mixture of TEA026 and wild-type strain EDL933 or TEA040 containing 2.5 x 10⁸ bacteria which were washed one time and resuspended in PBS. Seven days after inoculation, rabbits were sacrificed, and their gastrointestinal tracts were removed. Portions of the ileum, midcolon, and cecum were then homogenized, diluted, and plated on MacConkey agar containing sorbitol. After overnight growth, bacteria were replica plated on LB agar containing spectinomycin (100 µg/ml) to determine the number of TEA026 bacteria.

BPI sensitivity assays. We used the BPI-derived peptide P2, which has the antibacterial activity of whole protein (10, 13), to assess EHEC sensitivity. For these assays, bacteria grown overnight in LB broth were washed once in PBS and resuspended in PBS (pH 6.2). Then 5 x 10⁷ bacteria with or without 30 µg P2 (Tufts University Core Facility) were incubated in 0.5 ml PBS (pH 6.2) for 45 min at 37°C. After incubation, each assay mixture was placed on ice, diluted, and plated on LB agar. Each assay was performed in triplicate and repeated in three independent experiments.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

 
O157:H7 gal mutants can adsorb P1 and are sensitive to P1 lysis. In many enteric bacteria, extensive LPS O antigens obscure the LPS core oligosaccharide and thereby prevent adsorption of and lysis by phage P1. Since synthesis of the O157:H7 O antigen requires modification of galactose by the galE, galT, galK, and galU gene products, we hypothesized that EHEC strains with mutations in these genes would produce no O antigen and therefore would be far more susceptible to P1 infection. To test this hypothesis, we compared phage infection of and adsorption by the sequenced O157:H7 isolate EDL933 to those of four gal derivatives: the galETKM::aad-7 (TEA026), galETKM::tetA (TEA028), and galU::aad-7 (TEA023) deletion mutants and the galE::pTHE001 (TEA007) insertion mutant.

To assess whether gal loci influence infection of EHEC by phage P1, we performed cross-streak experiments. A streak of each bacterial strain was drawn perpendicular to a line of P1, and the consequences of encountering phage were assessed. There was no change in the growth of wild-type strain EDL933 in response to P1, indicating that this strain is resistant to phage infection. In contrast, all four gal mutants were lysed by phage P1 (data not shown).

To explore why the gal mutants are more susceptible to P1 infection, we examined whether the mutants showed enhanced adsorption of P1. Phage adsorption to the host bacterium is an essential step in phage infection. In these assays, each gal mutant was incubated with P1 and then centrifuged to pellet any phage adsorbed to the bacteria. Supernatants were then assayed to determine the number of unadsorbed P1 particles. As shown in Table 2, the wild-type strain adsorbed 30% of the phage, suggesting that there may be some nonspecific interactions between EHEC and P1, since the wild-type strain is resistant to P1 infection and therefore is presumed to have inaccessible core oligosaccharides. However, all four gal mutants adsorbed 95% of the P1, which is consistent with the hypothesis that access to P1's receptor is less impeded in these strains.

Reversion of the gal mutant using P1 transduction. We devised a simple strategy to change Gal^– strains back to Gal⁺ strains in order to enable study of Gal⁺ EHEC strains that have been engineered using P1. We were unable to revert the galETKM::aad-7 mutant (TEA026) by transducing the galETKM⁺ allele from TEA023, perhaps because the frequency of transduction was too low to obtain a revertant. Therefore, we utilized a two-step reversion method. We first moved the galE::pTHE001 mutation from TEA007 into the galETKM strain (TEA026). This strain was then used as a recipient for P1 transduction of the galETKM⁺ allele from a donor lysate grown on the galU strain (TEA023). Gal⁺ transductants were selected on agar plates containing M63 minimal medium supplemented with 0.2% galactose and 0.1% Casamino Acids and were verified to be Gal⁺ strains on MacConkey agar containing 1% galactose. This transduction was as efficient as transfer of other EHEC chromosomal markers. We chose one Gal⁺ revertant strain (TEA040) to test for P1 sensitivity. Like the isogenic wild-type strain, this Gal⁺ revertant had a reduced capacity to adsorb P1 phage compared to the gal mutants (Table 2), was resistant to P1 lysis in a P1 cross-streak experiment, and was not able to serve as a recipient for P1 transduction (data not shown).

