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Infection and Immunity, March 2005, p. 1466-1474, Vol. 73, No. 3
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.3.1466-1474.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

The Locus of Enterocyte Effacement-Encoded Effector Proteins All Promote Enterohemorrhagic Escherichia coli Pathogenicity in Infant Rabbits

Jennifer M. Ritchie1 and Matthew K. Waldor1,2*

Tufts University School of Medicine,1 Howard Hughes Medical Institute, Boston, Massachusetts2

Received 4 October 2004/ Returned for modification 8 November 2004/ Accepted 22 November 2004


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ABSTRACT
 
The genes encoding the enterohemorrhagic Escherichia coli (EHEC) type III secretion system (TTSS) and five effector proteins secreted by the TTSS are located on the locus of enterocyte effacement (LEE) pathogenicity island. Deletion of tir, which encodes one of these effector proteins, results in a profound reduction (~10,000-fold) in EHEC colonization of the infant rabbit intestine, but the in vivo phenotypes of other LEE genes are unknown. Here, we constructed in-frame deletions in escN, the putative ATPase component of the TTSS, and the genes encoding the four other LEE-encoded effector proteins, EspH, Map, EspF, and EspG, to investigate the contributions of the TTSS and the translocated effector proteins to EHEC pathogenicity in infant rabbits. We found that the TTSS is required for EHEC colonization and attaching and effacing (A/E) lesion formation in the rabbit intestine. Deletion of escN reduced EHEC recovery from the rabbit intestine by ~10,000-fold. Although EspH, Map, EspF, and EspG were not required for A/E lesion formation in the rabbit intestine or in HeLa cells, these effector proteins promote EHEC colonization. Colonization by the espH and espF mutants was reduced throughout the intestine. In contrast, colonization by the map and espG mutants was reduced only in the small intestine, indicating that Map and EspG have organ-specific effects. EspF appears to down-regulate the host response to EHEC, since we observed increased accumulation of polymorphonuclear leukocytes in the colonic mucosa of rabbits infected with the EHEC espF mutant. Thus, all the known LEE-encoded effector proteins influence EHEC pathogenicity.


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INTRODUCTION
 
Enterohemorrhagic Escherichia coli (EHEC) is a food- and waterborne pathogen that can cause diseases ranging from mild diarrhea to the potentially fatal hemolytic-uremic syndrome. EHEC includes several serotypes, but E. coli O157:H7 has been the predominant serotype isolated from outbreaks in developed countries, such as the United States and United Kingdom (17, 53). The key determinants of virulence in EHEC are production of Shiga toxins (Stx), a family of potent cytotoxins, such as Stx1 and Stx2, that inhibit protein synthesis in eukaryotic cells and colonization of the human intestine. Study of EHEC pathogenicity has been hindered by the lack of an animal model that recapitulates EHEC-mediated disease. Recently, Citrobacter rodentium infection of mice has become a surrogate model for the study of the pathogenic mechanisms of attaching and effacing (A/E) pathogens, such as EHEC and enteropathogenic E. coli (EPEC) (11, 12, 37, 49), but it is unclear whether C. rodentium pathogenicity accurately reflects all A/E pathogens.

EHEC, like EPEC and C. rodentium, forms characteristic histopathologic lesions termed A/E lesions on tissue culture cells and in animal intestines (25, 36, 49). The A/E lesion is defined by the intimate attachment of bacteria to the epithelial surface along with the localized loss (effacement) of microvilli. Marked cytoskeletal rearrangements, including the accumulation of polymerized actin and other host proteins, occur underneath the adherent bacteria, resulting in the formation of an actin-rich pedestal (reviewed in reference 15). The contribution of these histologic lesions to disease has not been determined. In EHEC or EPEC infection, it has been proposed that A/E lesions may promote diarrhea via malabsorption due to the loss of absorptive microvilli and/or that they enable the pathogen to modulate host signaling processes in a manner that also contributes to the development of diarrhea (27, 38).

