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Infection and Immunity, January 2001, p. 315-324, Vol. 69, No. 1
Biology Department, Reed College, Portland,
Oregon 972021; Center for Vaccine
Development,3 Department of Microbiology
& Immunology,4 and Departments of
Medicine and Pediatrics,5 University of
Maryland School of Medicine, Baltimore, Maryland 21201; and
Department of Public Health, Department of Cell Biology,
CINVESTAV-IPN, 07000 Mexico DF, Mexico2
Received 13 September 2000/Accepted 28 September 2000
At least five proteins are secreted extracellularly by
enteropathogenic Escherichia coli (EPEC), a leading cause
of infant diarrhea in developing countries. However only one, EspC, is
known to be secreted independently of the type III secretion apparatus encoded by genes located within the 35.6-kb locus of enterocyte effacement pathogenicity island. EspC is a member of the
autotransporter family of proteins, and the secreted portion of the
molecule is 110 kDa. Here we determine that the espC gene
is located within a second EPEC pathogenicity island at 60 min on the
chromosome of E. coli. We also show that EspC is an
enterotoxin, indicated by rises in short-circuit current and potential
difference in rat jejunal tissue mounted in Ussing chambers. In
addition, preincubation with antiserum against the homologous Pet
enterotoxin of enteroaggregative E. coli eliminated EspC
enterotoxin activity. Like the EAF plasmid, the espC
pathogenicity island was found only in a subset of EPEC, suggesting
that EspC may play a role as an accessory virulence factor in some but
not all EPEC strains.
Secretion of effector molecules
allows pathogenic bacteria to interact with their host and cause
disease. Enteropathogenic Escherichia coli (EPEC), a leading
cause of infantile diarrhea in developing countries, secretes at least
six proteins (24, 28). Four of these proteins, EspA
(29), EspD (31), EspB (46), and
EspF (36) (E. coli secreted protein), are
secreted by a type III secretion system, and the Esp molecules as well as the secretion apparatus are encoded within a 35.6-kb pathogenicity island termed the locus of enterocyte effacement (LEE) (11, 35). Recently, it was demonstrated that the EspA, EspD, and EspB
proteins form a translocon for delivering effector molecules into the
host cytoskeleton (15, 30). Another secreted molecule, Tir
(translocated intimin receptor) (27), is hypothesized to pass through this structure en route to translocation into the host
cell membrane, where it serves as a receptor for the EPEC adhesin
intimin, also encoded within the LEE (25). Tir is involved in host cell signaling and disruption of the cytoskeleton
(27). These secreted effector molecules are involved in
the formation of attaching and effacing (AE) intestinal lesions, a
hallmark of EPEC disease.
EPEC also secretes a 110-kDa protein which does not require the type
III secretion system for delivery into the extracellular milieu
(24, 45). This protein, EspC, shows amino acid homology to
members of the immunoglobulin A (IgA) protease family of
autotransporters which include, among others, the IgA protease of
Neisseria gonorrhoeae (38), Hap of
Haemophilus influenzae (23), Tsh of
avian-pathogenic E. coli (39), the SepA and
ShMu proteins of Shigella flexneri (4, 40), Pic
of enteroaggregative E. coli (EAEC) and Shigella flexneri 2457T (22), Pet of EAEC (12),
and EspP of enterohemorrhagic E. coli (EHEC)
(7). For a review, see reference 21.
The mechanism of autotransport was first described for the IgA protease
of N. gonorrhoeae (38). The precursor protein
is exported beyond the cytoplasmic membrane in a Sec-dependent manner, coupled to the cleavage of a signal peptide. The mature protein possesses a C-terminal domain that is thought to form a Several members of the autotransporter family of proteins, including
Tsh, SepA, ShMu, EspP, Pet, Pic, and EspC, have a conserved serine
protease motif. None of these proteins, however, cleave IgA. This
subfamily of autotransporters have thus been termed SPATE, for serine
protease autotransporters of Enterobacteriaceae (21). It was recently demonstrated that Pet of EAEC is an
enterotoxin that induces loss of actin microfilaments (12,
37). The function of EspC of EPEC, however, is unknown. A
mutation in espC did not affect the ability of EPEC to
disrupt cytoskeletal rearrangement, phosphorylate a 90-kDa host protein
(now known to be the bacterium-derived Tir protein), or adhere to or
invade three different tissue culture cell lines (45).
