1Division of Infectious Diseases, University of Maryland, Baltimore, 10 S. Pine Street, Baltimore, MD 21201.
2Institute of Molecular Evolutionary Genetics, Pennsylvania State University, University Park, PA 16802
Escherichia coli, a venerable workhorse for biochemical and genetic studies, and for the large-scale production of recombinant proteins, is one of the most intensively studied organisms on Earth. The natural habitat of E. coli is the gastrointestinal tract of warm-blooded animals and in humans, this species is the most common facultative anaerobe in the gut. Although most strains exist as harmless symbionts, there are many pathogenic E. coli strains that are capable of causing a variety of diseases in animals and humans. In addition, from an evolutionary perspective, strains of the genus Shigella are so closely related phylogenetically that they should also be included in the group of organisms recognized as E. coli (1;2). Pathogenic E. coli strains differ from those that predominate in the feces of healthy individuals in that they are more likely to express virulence factors (molecules directly involved in pathogenic processes but ancillary to normal metabolic functions). In addition to their role in disease processes, these virulence factors presumably enable the pathogenic strains to explore niches unavailable to commensal strains, and thus to spread and persist in the bacterial community. Yet, for all the differences among pathogenic and non-pathogenic E. coli, they all evolved from a common ancestor with Salmonella enterica some 100 million years ago.
It is therefore a mistake to think of E. coli as a homogeneous species. Most genes, even those encoding conserved metabolic functions, are polymorphic with multiple alleles (or sequence variants) found among different isolates (1). The composition of the genome of E. coli is also highly dynamic. The fully sequenced genome of the laboratory K-12 strain, whose derivatives have served an indispensable role in the laboratories of countless scientists, shows evidence of tremendous plasticity (3). It has been estimated that 18% of its genes were obtained horizontally from other species (4). Pathogenic E. coli strains harbor genomes that range in size from 4.9 to 5.3 megabases, 10 – 20% larger than that of E. coli K-12 (5). Many of the virulence factors that distinguish the various E. coli pathotypes were acquired by a variety of genetic processes. These virulence factors arrived either via plasmids, within the genomes of bacteriophages, or on pathogenicity islands (relatively large (>10 kb) genetic elements that encode virulence factors and are found inserted in the genomes of pathogenic strains, but absent from those of non-pathogenic strains). These elements frequently have base compositions that differ drastically from that of the content of the rest of the E. coli genome, indicating that they were acquired from another species. The purpose of this review is to explore some of the known virulence factors that contribute to the heterogeneity of E. coli strains and to review what is known regarding the origin and distribution of these factors.
There is a remarkable variety of pathogenic forms of E. coli associated with a spectrum of human and animal diseases. Certain pathogenic strains cause enteric diseases ranging in symptoms from cholera-like diarrhea to severe dysentery, whereas other E. coli have the ability to colonize the urinary tract, resulting in cystitis or pyelonephritis, or to cause extraintestinal infections, such as septicemia and meningitis. Two basic concepts have facilitated our understanding of the diversity of pathogenic forms of this versatile species. First, is the concept of the pathotype. Here, we use pathotype to refer to a group of E. coli isolates that have a similar mode of pathogenesis, as defined by distinct virulence factors, and thus cause clinically distinct disease. There are at least eight recognized pathotypes of E. coli that are capable of causing a variety of diseases in humans (Table 1). However, because virulence is often determined by transmissible genetic traits, it is also useful to have knowledge of the genetic relatedness and chromosomal background of bacteria that harbor similar virulence traits. This leads to the second concept, the idea of the pathogenic clone. We use the term clone to refer to an extant group of bacteria within a species that shares many similarities because of recent descent from a common ancestral cell. Bacteria of the same pathogenic clone or clonal group thus represent a monophyletic branch of an evolutionary tree. Early evidence for the clonal nature of pathogenic E. coli was seen in the repeated recovery of identical serotypes and biotypes from separate outbreaks of disease (6). The idea of widespread pathogenic clones gained support from the study of protein polymorphisms, first with patterns of the major outer proteins (7) and then through the broad application of multilocus enzyme electrophoresis (1). Recent sequence comparisons have shown that a phylogenetic approach based on the clone concept, however, is complicated by recombination events, which in addition to mutation, play a role in the divergence of bacterial genomes in nature (see (8) for a review).