A locus of enterocyte effacement (LEE) pathogenicity island is required for the formation of A/E lesions. The EHEC, EPEC, and C. rodentium LEE pathogenicity islands are similar and encode type III secretion systems (TTSSs) that inject effector proteins directly into host cells (19). These three LEE pathogenicity islands share 41 genes organized into five major operons, LEE 1 to 5 (11, 35, 47). A/E lesion formation in EPEC requires two LEE-encoded proteins, Tir (translocated intimin receptor), which is secreted by the TTSS, and intimin (46). An additional factor, EspFu, a non-LEE-encoded protein that is secreted by the LEE TTSS, is required for EHEC A/E lesion formation (4). The importance of Tir and intimin for intestinal colonization in animal infection studies has been established for EHEC (5, 10, 21, 32, 44, 56, 58), REPEC (rabbit EPEC) (30), and C. rodentium (13, 50).

In addition to Tir, four other effector proteins, EspH, Map, EspF, and EspG are encoded on the LEE pathogenicity island (Fig. 1) and translocated by the TTSS into the host cytosol, where they are thought to interfere with host signaling pathways (14, 22, 24, 33, 55). The function of some effector proteins is known for EPEC. EspH is a cytoskeleton-modulating protein that down-regulates filopodium formation and promotes pedestal formation in HeLa cells infected with EPEC or EHEC (55). Several cellular activities have been reported for EPEC Map: Map targets mitochondria, where it affects membrane potential (24), it promotes the transient formation of Cdc42-dependent filopodia in host cells (23), and it disrupts intestinal barrier function (9). EPEC EspF possesses at least two functions. EspF disrupts epithelial barrier function by altering the distribution of occludin, a protein found in tight junctions (34), and it induces apoptosis in HeLa cells (6). Finally, recent evidence indicates that EPEC EspG promotes host cytoskeletal rearrangements by destabilizing microtubule networks and triggering actin stress fiber assembly (31). EspG also exhibits significant homology to the VirA protein of Shigella flexneri, and cloned espG can rescue the invasion defect in a Shigella virA mutant (14). Besides Tir, the contribution of the LEE-encoded effector proteins in EHEC pathogenicity has not been explored in animal models.



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  		FIG. 1. LEE pathogenicity island of E. coli O157:H7 strain EDL933. The open reading frames encoding the translocated effector proteins are shown as light grey arrows, and escN is depicted as a white arrow. This figure incorporates information reported in references 12, 26, 28, 41, and 42. The putative polycistronic operons of EHEC LEE are shown as thin dotted lines.

We recently reported that infant rabbits are useful animal models in which to study the intestinal manifestations of EHEC infection (44). Infant rabbits inoculated with human clinical EHEC isolates develop severe diarrhea and intestinal inflammation, but not the systemic signs of disease, such as hemolytic-uremic syndrome, sometimes seen in humans naturally infected with this pathogen (39, 40, 44). In this model, Shiga toxin is critical for the development of EHEC-induced severe diarrhea and the predominantly heterophilic (polymorphonuclear leukocyte [PMN]) inflammation seen in the colon, while Tir and intimin are crucial for EHEC colonization, as deletions in tir or eae (the gene encoding intimin) result in up to ~10,000-fold reductions in colonization (44).

In this study, we examined the contribution of the LEE-encoded effector proteins EspH, Map, EspF, and EspG to EHEC pathogenicity in infant rabbits. We constructed a set of isogenic derivatives of EHEC strain 905, the human Stx2-producing E. coli O157:H7 clinical isolate that we used previously (44), with deletions in espH, map, espF, or espG. The espH, map, espF, and espG mutants formed A/E lesions in vitro and in vivo but each deletion reduced 905 intestinal colonization by approximately 10-fold, indicating that these LEE-encoded effectors function as accessory colonization factors. Deletion of espF also promoted the accumulation of PMNs in the intestinal mucosa, which suggests that EspF may dampen the host inflammatory response to EHEC.