Virulence genes of pathogenic bacteria are often located on
transmissible genetic elements such as plasmids, transposons, or
bacteriophages, and many of these proteins are encoded within specific
regions of the chromosome termed pathogenicity islands (17, 20,
32); the SPATE proteins adhere to this observation. EspP of
E. coli O157:H7 (7) and Pet of EAEC
(12) are encoded on virulence plasmids, pO157 and pAA2,
respectively, and ShMu of Shigella flexneri and Pic of EAEC
are located within a pathogenicity island (22, 40). The
location of espC in EPEC, however, is yet to be determined.
The gene encoding EspC has been cloned and sequenced. Previous studies
indicated that espC was not located within the LEE pathogenicity island of EPEC strain E2348/69 (11, 35) or
on the EAF plasmid (45), which contains genes necessary
for bundle-forming pilus biogenesis and the Per regulator. We therefore
hypothesized that espC was chromosomally located, perhaps
associated with other loci found only in pathogenic strains of
E. coli. In light of an earlier report that EspC was
not involved in the AE phenotype (45), we wanted to
further investigate a potential function for this protein. In this
study, we determine that espC is encoded within a
pathogenicity island located at 60 min on the chromosome of EPEC and
that the EspC protein has enterotoxic activity.
Bacterial strains and growth conditions.
The EPEC strain
containing espC used in this study was the
well-characterized, prototype AE pathogen E2348/69 (O127:H6)
(33). JPN15 is an EAF plasmid-cured derivative of strain
E2348/69 (25). Strains used in DNA colony hybridization
studies are listed in Table 1. For
isolating secreted proteins, recombinant plasmids were transformed into
laboratory strain HB101. All strains were grown in Luria-Burtani (LB)
broth aerobically at 37°C unless otherwise stated. Where appropriate,
culture media were supplemented with ampicillin (100 µg/ml).
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.1.315-324.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
espC Pathogenicity Island of
Enteropathogenic Escherichia coli Encodes an
Enterotoxin
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
-barrel in
the outer membrane, and the N-terminal passenger domain is exported
beyond the outer membrane through the -barrel. The passenger domain
is then clipped autocatalytically from the -barrel and released. In
the case of EspC of EPEC, the molecular mass of the exported passenger
protein is 110 kDa (45).
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Colony hybridization studiesa
Recombinant DNA techniques.
All genetic manipulations were
performed using DH5
as a host strain. Unless otherwise stated,
enzymes were purchased from BRL and procedures were performed as
described previously (41). A previously constructed cosmid
library containing EPEC DNA in pHC79 (34) was screened
using an internal espC SalI fragment (see Fig. 1) and the
colony hybridization procedure described below. For
electrophysiological measurements in rat jejunum tissue, a minimal DNA
fragment containing espC was directionally ligated into the
pBAD30 vector (19) after PCR amplification with
Pwo enzyme (Boehringer Mannheim) using the oligonucleotide
primers K1314B (5'-CCGGAATTCTGGACTTCAGATCTGGTGATA-3')
and 1315B
(5'-CTACGAAGCTTGCGAGCTTCTTGTGAGAAAGA-3') containing EcoRI and HindIII
restriction sites (underlined), respectively. Cosmid DNA was used as
the template for PCR. The plasmid containing the 4-kb
espC minimal fragment was designated pJLM174.