The diversity of pathotypes and their phylogenetic
relationship is illustrated in the dendrogram (Fig. 1A). This analysis includes strains of the
pathotypes associated with enteric disease and strains representing the major
phylogenetic groups (Groups A, B1, B2, and D) of the ECOR collection, a set of
natural isolates chosen to represent genetic variation in the E. coli species as a whole (9). The dendrogram includes pathogenic strains of the most common
clones of five serogroups (O26, O111, O55, O128, and O157) associated with
infectious diarrheal disease; these widespread clones are referred to as the
DEC (diarrheagenic E. coli) clones (10). In addition, there are representatives of the common clones of
enteroinvasive E. coli (11).
Pathotypes of E. coli
are concentrated in clonal groups, although some pathotypes are found in
multiple lineages (Fig. 1A). In
particular, there are two clusters of enteropathogenic E. coli (EPEC) that are associated with infantile diarrhea and two
clusters of enterohemorrhagic E. coli
(EHEC) that are associated with hemorrhagic colitis. The EPEC 1 and EHEC 1 clonal lineages are highly divergent
whereas both EPEC 2 and EHEC 2 are more closely related to one another and fall
into the B1 group of ECOR. The finding
of independent lineages harboring the same virulence factors and causing
clinically similar disease indicates that these pathotypes have evolved
multiple times in different clonal groups (12). It is noteworthy that the EPEC and EHEC groups are
phylogenetically distinct from the enteroinvasive E. coli (EIEC), bacteria that cause dysentery and are most closely related
to strains of the ECOR group A. The
clonal groups associated with enteric diseases are also different from those
recovered in extra-intestinal infections including uropathogenic E. coli (UPEC), which are found near
the bottom of the dendrogram in the B2 and D groups of ECOR (13;14).
It is beyond the scope of this review to detail the variety and intricacies of pathogenic mechanisms, as far as they are known, for the many classes of E. coli that cause disease. The reader is referred elsewhere for reviews of diarrheagenic (15) and extra-intestinal E. coli pathogenesis (16). Instead, we will provide an overview of the virulence factors and pathogenic mechanisms of two pathotypes, EPEC and EHEC, for which data exist both on the genetic basis of disease and on the phylogenetic history of the strains. These examples are put forward to demonstrate how genetic polymorphisms among E. coli strains profoundly influence disease.
Enteropathogenic E. coli
Enteropathogenic E. coli (EPEC) were the first group of strains recognized to be pathogenic when it was appreciated that strains cultured from devastating outbreaks of neonatal diarrhea differed serologically from strains isolated from healthy infants (17). Although these outbreaks are now rare in developed countries, EPEC strains continue to be a leading cause of diarrhea among infants from developing countries worldwide (15). In recent years, the pathogenesis of EPEC infection has proved to be amenable to genetic dissection and several themes have emerged.
A plasmid-encoded type IV bundle-forming pilus critical for virulence. In the early 1980s, investigators in the laboratory of James Kaper reported that a particular pattern of adherence to tissue culture cells by EPEC strains was associated with the presence of a large, EPEC adherence factor (EAF) plasmid (18). Rather than covering tissue culture cells uniformly, EPEC strains form densely packed three-dimensional clusters on the surface of the cells, a pattern known as localized adherence. This pattern of adherence is so characteristic of and specific to EPEC strains that it can be used as the basis for diagnosis (19). The ability to perform localized adherence can be transferred to non-pathogenic laboratory E. coli strains by transformation with a large plasmid found only in EPEC strains. Similarly, EPEC strains lose this ability and demonstrate attenuated pathogenicity when cured of the plasmid (20).