O157:H7 galETKM mutant is dramatically impaired in colonization of the infant rabbit intestine. Previous studies have shown that galE mutants of S. enterica serovars are defective in intestinal colonization (9, 12). We were interested in determining whether the EHEC galETKM deletion resulted in a similar defect in intestinal colonization. To do this, we tested the galETKM::aad-7 deletion mutant (TEA026) in a competition assay with the isogenic wild-type strain (EDL933) using the EHEC-infant rabbit model. We found that the galETKM deletion mutant (TEA026) was 500-fold less able to colonize the infant rabbit ileum, cecum, and midcolon (Fig. 1A). To demonstrate that this dramatic defect in intestinal colonization was due to the galETKM deletion, we performed a competition experiment with the Gal⁺ reverted strain and its galETKM::aad-7 parent strain. The Gal⁺ revertant (TEA040) outcompeted the galETKM mutant (TEA023) to an extent similar to the extent observed for the wild type (Fig. 1B), strongly suggesting that the galETKM deletion accounts for the colonization defect of TEA026. In vitro competition assays in which the galETKM strain and the wild type or the Gal⁺ revertant were grown in LB broth at 37°C revealed that the galETKM mutation resulted in a slight (2-fold) but statistically significant growth defect in rich medium (Fig. 1). This minor in vitro growth defect could not account for the drastic colonization defect of the gal mutant. Overall, these findings suggest that the O157 O antigen is critical for EHEC intestinal colonization.

View larger version (22K): [in this window] [in a new window]   FIG. 1. In vivo and in vitro competition assays with an EHEC galETKM mutant (TEA026) and wild-type strain EDL933 or with TEA026 and a Gal+ revertant of TEA026 (TEA040). Equal numbers of TEA026 and EDL933 (A) or TEA026 and TEA040 (B) were coinoculated into infant rabbits or into LB broth. After 7 days of infection, sections of the ileum, midcolon, and cecum and samples of stools were processed to determine competitive indices. The in vitro competition assays in LB broth were carried out for 16 h. The competitive index was determined as follows: [(number of output mutant bacteria)/(number of output wild-type or revertant bacteria)]/[(number of input mutant bacteria)/(number of input wild-type or revertant bacteria)]. Each of the competitive indices shown is statistically different from 1 (P < 0.00001, as determined by Student's t test).

Conclusions. EHEC strains, especially E. coli O157 strains, are significant causes of disease in many developed nations, yet there are no methods for transducing markers in this important group of pathogenic E. coli. It has been known for a long time that S. enterica serovar Typhimurium gal mutants have little or no O antigen, which makes these strains sensitive to P1 (18). Our findings strongly suggest that the O157 O antigen obscures the EHEC core oligosaccharide, the P1 receptor, in a similar fashion, since we found that O157:H7 gal mutants are sensitive to P1 infection. We also developed a relatively simple P1 transduction-based means to revert gal mutants to a Gal⁺ phenotype. Although we describe a method to genetically manipulate O157:H7 with P1, this technique can likely be adapted for use with other EHEC serotypes and probably with other pathogenic E. coli strains (such as enteropathogenic E. coli and uropathogenic E. coli) as well. Figure 2 outlines a general scheme for using P1 transduction for genetic manipulation of EHEC. This figure shows the method that our lab routinely uses to generate isogenic EHEC strains. This ability to move chromosomal markers enables generation of isogenic strains containing single or multiple mutations. Given the relative ease of the P1 transduction technique outlined here, it should be possible to backcross mutations generated with the lambda red system into genetically identical backgrounds.