-32P]dCTP using Ready-To-Go DNA Labeling beads, and
unincorporated nucleotides were removed using ProbeQuant G-50 Micro
columns following the manufacturer's instructions. Both kits were
purchased from Pharmacia Biotech. After hybridization, approximately
106 cpm of each radiolabeled probe was added to filters,
and incubation proceeded at 65°C overnight with shaking. Filters were
washed twice for 30 min each at 65°C and exposed to autoradiograph
film.
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DNA sequencing. DNA sequence analysis was performed by the University of Maryland Biopolymer Facility on an ABI automated sequencer with cosmid DNA templates purified using Qiagen midicolumns. Double-stranded sequence was aligned and analyzed using Sequencher software.
Preparation of concentrated supernatants. Wild-type E2348/69, HB101, HB101 (p2R) harboring espC in a cosmid clone, and the cosmid vector-only control HB101 (pHC79) were grown overnight at 37°C in 100 ml of LB broth. HB101(pJLM174) harboring the 4-kb espC minimal fragment in pBAD30 was also grown overnight at 37°C in 100 ml of LB broth supplemented with 0.2% glycerol and 0.2% glucose (repression) or 0.2% arabinose (induction). The bacterial cultures were centrifuged at 12,000 × g for 10 min and filtered through a 0.22-µm cellulose acetate filter, and the supernatants were concentrated 100 × and size fractionated (100-kDa cut off) using Ultrafilters (Biomax-100; Ultrafree; Millipore, Bedford, Mass.) according to the manufacturer's instructions.
Western immunoblot.
Proteins present in concentrated
supernatants from HB101, E2348/69, HB101(p2R), and the
espC minimal clone in HB101(pJLM174), grown with
glycerol and glucose or arabinose, were analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing
conditions (boiling for 5 min in the presence of -mercaptoethanol).
Proteins separated by SDS-PAGE were transferred to nitrocellulose BA85
membranes (Schleicher and Schuell, Keene, N.H.) by standard methods.
The membranes were incubated with rabbit antibodies against Pet protein
(37) diluted 1:100. Immunostaining was performed with
alkaline phosphatase-labeled polyclonal antibodies against rabbit
immunoglobulins (dilution, 1:2,000) and developed with
5-bromo-4-chloro-3-indolylphosphate and Nitro Blue Tetrazolium by
standard methods.
Electrophysiological measurements in rat jejunum. Ussing chamber experiments were performed as described previously (37). Jejunal segments removed from adult male Sprague-Dawley rats under sodium pentobarbital anesthesia were placed in ice-cold Ringer's solution for mammals and gassed with an O2-CO2 (95%-5%) mixture. The excised segments were cut open along their mesenteric border, washed with cold Ringer's solution, divided into six fragments, and mounted between the circular openings of two adjacent Ussing hemichambers. Each hemichamber was filled with 10 ml of Ringer's solution and kept at 37°C under constant O2-CO2 bubbling. Supernatants (100 µl) from HB101, HB101(p2R), HB101(pHC79), and HB101(pJLM174) were added to the mucosal hemichamber of rat jejunum preparations after 30 min of equilibration. Transepithelial electrical potential difference (PD) and total tissue conductance (Isc) were measured at 10-min intervals using a voltage clamp apparatus. Short-circuit current was calculated using Ohm's law (18). Statistical analyses were performed with Student's t test on data recorded from at least four experiments.
Ussing chamber experiments in which enterotoxic activity was inhibited by antibodies against Pet protein were performed by methods described previously (37). Concentrated supernatants (100 µl) were incubated for 30 min at room temperature with rabbit polyclonal antibodies directed against the Pet protein (diluted 1:25) prior to addition to the luminal hemichamber.Nucleotide sequence accession number. The nucleotide sequence of the EPEC pathogenicity island containing espC will appear in the EMBL/GenBank/DDBJ data library under accession number AF297061.
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RESULTS |
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Materials and Methods
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EspC is encoded within a region of unique DNA on the EPEC
chromosome.