The principal factor responsible for the localized adherence phenotype is a surface appendage known as the bundle-forming pilus (BFP), a member of the type IV fimbria family encoded on the EPEC plasmid (Figure 2A) (21). Localized adherence is now appreciated to result from dynamic aggregates of EPEC bacteria that are in turn due to the ability of BFP to reversibly aggregate into rope-like bundles. If any of the genes required for the formation of BFP are inactivated by mutation, the bacteria fail to form aggregates and do not display localized adherence (22). The major structural subunit of BFP is bundlin, a highly polymorphic protein encoded by bfpA of the plasmid-borne bfp operon (see below). Another protein, BfpF, which is predicted to be a cytoplasmic nucleotide-binding protein, plays a special role in aggregation. When bfpF is mutated, the bacteria continue to make pili that aggregate and allow the bacteria to do the same (23); however, the pili fail to form higher-order bundles and the bacteria remain trapped in aggregates (24). Interestingly, despite the fact that they remain capable of further steps in pathogenesis (see below), bfpF mutants are significantly attenuated in their ability to cause diarrhea in volunteers (25). Thus it appears that not only the BFP structure, but also intact BFP function is required for full virulence.
A chromosomal pathogenicity island that encodes a type III secretion system and the ability to alter the host cytoskeleton. The hallmark of EPEC infection is the ability to attach intimately to the host cell membrane, destroy microvilli, and induce the formation of cup-like pedestals composed of cytoskeletal proteins upon which the bacteria sit (Figure 2B). This ability, known as attaching and effacing activity (26), has been observed in vitro and in duodenal and rectal biopsies from infants with EPEC infection (27;28). A 35,624 bp genetic element known as the locus of enterocyte effacement (LEE) is necessary for this effect and, when cloned from EPEC strain E2348/69 into a non-pathogenic E. coli strain, is sufficient to confer attaching and effacing activity (29). The LEE is considered to be a pathogenicity island because it contains virulence loci, it is not found in non-pathogenic E. coli strains, it is inserted into the genome of E. coli at restricted sites (tRNA genes), and it contains a G+C content (38%) considerably lower than that of the K-12 genome that indicates its foreign origin in another species. The LEE is inserted near different tRNA loci in different EPEC strains (30;31). The LEE from strain E2348/69 carries 41 genes including those encoding a type III secretion system, the proteins secreted via this system, an adhesin and its cognate receptor, a regulator, and several genes of unknown function (32). Type III secretion systems are found in bacteria from several Gram-negative genera that have close relationships with eukaryotic hosts (33). Their function is to deliver effector proteins to the surface or interior of host cells. Thus, they are capable of transporting bacterial proteins across three membranes: the inner and outer membranes of the bacteria and the host cell plasma membrane.
The proteins secreted via type III systems can be divided into two classes, the effector proteins, which are translocated to the host cell, and the components of the translocation apparatus, which are required to deliver the effector proteins into the host cell. The best-characterized EPEC effector protein thus far is called Tir, for translocated intimin receptor. Tir is encoded by the LEE and translocated via the type III system into host cells where it is inserted in the plasma membrane (34). Mutations in components of the type III secretion system or in the genes encoding two of the secreted proteins, EspA and EspB, prevent the translocation of Tir. Thus, EspA and EspB can be classified as part of the translocation apparatus. Tir has two membrane-spanning domains and is oriented so that both the amino and carboxyl termini protrude into the host cell cytoplasm (35). Once Tir is inserted into the host cell, it serves as the receptor for intimin, an outer membrane protein required for virulence that is encoded by the eae gene on the same operon as tir in the LEE (36). Thus EPEC have evolved an adherence mechanism in which the bacteria synthesize both the adhesin (intimin) and its receptor (Tir) that is inserted directly into the host cell by the LEE secretion apparatus.
The three-dimensional structure of the extracellular domain of intimin bound to the extracellular domain of Tir has recently been solved (37). Intimin consists of a series of immunoglobulin-like domains (D0 – D2) that give the protein a rigid roughly cylindrical shape. The distal, carboxyl terminal domain (D3) consists of an incomplete C-lectin structure. In the cell membrane, Tir forms a dimer with each molecule consisting of a pair of anti-parallel alpha helices separated by a hairpin turn. The entire structure is a four-helix bundle with the hairpin loops protruding from either side (Figure 2C). Intimin binds to Tir principally at the loops, such that each Tir dimer binds to two intimin molecules. Tir forms contacts with intimin in a groove between the last immunoglobulin domain and the C-lectin domain. To achieve this configuration, both intimin and Tir appear to be oriented roughly parallel to both the bacterial and eukaryotic cell membranes. This orientation would account for the distance of only 10 nm separating the bacteria and host cells.