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MATERIALS AND METHODS
 
Bacterial strain construction. Deletions of espH, map, espF, espG, or escN were introduced into 905, an Stx2-producing E. coli O157:H7 clinical isolate from a patient with hemolytic-uremic syndrome (45). These isolate 905 derivatives were constructed using the PCR-based one-step gene inactivation system (8) as described previously (44). PCR primers were designed using DNA sequences from the E. coli O157:H7 reference strain EDL933 (42). Gene amplifications were performed using the following primer pairs: primers espH-F and espH-R for the espH gene, primers map-F and map-R for map, espF-F and espF-R for espF, espG-F and espG-R for espG, and finally, escN-F and escN-R for escN (Table 1). PCR products were electroporated into 905 containing the lambda RED-encoding plasmid, pKD46 (8). Recombinants containing the kanamycin resistance gene in place of the gene of interest were selected on L-agar plates containing 50 µg of kanamycin ml–1 and confirmed by PCR analyses. The kanamycin resistance gene, which was flanked by FRT sites, was subsequently removed using the FLP recombinase as described previously (8). Removal of the kanamycin resistance gene was confirmed by PCR analyses and sequencing. The growth of each of the 905 derivatives in L broth at 37°C was the same as that of the wild-type strain.


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  		TABLE 1. Oligonucleotide primers used in this study

Rabbit experiments. Infant rabbits were infected as described previously (44). Briefly, litters of 3-day-old infant rabbits, which were housed with their mothers, were intragastrically inoculated (~5 x 108 CFU per 90-g rabbit) with EHEC strain 905 or one of its derivatives. After inoculation, the infant rabbits were monitored twice daily for clinical signs of disease. Diarrhea was scored as follows: none, no diarrhea (normal pellets are dark green, hard, and formed); mild, diarrhea consisting of a mix of soft formed and unformed pellets, resulting in light staining of the hind legs; severe, diarrhea consisting of unformed or liquid stool, resulting in significant staining of the perineum and hind legs. Most rabbits were necropsied 7 days postinoculation, and samples were collected for histologic and microbiologic analyses, as well as for the measurement of Stx2 and interleukin 8 (IL-8) concentrations. Some rabbits were sacrificed at 2 days postinoculation to obtain tissue samples for transmission electron microscopy.

Histologic and microbiologic analyses. Tissues were fixed in 10% neutral-buffered formalin, routinely processed, and stained with hematoxylin and eosin. The semiquantitative assessment of tissue infiltration with heterophils (PMNs) and edema or congestion was performed as described previously (44). Mucosal changes in tissue samples were assessed as described previously (52). Tissue samples for transmission electron microscopy were fixed in 2.5% glutaraldehyde (pH 7.3) buffered in 0.1 M sodium cacodylate and processed as described previously (44). The numbers of EHEC CFU in tissue and stool samples were determined by plating tissue homogenates on plates containing MacConkey agar and sorbitol. Feces present in tissue samples were removed prior to the determination of bacterial CFU as described previously (44).

FAS assay. The fluorescent actin staining (FAS) assay was performed as described previously (4, 25), except that HeLa cells were infected with 107 CFU of EHEC strain 905 or one of its derivatives. Fixed monolayers were treated with 4',6'-diamidino-2-phenylindole (DAPI) (1 µg ml–1) to detect bacteria and with AlexaFluor568-phalloidin (1:100; Molecular Probes, Inc., Eugene, Oreg.) to detect F-actin and examined using a Zeiss Axioplan2 imaging fluorescence microscope. Pedestal formation efficiency was quantified by counting the cell-associated bacteria that were colocalized with intense F-actin staining of the host cell. Approximately 50 HeLa cells harboring 5 to 20 bacteria per cell were examined for each strain in three independent experiments.