An internal 1.6-kb SalI espC
fragment (Fig. 1) was used to identify
cosmids containing espC and flanking sequences from a previously constructed cosmid library of EPEC E2348/69 DNA
(34). Four independent, nonidentical cosmid clones (p2R,
p2L, p9R, and p10R) were obtained by colony hybridization; cosmids were
isolated, and restriction digests demonstrated that all contained the
1.6-kb SalI fragment, a 4-kb KpnI fragment, and
other identical restriction fragments (Fig. 1 and data not shown).
Therefore, all four cosmids contained espC and surrounding
DNA. Two of the cosmids, p2R and p10R, were subjected to sequence
analysis.
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Junctions of unique DNA sequences.
The left junction of the
unique DNA sequences was defined by Blast search and PCR analysis.
Blast search identified DNA sequences within cosmid p9R identical to
those located adjacent to the orf360 gene at 60.06 min of
the E. coli strain MG1655 chromosome (Fig. 2A).
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Distribution of unique DNA sequences. Colony hybridization studies were used to determine the distribution in 42 E. coli strains of espC and other DNA elements found within the unique 15.2-kb region, as well as the E. coli K-12 genes ygaF and ssrA, located near the left and right junctions, respectively. The majority of these strains were previously categorized into diarrheagenic E. coli clones (DEC) groups according to the phylogenetic analyses of Whittam et al. (48, 49). Probes B, C, and D (Fig. 1), directed to orf3 and espC, only hybridized to DNA from pathogenic strains of the EPEC1 category of DEC clones, which includes DEC1 and DEC2 (Table 1) (49). DNA sequences homologous to orf3 and espC were found only in EPEC E2348/69 (O127:H6), the EAF plasmid-cured derivative of this strain, JPN15, and serotypes O142:H6, O55:H6, and O55:NM. Probes B, C, and D did not hybridize to serotypes of the DEC collection belonging to the EPEC2, EHEC1, or EHEC2 categories. We looked at other AE pathogens, specifically Citrobacter rodentium biotype 4280, RDEC1, and other strains of both EHEC and EPEC categories, and found that probes B, C, and D did not hybridize to DNA from these strains.
PCR was performed to determine whether other EPEC or EHEC strains related to E2348/69 possessed insertions at the left and right junctions. Identical to the PCR results obtained with E2348/69, the DNA template from another EPEC strain, serotype O142:H6, generated a 716-bp amplicon with primer pair 2 and no product with primer pair 1 (Fig. 3A), a 1,416-bp amplicon with primer pair 4, and no product using primer pair 3 (Fig. 3B). EPEC E2348/69 and O142:H6 are both members of the EPEC1 diarrheagenic E. coli category (49). Serotypes O157:H7, O111:H2, and O26:H11 from diarrheagenic E. coli categories EHEC1, EPEC2, and EHEC2, respectively, were also subjected to this analysis. DNA templates from these strains generated K-12-specific amplicons of 584 bp using primer pair 1 and no product using primer pair 2. These results indicated that there were no insertions located at 60.06 min on the chromosomes of these strains (Fig. 3A), consistent with the results of the DNA hybridization studies (Table 1). PCR using EHEC1, EPEC2, and EHEC2 DNA as templates generated no products with either primer pair 3 or 4 (Fig. 3B). In addition, no PCR products were visualized using the same DNA templates and primers J9, which is specific to smpB, and K1062 or the primer pair J9 and J10. These data suggested that DNA sequences located at 59.35 min in the EHEC1, EPEC2, and EHEC2 strains differed from the DNA sequences at the same location of the chromosome in E. coli K-12 strain MG1655. The 15.2-kb region of unique DNA contained several mobile genetic elements, most of which appeared to be nonfunctional due to deletions or frameshift mutations (Fig. 1). Hybridization probes E and F corresponded to orfs with predicted amino acid homology to putative transposases found in E. coli and Y. pestis, respectively (13, 43). DNA sequences homologous to probes E and F, compared to those homologous to probes B, C, and D, were more commonly found in pathogenic strains of E. coli, including several of the AE pathogens (Table 1). In addition, probe F hybridized to DNA from laboratory strain HB101. We were unable, however, to observe any correlation between the existence of sequences homologous to genes encoding the putative transposases and a specific category of E. coli pathogen. The E. coli K-12 gene ygaF located at 60.08 min is near the left junction at 60.06 min of the chromosome. Probe A, corresponding to ygaF, also hybridized to all strains of E. coli tested, including K-12 laboratory strains and enteric pathogens (Table 1). The only exception was that probe A did not hybridize with one strain of DEC clone 11; however, this probe did hybridize to three out of four strains of the DEC clone 11 tested. Probe G, corresponding to ssrA, located at 59.35 min of the chromosome, also hybridized to all strains of E. coli tested (Table 1).Phenotypic analysis of the EspC protein.