While Tir is clearly an effector protein, the roles of three other proteins, EspA, EspB, and EspD, which are encoded in an operon in the LEE and are secreted by EPEC via the type III system, are still being defined. EspA appears to be purely a component of the translocation apparatus. EspA molecules form a surface appendage that can be seen by electron microscopy bridging the bacteria and host cells (38). There is no evidence that EspA molecules end up in the host cell cytoplasm or membranes (39). EspD has several putative transmembrane domains and has been observed in the host cell membrane (40). Because it is required for the translocation of EspB (40;41), EspD is also a part of the translocation apparatus. Interestingly, when espD is mutated, EspA filaments are much shorter than normal, suggesting a role for EspD in formation or stabilization of the translocation apparatus (38).
The function of the EspB protein is more enigmatic. While EspB is required for the translocation of Tir, indicating that it is a component of the translocation apparatus, EspB is itself translocated to the host cytoplasm (41;42). The protein has hydrophobic stretches that could act as transmembrane domains, and EspB molecules have been detected in the host cell membrane. From these observations, some investigators have suggested that EspB forms part of a pore that enables the passage of Tir into the host cell (43). However when host cells are transfected with a vector that enables them to express EspB, their shape is radically altered and they lose stress fibers, suggesting that EspB can affect cytoskeletal regulation and may also act as an effector protein (44).
What triggers the molecular events in the host cells that lead to the attaching and effacing activity? A recent study shows that the Arp2/3 complex, which nucleates and polymerizes actin, is localized within the actin rich pedestals of attaching and effacing lesions (45). Members of the Wiscott-Aldrich syndrome protein (WASP) family, which activate the Arp 2/3 complex, are also localized within the pedestals and dominant-negative forms of WASP prevent attaching and effacing activity. Thus it has been proposed that EPEC activates WASP to stimulate the polymerization of actin (Figure 2C).
Recently, light has been shed on the role of another secreted protein in pathogenesis. EspF is encoded downstream of the other esp genes by the last gene in the LEE pathogenicity island and is secreted via the type III apparatus. When espF was mutated no defect in attaching and effacing activity was detected (46). However, an espF mutant fails to cause a decrease in transepithelial electrical resistance, a phenotype associated with wild type EPEC strains that may be related to loss of intestinal barrier function and diarrhea in vivo (McNamara et al., submitted). In addition, the espF mutant fails to induce apoptosis in host cells, another feature of the EPEC-host cell interaction (Crane et al., submitted). Application of EspF to the exterior of cells has no effect, but synthesis of EspF within cells after transfection with an expression vector results in rapid cell death. Examination by confocal microscopy and by expression of a reporter fusion protein confirmed that EspF is translocated to the interior of host cells by the type III secretion apparatus. Interestingly, EspF contains proline rich repeats that may serve as Src-homology 3 binding domains to interact with an as yet unidentified host protein(s). Thus, EspF is a novel EPEC effector protein that plays a role in loss of intestinal barrier function and host cell apoptosis.
A large toxin that inhibits lymphocyte activation. Several years ago a factor produced by EPEC and related strains of E. coli that inhibits lymphocyte activation was described (47). This heat-labile factor blocks lymphocyte proliferation and production of interferon-g, IL-2, IL-4 and IL-5 in response to a variety of stimuli including phorbol esters, mitogens, CD3 cross-linking, and antigens (48;49). Though lymphocytes exposed to the factor are non-responsive, there is no evidence that they undergo apoptosis or are killed. When the gene encoding this factor was cloned and mutated, the resulting strain no longer had the activity (50). The product of the gene, lymphostatin, is a huge protein, 366 kDa in mass. A relatively short stretch of the sequence is homologous to the enzymatic domain of the large Clostridial cytotoxins, which glycosylate and inactivate members of the Rho family of small mammalian GTPases. Sequences homologous to the lymphostatin gene are widespread, but distributed sporadically among EPEC and EHEC strains. The mechanism by which lymphostatin blocks lymphocyte activation and the role, if any, of lymphostatin in disease have not been established.