Measurement of Stx2 and IL-8. Total (extracellular and periplasmic) Stx2 concentrations in homogenized tissue and stool samples treated with polymyxin B (2 mg ml–1) were determined using an enzyme-linked immunoassay as described previously (2). IL-8 concentrations in homogenates of midcolon tissue samples were determined using an enzyme-linked immunoassay developed to detect rabbit IL-8 (18). Briefly, midcolon tissue samples were washed twice in ice-cold phosphate-buffered saline containing a mixture of protease inhibitors (10 µg of leupeptin ml–1, 1 mM phenylmethylsulfonyl fluoride [PMSF], and 0.5 mM dithiothreitol [DTT]) and then homogenized in 1 ml of lysate buffer (25 mM HEPES [pH 7.5], 300 mM NaCl, 1.5 mM MgCl2, 2 mM EDTA [pH 8.0], 1% Triton X-100, 0.1 mM Na3VO4, 20 mM ß-glycerol phosphate, 10 µg of leupeptin ml–1, 1 mM PMSF, and 0.5 mM DTT). The samples were filtered (QIAshredder; QIAGEN) and mixed at 4°C for 30 min. Cellular debris was removed by centrifugation at 4°C for 15 min, and the supernatants were stored at –80°C prior to analysis. The protein concentrations of the extracts were determined using the Coomassie protein assay reagent kit, according to the manufacturer's instructions (Pierce, Rockford, Ill.). Mouse anti-IL-8 monoclonal antibody, WS-4, and a guinea pig anti-rabbit IL-8 polyclonal antisera, were kind gifts from Shi Ishikawa (University of Tokyo, Tokyo, Japan).

Statistical analysis. Bacterial counts (after log transformation), Stx2 and IL-8 concentrations, and FAS assay data were analyzed using the Student t test, comparing each mutant to the wild type. In samples where no bacterial colonies were detected at the lowest dilution, the mean values presented in Fig. 3 were calculated using the lower limit of detection as a value. Histology scores are ordinal nonparametric data and were analyzed using the Mann-Whitney U test on Prism software (GraphPad, San Diego, Calif.).



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  		FIG. 3. Recovery of 905 or one of its derivatives from intestinal segments or stool samples from infected rabbits 7 days postinoculation. Samples below the detection limit (open squares) and scores for rabbits inoculated with the independently derived espH or espF deletion mutants (x) are indicated. The mean value for each strain is indicated by a bar.


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RESULTS AND DISCUSSION
 
A functional TTSS is required for EHEC-induced disease in infant rabbits. Initially, we investigated whether a functional TTSS is required for EHEC-induced disease and intestinal colonization in infant rabbits. This was likely since Tir, which is translocated by TTSS (16), plays a key role in EHEC-mediated disease and colonization of the infant rabbit intestine (44). To test this, we constructed 905{Delta}escN, a derivative of 905 that contains an in-frame deletion in escN. escN encodes the putative ATPase component of the TTSS, and its deletion renders the strain deficient for type III secretion (20). Using the FAS assay, we found that, as expected, 905{Delta}escN did not induce pedestal formation on HeLa cells (Table 2). Deletion of escN from 905 abolished its pathogenicity in infant rabbits. In marked contrast to the severe diarrhea observed in rabbits inoculated with 905, rabbits inoculated with 905{Delta}escN did not develop diarrhea (Table 3), perianal redness, or ruffled fur and exhibited no fecal contamination on their rear legs or perinea at necropsy. Consistent with our in vitro observations, no A/E lesions were detected in samples from colons of 905{Delta}escN-infected rabbits (Fig. 2A); A/E lesions were readily detected in midcolon samples from rabbits infected with strain 905 by transmission electron microscopy (Fig. 2B). 905{Delta}escN intestinal colonization was also markedly impaired. There were 3 to 4 orders of magnitude fewer CFU recovered from the ilea, ceca, and midcolons of rabbits infected with 905{Delta}escN compared to the numbers of CFU recovered from these regions of the intestinal tract in rabbits infected with 905 (Fig. 3A to C).


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  		TABLE 2. Formation of pedestals by 905 or one of its derivatives on HeLa cells assessed by the FAS assay


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  		TABLE 3. Diarrhea in rabbits inoculated with 905 and its derivatives



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  		FIG. 2. Transmission electron micrographs of colonic tissue taken from rabbits infected with EHEC 905{Delta}escN (A), 905 (B), 905{Delta}espH (C), 905{Delta}map (D), 905{Delta}espG (E), and 905{Delta}espF (F). Colon samples were taken 2 days postinoculation.