Comparison of the
deduced EspC amino acid sequence with those available in GenBank
databases revealed 52% identity (67% similarity) with the Pet protein
of enteroaggregative E. coli (EAEC) (12), a
toxin which produces enterotoxic activity on intestinal tissue mounted
in Ussing chambers (12, 37). To determine the possible enterotoxic activity of EspC protein, the > 100-kDa fraction of supernatants from HB101(p2R) harboring the cloned espC gene
and HB101(pHC79) containing the vector only were added to the luminal side of rat jejunal tissue mounted in the Ussing chamber. Concentrated HB101(p2R) supernatant fractions containing EspC produced an increase in jejunal PD and Isc (Fig. 4A and B),
whereas concentrated supernatants from HB101 (pHC79) failed to induce
increases in PD and Isc.
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In this study, we have demonstrated that the espC gene of the prototype EPEC strain E2348/69 is located within a unique region of DNA not found in commensal or laboratory strains of E. coli. Based on previously presented definitions (20), we have designated this region the espC pathogenicity island because (i) the region contains at least two loci associated with virulence, the previously described espC gene (45) and ORF3, which shows predicted amino acid similarity with VirA of Shigella flexneri (47), (ii) sequences homologous to espC and orf3 were present in pathogenic but not laboratory strains of E. coli, (iii) the G+C content of the unique 15, 195-bp region is 40.5%, substantially lower than that of E. coli K-12 (50.8%) (6), suggesting that this region was acquired by horizontal transfer (2), and (iv) several ORFs with predicted protein similarity to a variety of mobile genetic elements are contained in this region. The espC pathogenicity island inserted at a chromosomal site adjacent to a tRNA-like gene, ssrA, which is also the site of insertion of the pathogenicity island of V. cholerae (26).
By DNA sequence and PCR analysis, the left junction of the espC pathogenicity island was located at 60.06 min of the complete sequence of the E. coli strain MG1655 chromosome, adjacent to orf360. Similarly, by DNA sequence and PCR analysis, the right junction of the espC pathogenicity island of EPEC1 strains was located at 59.35 min, adjacent to the ssrA gene (6). It was recently reported that one of the so-called loops or genomic regions that differ between Salmonella enterica serovar Typhimurium and E. coli is located at 60 min, between the smpB and nrdE genes (3). There is approximately 91 kb of DNA between smpB and nrdE in S. enterica compared to 46 kb at this site in E. coli strain MG1655. Therefore, within this region of S. enterica lies 45 kb of DNA not found in E. coli, and it has been proposed that this region may contain loci that distinguish Salmonella from E. coli (3). Similarly, the espC pathogenicity island may distinguish the EPEC1 category from commensal strains and other categories of E. coli intestinal pathogens.