Two divergent
groups of EPEC. There are two phylogenetic
groups that have a concentration of EPEC (Fig. 1), and both of which contain
strains with serotypes first incriminated in outbreaks of infantile diarrhea in
the 1940s and 1950s (51-54). EPEC 1 includes some of
the original adherent strains identified by Cravioto et al. (55), most
notably, strain E2348/69 (serotype O127:H6), the widely used model organism of
human EPEC infection. This group also
includes widespread EPEC clones with serotypes O55:H6, O119:H6, O125:H6
O127:H6, and O142:H6 (10;56). Bacteria of these clones
are common in Brazil where they are usually probe-positive for eae (indicating the presence of the
LEE), bfpA (indicating the presence
of the EAF plasmid), and display typical localized adherence (57;58).
The second group is EPEC 2, which consists of the classical EPEC
serotypes O111:H2, O114:H2, O126:H2, and O128:H2. DEC 12 (O111:H2) marks a common EPEC clone with an
intercontinental range (10), which
historically has been the most common E.
coli recovered from outbreaks of infantile diarrhea in the US (59) and is the
most frequently recovered O111 clone associated with diarrheal disease in
Brazil (60). The EPEC 2 group also includes strain B171,
a non-motile O111 strain originally recovered from a diarrhea outbreak (61). This EPEC 2 strain has also been intensively
studied (25;62;63).
Although EPEC strains cause similar disease, the two EPEC groups
have different intimin alleles (64;65) and distinct sites of
insertion of the LEE pathogenicity island (31). Recently, Monteiro-Neto and Trabulsi (1999)
demonstrated that the two EPEC groups can be rapidly distinguished by their
ability to metabolize phenylpropionic acid (66).
In both EPEC groups the bfp operon is carried on highly related EAF plasmids. Some of these plasmids are self-transmissible (67), but the single member of the group that has been sequenced in its entirety lacks genes for transmission (63). The bfpA gene, which encodes bundlin, the major structural subunit of BFP shows considerable sequence variability. Among 19 bfpA genes that have been sequenced, there exist 8 distinct alleles, which can be separated into 2 groups (a and b). The a and b bundlin alleles are distributed in both EPEC groups suggesting that the plasmids have recently spread horizontally (Blank et al, submitted). Comparison of the sequences of a and b bundlin also indicates an excess of non-synonymous substitution in the 3’ end of the gene. This finding suggests the influence of positive selection for amino acid replacements and enhanced polymorphism in bundlin, which could be a source of variation in virulence among EPEC clones.
In summary, the interactions between EPEC and the host are complex (Figure 2C). A plasmid-encoded type IV bundle forming pilus is essential for full virulence, but exactly how it functions to facilitate infection is not clear. The LEE pathogenicity island encodes a type III secretion system, an outer membrane adhesin and its cognate receptor necessary for attaching and effacing activity. An additional protein translocated to host cells induces host cell death and a loss of intestinal barrier function. A large toxin with lymphocyte inhibitory activity may aid the bacteria in forestalling an immune response. Finally, the combination of virulence factors that define EPEC has emerged at least twice in the evolutionary radiation of pathogenic E. coli. The extent to which these clone complexes differ in virulence or epidemiological properties has not been fully explored.
Enterohemorrhagic E. coli
Enterohemorrhagic E. coli (EHEC) was first recognized as a
cause of infectious diarrheal disease as a result of several outbreaks of
severe bloody diarrhea (hemorrhagic colitis) in the early 1980s. Since then EHEC strains, particularly
serotype O157:H7, have been implicated worldwide in outbreaks of food and water
borne disease in developed countries.
The nomenclature for this group of organisms is confusing. EHEC belong to a larger group of pathogenic
strains known as Shiga toxin-producing E.
coli (STEC), which are defined by their ability to produce Shiga toxins
(Stx). (For historical reasons these
same toxins are alternatively referred to as verotoxins and the organisms that
produce them as VTEC.) EHEC are a
subset of STEC that in addition to producing Shiga toxins share with EPEC the
LEE and attaching and effacing activity.