Consistent with the lack of clinical signs of disease and the striking reduction in intestinal colonization in the rabbits infected with 905{Delta}escN, no histologic changes were observed in the intestinal tracts of these rabbits (Fig. 4). Furthermore, the levels of the proinflammatory chemokine IL-8 were also significantly lower in the 905{Delta}escN-infected rabbits (21 ± 2 pg of IL-8 mg of protein–1 [mean ± standard error {SE}] in homogenates of the midcolons of 905{Delta}escN-infected rabbits compared to 136 ± 23 pg of IL-8 mg of protein–1 in homogenates of the midcolons of 905-infected rabbits [P < 0.001]).



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  		FIG. 4. Colitis scores from the midcolons of infant rabbits inoculated with 905 or one of its derivatives. Sections stained with hematoxylin and eosin were scored for heterophils (A), edema and congestion (B), and mucosal damage (C) as described previously (44, 52). The scores for rabbits inoculated with the independently derived espF deletion mutant (x) are indicated. The median value for each strain is indicated by a bar.

These findings indicate that a functional TTSS is required for EHEC-mediated disease and intestinal colonization in infant rabbits and are consistent with the results of studies investigating the role of the TTSS in other A/E pathogens, such as C. rodentium (12) and EPEC (1, 54). Interestingly, we observed greater numbers of EHEC 905{Delta}tir in the midcolons of infected rabbits (44) than 905{Delta}escN from the same tissue section. Although these differences were small (less than 1 order of magnitude), they suggested that type III secreted effector proteins other than Tir play a role in the pathogenicity of strain 905.

EspH promotes EHEC colonization of the intestinal tract. espH does not influence C. rodentium colonization of the murine intestine, nor is it required for C. rodentium-induced pedestal formation on HeLa cells (12, 37). To examine whether EspH influenced EHEC pathogenicity, we constructed 905{Delta}espH, a derivative of 905 that contains an in-frame deletion in espH. espH was not required for EHEC-induced pedestal formation on HeLa cells (Table 2) or for pedestal formation in the colons of infant rabbits (Fig. 2C).

Even though 905{Delta}espH induced pedestal formation in vitro and in vivo, deletion of espH attentuated the pathogenicity of 905. In contrast to infection with 905, not all infant rabbits infected with 905{Delta}espH developed severe diarrhea (Table 3). Nine of 17 rabbits infected with 905{Delta}espH had mild diarrhea; these rabbits exhibited limited fecal staining on their rear legs and perinea and contained a mix of formed and unformed stool in their distal colons at necropsy. The espH deletion reduced 905 colonization throughout the intestinal tract (Fig. 3). Compared to the numbers of 905 CFU recovered from the intestine, fewer 905{Delta}espH CFU (~1 log unit fewer) were recovered from each of the three intestinal sections sampled. Despite the reduced severity of diarrhea observed in some rabbits and the reduced numbers of 905{Delta}espH recovered from the intestinal tracts of all infected rabbits, the levels of colonic pathology in these animals were not reduced. The scores for PMN infiltration, edema, and congestion and the level of mucosal damage in rabbits infected with 905{Delta}espH were similar to those observed in rabbits infected with 905 (Fig. 4). Similar levels of IL-8 were also measured in midcolon homogenates from both these groups of rabbits (data not shown).

The location of espH at the end of the LEE 3 operon (Fig. 1) makes it unlikely that a polar effect would result from the espH deletion. To rule out the possibility that a second, unlinked mutation in the 905{Delta}espH strain was responsible for its colonization defect, we constructed another espH deletion mutant in the 905 background. The phenotypes of infant rabbits infected with this independently derived 905{Delta}espH deletion mutant were very similar to the phenotypes of rabbits infected with the original 905{Delta}espH mutant. The infected rabbits displayed either mild or severe diarrhea (Table 3), and their levels of intestinal colonization were lower than those of rabbits infected with strain 905 (Fig. 3). We did not attempt to complement the espH deletion because of the problems that have been associated with complementation in other animal studies (13, 37).