The observation that the left and right junctions were at 60.06 and 59.35 min, respectively, of the E. coli chromosome suggests that approximately 33 kb of DNA found between these locations in the K-12 strain MG1655 chromosome are missing from the EPEC1 strains or that chromosomal rearrangements have occurred in this region. Rearrangements or sequence differences may also be present in the EPEC2, EHEC1, and EHEC2 pathogens at these locations, because oligonucleotide primers directed to neither the ssrA nor smpB sequences of strain MG1655 amplified predicted fragments by PCR when EPEC2, EHEC1, or EHEC2 DNA was used as the template. However, a 407-bp DNA probe directed to the ssrA sequences of strain MG1655 hybridized to DNA from all three of these pathotypes.
DNA probes directed to espC hybridized to DNA from strains of the EPEC1 category (E2348/69 and DEC clones 1 and 2 (48, 49), but not to other AE pathogens, including representatives of the EPEC2, EHEC1, and EHEC2 categories of diarrheagenic E. coli, RDEC1, and Citrobacter rodentium biotype 4280. This observation and the fact that the cloned LEE from strain E2348/69 (EPEC1) in the E. coli laboratory strain HB101 is sufficient to form AE lesions in vitro (35) are consistent with the conclusion that EspC is not associated with the intestinal AE phenotype (45).
espC and adjacent non-K-12 sequences may have been acquired by EPEC1 in a single horizontal transfer event. Alternatively, espC and the flanking sequences may have been acquired via multiple events. Evidence supporting the latter hypothesis comes from analysis of the G+C content of the espC pathogenicity island. The G+C content of the espC structural gene is 42.5%, whereas that of the sequences spanning from rorf1 to orf3 is approximately 35% and of those including orf5 to orf8 is 46%. These data might suggest that the island was acquired by multiple events of horizontal transfer, since bacteria develop characteristic G+C contents during evolution. These data are also consistent with the PCR analyses performed on a distribution of pathogenic E. coli serotypes. We observed a decrease in the G+C percentage from the right to left junction that likely signifies that the DNA closer to the left junction was horizontally acquired earlier than the DNA towards the right junction. A similar mosaic structure, with DNA sequences acquired from distinct sources, has been described for the smpB-nrdE intergenic region of S. enterica (3).
Downstream of espC we identified DNA sequences similar to IS1351 and IS200. Remnants of IS elements flank the pet gene on the pAA2 plasmid of EAEC, and IS1203-like and iso-IS1-like elements flank espP on the pO157 plasmid of E. coli serotype O157:H7 (7, 9, 12). The 102-kb high pathogenicity island of Y. pestis can be deleted by a recombination event occurring between two flanking IS100 element (14), and it was proposed that the IS629 and IS911 elements may have played a role in the insertion of pet onto plasmid pAA2 (12). Similarly, IS911 and IS629 elements are found upstream of the chromosome-located pic gene of EAEC (22). From these observations, we hypothesize that the IS1351 or IS200 elements or perhaps the IS100 putative transposase and associated repetitive sequence may have played a role in the acquisition of espC by the EPEC1 strains.
Members of the autotransporter family have been identified in a number of pathotypes of E. coli. Surprisingly, DNA probes directed to the conserved C terminus of the espC structural gene did not hybridize to DNA from EAEC, which contain the gene encoding the homologous Pet autotransported enterotoxin, and a pet probe did not hybridize to EPEC DNA (data not shown). Similar to these results, an espP DNA probe encoding the homologous EspP protein in EHEC O157:H7 did not hybridize to EPEC strain E2348/69 in a Southern analysis, though it did hybridize to DNA from several isolates of serotype O157:H7 and one isolate of serotype O26 (7). However, Brunder et al. (7) visualized secreted proteins from two EPEC strains, including E2348/69, that cross-reacted with EspP antiserum. These data might suggest that, due to divergent DNA sequences, hybridization studies are not appropriate for identifying genes encoding related autotransporters of the SPATE subfamily, whereas antibody cross-reactivity in immunoblot procedures more readily detects the structural relatedness of these proteins.