View larger version (22K): [in this window] [in a new window]   FIG. 2. General scheme for P1-facilitated genetic engineering of EHEC. (A) The desired mutation in the galETKM::tetA or galETKM::aad-7 background is generated (e.g., using the lambda red recombination system) so that the mutation can be moved by P1 transduction. (B) The desired mutation is moved into the galE::pTHE001 background using P1 transduction, selecting for the antibiotic resistance gene to generate an isogenic backcrossed mutant. (C) A second P1 transduction into the backcrossed strain is performed, selecting for growth on galactose minimal plates. Abbreviations: x, gene of interest; abR, antibiotic resistance gene; tetR, tetracycline resistance gene.

O antigens likely act as important barriers against extracellular assaults mounted by the host immune system. O-antigen mutants of enteric bacteria like V. cholerae, Klebsiella serotype O1:K20, and Shigella flexneri are more susceptible to antimicrobial peptides and complement killing (14, 17, 29). S. enterica serovar Typhi, Brucella melitensis, and V. cholerae O-antigen mutants have been shown to be impaired in intestinal colonization as well (3, 9, 17, 26). In fact, the only oral live attenuated vaccine against S. enterica serovar Typhi used in the United States is a galE mutant. Given the pronounced attenuation of the O157:H7 galETKM::aad-7 mutant, perhaps EHEC gal mutants could also be developed into useful vaccines.

	ACKNOWLEDGMENTS

 
We thank James Slauch for helpful comments and Brigid Davis and Craig D. Ellermeier for critical reading of the manuscript.

We thank the NIH and Howard Hughes Medical Institute for funding this work.

	FOOTNOTES

 
* Corresponding author. Present address: Channing Laboratories, Brigham and Women's Hospital, 181 Longwood Ave., Boston, MA 02115. Phone: (617) 525-4650. Fax: (617) 525-4660. E-mail: mwaldor{at}rics.bwh.harvard.edu.

Published ahead of print on 11 December 2006.

Editor: A. D. O'Brien

	REFERENCES

Top Abstract Introduction Materials and Methods Results and Discussion References

 