These findings indicate that EspH serves as an EHEC intestinal colonization factor and suggest that EspH promotes EHEC-induced diarrhea. The mechanism by which EspH promotes EHEC intestinal colonization is not known. EspH may augment the initial adherence of EHEC to intestinal epithelial cells, since an espH mutant induced fewer and more-fragmented actin pedestals on HeLa cells after short infection periods than the wild-type strain (55). Since deletion of espH from the C. rodentium LEE had no effect on this pathogen's colonization of the murine intestine, our findings suggest that the LEE-encoded effectors have different functions in different A/E pathogens.

Map and EspG promote EHEC colonization of the small intestine. Deletion of either map or espG had no effect on C. rodentium colonization of the murine colon at late time points postinoculation (12, 37); however, at earlier time points, Mundy et al. (37) found that a C. rodentium map mutant had reduced intestinal colonization. In vitro studies using EPEC have revealed that Map and EspG are not required for pedestal formation but that both proteins interfere with host cytoskeleton rearrangements (9, 23). Map promotes the formation of filopodia over that of pedestals in a dose-dependent manner in HeLa cells; in the absence of Map, there was accelerated EPEC-induced pedestal formation (23). EspG triggers actin stress fiber formation and stimulates microtubule destabilization, events that may alter intestinal barrier permeability (9).

To investigate the contribution of Map to EHEC pathogenesis, we constructed 905{Delta}map. As reported for EPEC (23), 905{Delta}map induced pedestal formation to a level similar to that of 905 in HeLa cells (Table 2). Furthermore, 905{Delta}map formed pedestals in the colons of infant rabbits (Fig. 2D). Infant rabbits infected with 905{Delta}map had clinical signs of disease, including severe diarrhea, that were indistinguishable from those observed in rabbits inoculated with 905 (Table 3). However, map proved to be important for 905 colonization of the small intestine. There was an 11-fold reduction in 905{Delta}map colonization of the small intestine (Fig. 3A). Map did not influence 905 colonization of the ceca and midcolons, as equal numbers of 905{Delta}map and 905 were recovered from these intestinal segments (Fig. 3B and C). Finally, the levels of intestinal inflammation, Stx2, and IL-8 in rabbits infected with 905{Delta}map were similar to those of rabbits infected with 905 (Fig. 4 and data not shown).

espG, like map, promoted EHEC colonization of the rabbit small intestine but did not otherwise influence the pathogenicity of 905 in infant rabbits. There were 12-fold-fewer 905{Delta}espG CFU recovered from the ilea of infected rabbits than 905 CFU (Fig. 3A). However, recovery of this 905 derivative containing an in-frame deletion of espG from the ceca and midcolon did not differ from that of 905 (Fig. 3B and C). Although a role for EspG in intestinal colonization was not seen in C. rodentium (12, 37), our results are consistent with observations of the espG homolog in REPEC. Elliot et al. (14) detected ~1-log-fewer CFU of an REPEC espG mutant than the wild-type REPEC strain from the feces of infected adult rabbits. Interestingly, while 905{Delta}espG formed A/E lesions in rabbits (Fig. 2E), it induced significantly (P < 0.01) fewer pedestals on HeLa cells than 905 (Table 2).

There are several possible explanations why EHEC 905{Delta}map and 905{Delta}espG exhibit an organ-specific defect in colonization of the small intestine. In infant rabbits, it is possible that the targets for Map and EspG are found only in the small intestine. Alternatively, Map and EspG may promote colonization of both the small and large intestines, but the activity of host factors present only in the large intestine obscure the actions of Map and EspG. Polar effects are unlikely to account for the phenotypes of 905{Delta}map and 905{Delta}espG that we observed, since map is located between LEE 3 and LEE 5 and is transcribed as a monocistronic unit (47) and espG lies outside the main LEE operons on the far left-hand side of the pathogenicity island (Fig. 1).