EHEC strains of serotype O157:H7 have caused both the largest number of
outbreaks and the outbreaks that have involved the greatest numbers of
patients. Strains with this serotype
have also caused the majority of sporadic STEC infections (68-70). Although EHEC O157:H7 strains contain large plasmids similar to
those of EPEC, they do not carry the genes required for synthesis of BFP. Instead these plasmids carry a homologue of
the lifA gene encoding lymphostatin,
genes encoding a type II secretion system,
catalase-peroxidase (katP), a
secreted serine protease (espP), and
a hemolysin operon (71-75). It is not clear
what, if any, proteins are secreted by the type II system. Indeed, the role of the EHEC plasmid in
pathogenesis has not been confirmed in animal models of infection (76).
The most serious complication of EHEC infection is hemolytic-uremic syndrome (HUS). HUS is a microangiopathic hemolytic anemia characterized by disseminated capillary thrombosis and ischemic necrosis (68). The kidneys are the end organs most severely affected, but ischemic necrosis of the intestines, central nervous system (stroke) and indeed any organ may occur. Approximately 15% of those with HUS either die or are left with chronic renal failure (69). Because of the danger of HUS, EHEC strains and mutants cannot be tested in volunteers. EHEC strains of serotype O157:H7 can tolerate acidic environments and have a very low infectious dose (77). These strains colonize the gastrointestinal tract of cattle and may contaminate ground beef during processing. A variety of other foods including milk, juices, lettuce and sprouts have been involved in outbreaks. The infection is acquired by ingestion of contaminated food or water or by person-to-person spread through close contact.
Production of Shiga toxins. The Shiga toxins are the most important factor that differentiates EHEC from EPEC. These toxins are encoded by bacteriophages related to the classic lambda phage, which lysogenize these strains (78). There are two forms of the toxin that are found in the majority of EHEC strains pathogenic for humans, Stx1 and Stx2, encoded by highly-related bacteriophages. Each toxin is composed of a single A subunit non-covalently associated with a pentamer composed of identical B subunits (79). The B subunits bind to the toxin receptor on host cells, which is globotrioacyl ceramide and related members of the globo series of glycolipids (80). The A subunit is taken up by endocytosis and transported retrograde to the endoplasmic reticulum (81). The toxin target is the 28S rRNA, which is depurinated by the toxin at a specific adenine residue. The result is a cessation of protein synthesis and death of the cell by apoptosis (82). Receptors for Shiga toxins are found on endothelial cells. Renal microvascular endothelial cells appear to be particularly sensitive to the toxin (83). It is presumed that Shiga toxins enter the systemic circulation after translocation across the intestinal epithelium (84) and damage endothelial cells, which leads to activation of coagulation cascades, formation of microthrombi, intravascular hemolysis, and ischemia.
The EHEC LEE. The composition of the EHEC LEE from a serotype O157:H7 strain is very similar to that of a distantly related EPEC O127:H6 strain (85). The gene order of the elements from the two strains is identical and the predicted amino acid sequences of most of the proteins that compose the type III secretion systems are nearly identical. Three differences between the LEE elements of EPEC and EHEC stand out. The EHEC LEE is larger than that of EPEC due to the presence in the former of the remnants of a lysogenic bacteriophage at one end. It appears that this phage entered the EHEC LEE subsequent to acquisition of the LEE by a progenitor of the EHEC strain. Another striking difference between the genes of the EPEC and EHEC LEE are that those encoding intimin and the secreted proteins are much more divergent than those encoding most of the components of the secretion system per se (Figure 3). It has been suggested that this divergence could be the result of selective pressure exerted by the immune system of the host on the bacteria. Like that of EPEC, the EHEC LEE has an espF gene, which could be involved in cell death and loss of intestinal barrier function. Interestingly, the predicted EHEC EspF protein has four proline-rich motifs, rather than three, as does the EPEC protein. Finally, while the LEE from EPEC strain E2348/69 is sufficient to confer attaching and effacing activity upon non-pathogenic strains of E. coli, the LEE from O157:H7 EHEC is not (86). Presumably, this difference is due to polymorphisms within the LEE sequences.
Evolution of EHEC groups. Like EPEC, EHEC strains
fall into two divergent clonal groups.
EHEC 1 includes the O157:H7 clone complex and the closely related O55:H7
clone (DEC 5), an atypical EPEC clone.