1.	Bilge, S. S., J. C. Vary, Jr., S. F. Dowell, and P. I. Tarr. 1996. Role of the Escherichia coli O157:H7 O side chain in adherence and analysis of an rfb locus. Infect. Immun. 64:4795-4801.[Abstract]
2.	Canny, G., O. Levy, G. T. Furuta, S. Narravula-Alipati, R. B. Sisson, C. N. Serhan, and S. P. Colgan. 2002. Lipid mediator-induced expression of bactericidal/permeability-increasing protein (BPI) in human mucosal epithelia. Proc. Natl. Acad. Sci. USA. 99:3902-3907.[Abstract/Free Full Text]
3.	Chiang, S. L., and J. J. Mekalanos. 1999. rfb mutations in Vibrio cholerae do not affect surface production of toxin-coregulated pili but still inhibit intestinal colonization. Infect. Immun. 67:976-980.[Abstract/Free Full Text]
4.	Cockerill, F. III, G. Beebakhee, R. Soni, and P. Sherman. 1996. Polysaccharide side chains are not required for attaching and effacing adhesion of Escherichia coli O157:H7. Infect. Immun. 64:3196-3200.[Abstract]
5.	Datsenko, K. A., and B. L. Wanner. 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97:6640-6645.[Abstract/Free Full Text]
6.	Feng, P. 2001. Escherichia coli, p. 143-162. In R. G. Labbe and S. Garcia (ed.), Guide to foodborne pathogens. John Wiley and Sons, Inc., New York, NY.
7.	Gazzano-Santoro, H., J. B. Parent, L. Grinna, A. Horwitz, T. Parsons, G. Theofan, P. Elsbach, J. Weiss, and P. J. Conlon. 1992. High-affinity binding of the bactericidal/permeability-increasing protein and a recombinant amino-terminal fragment to the lipid A region of lipopolysaccharide. Infect. Immun. 60:4754-4761.[Abstract/Free Full Text]
8.	Genevaux, P., P. Bauda, M. S. DuBow, and B. Oudega. 1999. Identification of Tn10 insertions in the rfaG, rfaP, and galU genes involved in lipopolysaccharide core biosynthesis that affect Escherichia coli adhesion. Arch. Microbiol. 172:1-8.[CrossRef][Medline]
9.	Gilman, R. H., R. B. Hornick, W. E. Woodard, H. L. DuPont, M. J. Snyder, M. M. Levine, and J. P. Libonati. 1977. Evaluation of a UDP-glucose-4-epimeraseless mutant of Salmonella typhi as a liver oral vaccine. J. Infect. Dis. 136:717-723.[Medline]
10.	Gray, B. H., and J. R. Haseman. 1994. Bactericidal activity of synthetic peptides based on the structure of the 55-kilodalton bactericidal protein from human neutrophils. Infect. Immun. 62:2732-2739.[Abstract/Free Full Text]
11.	Haldimann, A., and B. L. Wanner. 2001. Conditional-replication, integration, excision, and retrieval plasmid-host systems for gene structure-function studies of bacteria. J. Bacteriol. 183:6384-6393.[Abstract/Free Full Text]
12.	Hohmann, A., G. Schmidt, and D. Rowley. 1979. Intestinal and serum antibody responses in mice after oral immunization with Salmonella, Escherichia coli, and Salmonella-Escherichia coli hybrid strains. Infect. Immun. 25:27-33.[Abstract/Free Full Text]
13.	Little, R. G., D. N. Kelner, E. Lim, D. J. Burke, and P. J. Conlon. 1994. Functional domains of recombinant bactericidal/permeability increasing protein (rBPI23). J. Biol. Chem. 269:1865-1872.[Abstract/Free Full Text]
14.	McCallum, K. L., G. Schoenhals, D. Laakso, B. Clarke, and C. Whitfield. 1989. A high-molecular-weight fraction of smooth lipopolysaccharide in Klebsiella serotype O1:K20 contains a unique O-antigen epitope and determines resistance to nonspecific serum killing. Infect. Immun. 57:3816-3822.[Abstract/Free Full Text]
15.	Mead, P. S., L. Slutsker, V. Dietz, L. F. McCaig, J. S. Bresee, C. Shapiro, P. M. Griffin, and R. V. Tauxe. 1999. Food-related illness and death in the United States. Emerg. Infect. Dis. 5:607-625.[Medline]
16.	Miller, V. L., and J. J. Mekalanos. 1988. A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J. Bacteriol. 170:2575-2583.[Abstract/Free Full Text]
17.	Nesper, J., C. M. Lauriano, K. E. Klose, D. Kapfhammer, A. Kraiss, and J. Reidl. 2001. Characterization of Vibrio cholerae O1 El tor galU and galE mutants: influence on lipopolysaccharide structure, colonization, and biofilm formation. Infect. Immun. 69:435-445.[Abstract/Free Full Text]
18.	Ornellas, E. P., and B. A. Stocker. 1974. Relation of lipopolysaccharide character to P1 sensitivity in Salmonella typhimurium. Virology 60:491-502.[CrossRef][Medline]
19.	Paton, A. W., E. Voss, P. A. Manning, and J. C. Paton. 1998. Antibodies to lipopolysaccharide block adherence of Shiga toxin-producing Escherichia coli to human intestinal epithelial (Henle 407) cells. Microb. Pathog. 24:57-63.[CrossRef][Medline]
20.	Paton, J. C., and A. W. Paton. 1998. Pathogenesis and diagnosis of Shiga toxin-producing Escherichia coli infections. Clin. Microbiol. Rev. 11:450-479.[Abstract/Free Full Text]
21.	Perna, N. T., G. Plunkett III, V. Burland, B. Mau, J. D. Glasner, D. J. Rose, G. F. Mayhew, P. S. Evans, J. Gregor, H. A. Kirkpatrick, G. Posfai, J. Hackett, S. Klink, A. Boutin, Y. Shao, L. Miller, E. J. Grotbeck, N. W. Davis, A. Lim, E. T. Dimalanta, K. D. Potamousis, J. Apodaca, T. S. Anantharaman, J. Lin, G. Yen, D. C. Schwartz, R. A. Welch, and F. R. Blattner. 2001. Genome sequence of enterohaemorrhagic Escherichia coli O157:H7. Nature 409:529-533.[CrossRef][Medline]
22.	Perry, M. B., L. MacLean, and D. W. Griffith. 1986. Structure of the O-chain polysaccharide of the phenol-phase soluble lipopolysaccharide of Escherichia coli 0:157:H7. Biochem. Cell Biol. 64:21-28.[Medline]
23.	Peschel, A. 2002. How do bacteria resist human antimicrobial peptides? Trends Microbiol. 10:179-186.[CrossRef][Medline]
24.	Pluschke, G., and M. Achtman. 1984. Degree of antibody-independent activation of the classical complement pathway by K1 Escherichia coli differs with O antigen type and correlates with virulence of meningitis in newborns. Infect. Immun. 43:684-692.[Abstract/Free Full Text]
25.	Raetz, C. R. H. 1996. Bacterial lipopolysaccharides: a remarkable family of bioactive macroamphiles, p. 1035-1063. In F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed., vol. 1. ASM Press, Washington, DC.
26.	Rajashekara, G., D. A. Glover, M. Banai, D. O'Callaghan, and G. A. Splitter. 2006. Attenuated bioluminescent Brucella melitensis mutants GR019 (virB4), GR024 (galE), and GR026 (BMEI1090-BMEI1091) confer protection in mice. Infect. Immun. 74:2925-2936.[Abstract/Free Full Text]
27.	Ritchie, J. M., C. M. Thorpe, A. B. Rogers, and M. K. Waldor. 2003. Critical roles for stx2, eae, and tir in enterohemorrhagic Escherichia coli-induced diarrhea and intestinal inflammation in infant rabbits. Infect. Immun. 71:7129-7139.[Abstract/Free Full Text]
28.	Singer, M., T. A. Baker, G. Schnitzler, S. M. Deischel, M. Goel, W. Dove, K. J. Jaacks, A. D. Grossman, J. W. Erickson, and C. A. Gross. 1989. A collection of strains containing genetically linked alternating antibiotic resistance elements for genetic mapping of Escherichia coli. Microbiol. Mol. Biol. Rev. 53:1-24.[Abstract/Free Full Text]
29.	West, N. P., P. Sansonetti, J. Mounier, R. M. Exley, C. Parsot, S. Guadagnini, M. C. Prevost, A. Prochnicka-Chalufour, M. Delepierre, M. Tanguy, and C. M. Tang. 2005. Optimization of virulence functions through glucosylation of Shigella LPS. Science 307:1313-1317.[Abstract/Free Full Text]