EspF suppresses inflammation and promotes EHEC colonization. In mice, C. rodentium espF mutants exhibited reduced intestinal colonization and colonic hyperplasia but formed pedestals on tissue culture cells (12, 37). EHEC 905{Delta}espF induced pedestal formation in HeLa cells (Table 2) (4) and in the colons of infant rabbits (Fig. 2F). Interestingly, deletion of espF promoted the pathogenicity of 905 in infant rabbits. Although difficult to quantify, the diarrhea in the rabbits infected with 905{Delta}espF appeared more voluminous than in rabbits infected with 905 (Table 3). At necropsy, examination of the intestinal tracts of 905{Delta}espF-infected rabbits revealed that the ceca and colons contained more liquid feces and appeared more swollen than the intestinal tracts of 905-infected rabbits. Despite the increased severity of the clinical signs of disease in 905{Delta}espF-infected rabbits, intestinal colonization by 905{Delta}espF was reduced. The numbers of 905{Delta}espF CFU recovered from the ilea, ceca, and midcolons of infected rabbits were ~1 log lower than the numbers of 905 CFU recovered from these parts of the intestine (Fig. 3A to C).

Consistent with our visual impressions of the midcolon, histologic analysis revealed that there was significantly more inflammation, manifest as PMN infiltration, in the midcolons of 905{Delta}espF-infected rabbits than in 905-infected rabbits (Fig. 4A). The levels of edema, congestion, and mucosal damage in 905{Delta}espF- and 905-infected rabbits were the same (Fig. 4B and C). To further quantify the differences in PMN infiltration in 905{Delta}espF- and 905-infected rabbits, we counted the number of PMNs per high-power field (x400 magnification). There were significantly (P < 0.05) more PMNs in sections from the midcolon of rabbits infected with 905{Delta}espF than those infected with 905 (80 ± 8 and 61 ± 5 [mean ± SE], respectively).

Given the unexpected findings that the espF mutant elicited increased diarrhea and intestinal inflammation but had diminished intestinal colonization, we constructed another espF deletion mutant to rule out the possibility that a second, unlinked mutation in the 905{Delta}espF strain was responsible for these phenotypes. Since espF is the last gene of the LEE 4 operon, polar effects of the espF mutation are unlikely to account for the phenotypes we observed. Infection of infant rabbits with this independently derived 905{Delta}espF mutant resulted in phenotypes very similar to that of our original mutant (Table 3 and Fig. 3 and 4) with one exception; colonization of the ileum by the second 905{Delta}espF derivative was not statistically different from that of 905.

Overall, our findings suggest that EspF promotes EHEC colonization by modulating the host inflammatory response through inhibition of PMN accumulation in the colon by an unknown mechanism. Other enteric bacteria modulate the host immune response with effector proteins delivered to the host cell cytoplasm via TTSSs (48). For example, YopJ in Yersinia pseudotuberculosis (YopP in Yersinia enterocolitica) represses expression of inflammatory mediators, such as IL-8 (51). IL-8, a potent PMN chemoattractant, is one of the major chemokines induced after EHEC infection of intestinal epithelial cell lines (7, 43); elevated IL-8 levels are also found in the sera of patients infected with this pathogen (57). Although inhibition of IL-8 production by EspF could explain our findings, we did not detect differences in the levels of IL-8 in midcolon homogenates of rabbits infected with 905{Delta}espF or 905 (data not shown). However, the kinetics of IL-8 expression in infant rabbits after EHEC infection require further definition, and other factors besides IL-8 could influence PMN accumulation in infant rabbits.

Conclusions. Our findings indicate that the LEE pathogenicity island enables EHEC to colonize the intestinal tract. Disabling the LEE-encoded TTSS by deleting escN or genes encoding the individual effector proteins secreted by the TTSS reduced EHEC colonization of the rabbit intestine. Deletion of the effector proteins compromised EHEC intestinal colonization to various degrees. Tir plays a central role in colonization, since deletion of tir resulted in up to a ~10,000-fold reduction in colonization (44). In contrast, EspH, EspF, EspG, and Map are accessory colonization factors, since their removal resulted in only modest (~10-fold) reductions in colonization. Furthermore, EspG and Map influenced colonization of only the small intestine, suggesting that these effector proteins exhibit organ-specific effects. None of the four LEE-encoded effector proteins studied here were required for A/E lesion formation in vivo or in vitro, suggesting that formation of these lesions does not guarantee maximal intestinal colonization. The relatively subtle effects of espH, espF, espG, and map on colonization may indicate that there is some redundancy in their activities. Analyses of double mutants might uncover more dramatic colonization defects as is the case in Y. pseudotuberculosis. Colonization of the murine intestine by Y. pseudotuberculosis was dramatically reduced in a yopH yopE double mutant but not in single yopH or yopE mutant strains (29).