We have determined that EspC possesses enterotoxic activity, finding that concentrated supernatants from a laboratory strain of E. coli expressing EspC increased tissue PD and Isc using rat jejunum mounted in Ussing chambers. Antibodies directed against the homologous Pet protein of EAEC cross-reacted with EspC in Western blots, and preincubation with Pet antiserum eliminated EspC's ability to increase PD and Isc in Ussing chambers. A previous study examined the possible role of EspC in interactions with epithelial cells (45). These investigators found that EspC was not necessary for mediating EPEC-induced signal transduction necessary for formation of AE lesions in HeLa cells and did not play a role in either adherence or invasion of tissue culture cells. Enterotoxin activity was not tested in this previous study.
In addition to EspC, the orf3 gene product of the
espC pathogenicity island may also encode a virulence
factor. The predicted ORF3 protein shows similarity with VirA of
Shigella and rORF2 of EPEC. Both cloned ORF3 and rORF2 were
able to rescue a Shigella 
virA mutation that eliminates
this bacterium's ability to invade epithelial cells in culture (S. Elliott, E. O. Krejany, J. L. Mellies, R. M. Robins-Browne, C. Sasakawa, and J. B. Kaper, submitted for
publication). An orf3 and rorf2 double mutation
did not affect EPEC invasion, though it was demonstrated that the
protein product of rorf2 of EPEC was secreted by the type
III secretion system and translocated into host epithelial cells
(Elliott et al., submitted). Because of its homology with the product
of rorf2, the protein product of orf3 of the
espC pathogenicity island may also be secreted by the type
III system, but the function of these proteins encoded by
orf3 and rorf2 remains obscure.
Identification of autotransporters in at least 30 gram-negative
pathogens (21) which cause a variety of diseases suggests that these proteins play an important role in pathogenesis. The C-terminal -domains of autotransporters show a high degree of amino
acid homology and appear to perform a common function, whereas the
passenger domains are more divergent. Many functions have been
attributed to the passenger domains, including adhesion, protease and
toxin activity, and cellular invasion (5, 12, 22, 23, 39,
42). Of the SPATE autotransporters most closely related to EspC,
Pet of EAEC is also an enterotoxin (12, 37), whereas the
EspP protein of EHEC serotype O157:H7 cleaves human coaggulation factor
V and has been proposed to be a contributing factor in mucosal
hemorrhaging in patients with hemorrhagic colitis (7). Pic
of EAEC was implicated in mucinase activity, serum resistance, and
hemagglutination (22). Like the EAF plasmid of EPEC, the
espC pathogenicity island was found only in a subset of
these pathogens, though other strains may possess genes encoding comparable function. EspC most likely plays an accessory role in EPEC
pathogenesis, presumably as an enterotoxin, and the location of the
gene encoding EspC within a pathogenicity island supports this hypothesis.
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ACKNOWLEDGMENTS |
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We thank Lisa Sadzewicz and Nick Ambulos of the University of Maryland at Baltimore Biopolymer Laboratory for DNA sequencing and analysis and Maria Dubois for plasmid isolations, PCR, and other molecular procedures. We also thank Tom Whittam, Vanessa Sperandio, Simon Elliott, and David Karaolis for providing strains.
This work was supported by NIH grants AI21657 (J.B.K.) and AI43615 and AI33069 (J.P.N.). Work completed by J. F. was funded in part by an NSF AIRE Fellowship.
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FOOTNOTES |
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* Corresponding author. Mailing address: Center for Vaccine Development, University of Maryland School of Medicine, 685 W. Baltimore St., Baltimore, MD 21201. Phone: (410) 706-3004. Fax: (410) 706-0182. Email: jkaper{at}umaryland.edu.
Editor: V. J. DiRita
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Infection and Immunity, January 2001, p. 315-324, Vol. 69, No. 1
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.1.315-324.2001
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