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• Shifrin, Y., Peleg, A., Ilan, O., Nadler, C., Kobi, S., Baruch, K., Yerushalmi, G., Berdichevsky, T., Altuvia, S., Elgrably-Weiss, M., Abe, C., Knutton, S., Sasakawa, C., Ritchie, J. M., Waldor, M. K., Rosenshine, I. (2008). Transient Shielding of Intimin and the Type III Secretion System of Enterohemorrhagic and Enteropathogenic Escherichia coli by a Group 4 Capsule. J. Bacteriol. 190: 5063-5074 [Abstract] [Full Text]  
• Ho, T. D., Davis, B. M., Ritchie, J. M., Waldor, M. K. (2008). Type 2 Secretion Promotes Enterohemorrhagic Escherichia coli Adherence and Intestinal Colonization. Infect. Immun. 76: 1858-1865 [Abstract] [Full Text]  
• Fabich, A. J., Jones, S. A., Chowdhury, F. Z., Cernosek, A., Anderson, A., Smalley, D., McHargue, J. W., Hightower, G. A., Smith, J. T., Autieri, S. M., Leatham, M. P., Lins, J. J., Allen, R. L., Laux, D. C., Cohen, P. S., Conway, T. (2008). Comparison of Carbon Nutrition for Pathogenic and Commensal Escherichia coli Strains in the Mouse Intestine. Infect. Immun. 76: 1143-1152 [Abstract] [Full Text]  

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