The mechanisms by which EspH, EspF, EspG, and Map promote colonization remain to be elucidated. These effector proteins could influence any of the four types of processes—niche occupancy (such as chemotaxis [3]), metabolism, adherence, and resistance to host defenses—that control colonization. Since the effector proteins are translocated to the host cytoplasm, it is likely that they affect either adherence or resistance to host defenses. Given previous findings that EspH and Map influence host cytoskeletal processes in tissue culture cells (23, 55) and our observation that EspG promotes the formation of pedestals on HeLa cells, these three effector proteins may promote adherence. Besides promoting EHEC adherence, the LEE-encoded effector proteins may dampen or even subvert the host immune response to EHEC. EspF appears to act in this fashion, since in the absence of this effector protein, we observed increased PMN accumulation in the intestinal mucosa. This suggests that EspF reduces the amount or activity of a host cytokine.

Our observations indicate that several factors govern the severity of EHEC-induced diarrhea in infant rabbits. We previously established that Stx2 is critical for EHEC-induced diarrhea (44). Intestinal colonization itself may also promote diarrhea. Rabbits infected with EHEC 905{Delta}espH had reduced diarrhea, but their levels of Stx2 were similar to those measured in the intestines of rabbits infected with strain 905 (data not shown). Our findings also suggest that the host inflammatory response to EHEC contributes to EHEC-induced diarrhea. Even though deletion of espF lowered EHEC colonization, the heightened inflammation we found in rabbits infected with 905{Delta}espF was associated with more severe diarrhea in these animals. We previously found that Shiga toxin was also critical for the development of inflammation in EHEC-infected infant rabbits (44). In other pathogens, virulence factors that promote inflammation also heighten disease. For example, several effector proteins secreted by the Salmonella enterica serovar Typhimurium TTSS-1 are thought to promote infiltration of PMNs into the intestine and lead to the development of diarrhea (59). The proinflammatory effects of Shiga toxin may be at least partially offset by the anti-inflammatory effects of EspF. Often anti-inflammatory factors, like EspF, promote colonization, whereas proinflammatory factors, like Shiga toxin, augment dissemination. Ultimately, EHEC and other pathogens may control the host immune response to achieve a balance between growth in the host (colonization) and dissemination (via diarrhea) to new hosts.

Finally, our findings suggest that there are important differences in the pathogenicity of EHEC and C. rodentium. At least in infant rabbits, the functions of the EHEC effector proteins are not identical to those reported for the respective C. rodentium effector proteins (12, 37). For example, espH plays a role in colonization in EHEC but not in C. rodentium. These differences indicate that conclusions drawn from studies of C. rodentium infection of mice may not be applicable to EHEC.


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ACKNOWLEDGMENTS
 
We thank Cathy Linsenmayer and Cheleste Thorpe for help with electron microscopy and guidance with cell lysis protocols, respectively. We are indebted to Kenneth Campellone (University of Massachusetts) for teaching us the FAS assay. We thank Anne Kane and Doug Jefferson for preparing microbiological media and for providing HeLa cells, respectively. We are indebted to Arlin Rogers (MIT) for histopathologic assessment of rabbit tissue. We also thank Theresa Ho, Marion Shonn Dorer, Jonathan Livny, John Leong, and Kenneth Campellone for critical review of the manuscript.

This study was supported in part by the Howard Hughes Medical Institute and by grants AI-42347 and P30DK-34928 for the Center for Gastroenterology Research on Absorptive and Secretory Processes.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111. Phone: (617) 636-2730. Fax: (617) 636-2723. E-mail: matthew.waldor{at}tufts.edu. Back

Editor: A. D. O'Brien


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Infection and Immunity, March 2005, p. 1466-1474, Vol. 73, No. 3
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.3.1466-1474.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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