Bacteria of the DEC 5 EPEC clone have the eae gene encoding intimin (and therefore the LEE) but most lack the
EAF plasmid encoding BFP and they do not typically display localized adherence (87). A study of O55 strains from Brazil showed
that bacteria of this clone invariably carriy the eae gene, but otherwise display a diverse array of virulence traits
(57). The results suggest that DEC 5 is a
pathogenic clone with a propensity to acquire new virulence factors.
In addition to its distinct virulence traits, E. coli O157:H7 is unusual in that these
organisms do not ferment sorbitol rapidly (88) or exhibit b-glucuronidase (GUD) activity (89), in contrast to most commensal E. coli. In 1990, Karch and
colleagues discovered a nonmotile (H-) O157 clone that was implicated in an
outbreak of HUS in Germany (90). These
bacteria are eae gene positive and
produce Stx2; however, phenotypically they are dissimilar from typical O157:H7
in that they are sorbitol-positive (Sor+).
Because the restriction digests of Sor+ O157:H- strains differed from
typical O157:H7 in pulsed field gel electrophoresis, it was hypothesized that
the Sor+ O157:H- is a new clone with similar virulence properties to those of
O157:H7 (90). Feng and coworkers (91) tested this hypothesis using
multilocus enzyme electrophoresis to assess the clonal relationships among a
variety of Stx-producing O157 strains. The analysis revealed that these strains
comprise a cluster of 5 closely related ETs that differ from one another by
only one or two enzyme alleles. The
Sor+ O157:H- strains from Germany (92) were the most divergent clone
of the complex differing by two enzyme alleles from the common O157:H7
electrophoretic type.
Stepwise
Evolution of E. coli O157:H7. From the genotypic and
phenotypic data, Feng and colleagues (1998) formulated an evolutionary model
that posits a series of steps that have occurred in the emergence of O157:H7 (91). The model is based on the assumption that during divergence, the
probability of loss of function greatly exceeds that of gain of function for
metabolic genes; that the gain of function usually occurs via lateral transfer
of genes; and that the sequence of events invoking the fewest total steps is
the most likely model.
The evolutionary steps are
outlined in Figure 4, which begins at the left with the ancestral or primitive
states and progresses to the right to the contemporary or derived states. The model begins with an EPEC-like ancestor
that is assumed to resemble most present day E. coli in its ability to express ß-glucuronidase (GUD+) and
ferment sorbitol (SOR+). From this
EPEC-like ancestor, the immediate ancestor with the O55 somatic and the H7
flagellar antigens evolved. This ancestral cell, labeled A1, represents the
most recent common ancestor of the O55:H7 clone and O157:H7 and its
relatives. A1 is assumed to have
inherited the LEE at the selC site
from an early EPEC-like ancestor carrying the g
variant of the eae gene. The next step, A1 to A2, was the acquisition
of stx2, presumably by transduction
by a toxin-converting bacteriophage, resulting in Stx2-producing O55:H7
strains. The next stage involved two changes, the acquisition of the EHEC
plasmid (93) and a switch in somatic
antigen from O55 to O157 (94). From here, the model proposes that two distinct lines
evolved. In the lower path, the
bacterial lineage lost motility, but retained the Stx2 and the GUD+ SOR+
primitive phenotypes, to give rise to the German O157:H- clone. Along the upper path, the lineage lost GUD activity
and the ability to ferment sorbitol, and acquired the Stx1 gene (presumably by
phage conversion) to give rise to the phenotype of the common O157:H7 clone
that has spread globally. Recent loss
of Stx genes and motility in nature, or during isolation and culture, would
account for the variants among isolates of this clone.
The stepwise model of Feng et
al. (1998) makes specific predictions about the history of descent and the
order of acquisition of virulence factors in the emergence of the EHEC
pathotype. The model predicts that both
O157:H7 and the German O157:H- were derived from an EPEC-like O55:H7 ancestor
that carried the LEE and acquired the Stx2 gene. This proposition is supported by the similarities between these
strains in eae sequence (95) and by the presence of
identical mutations in the ß-glucoronidase gene (91). The German O157:H- clone, however, represents an early diverging
member of the EHEC clone complex, which retained the ancestral ability to
ferment sorbitol and to express ß-glucuronidase activity. The hypothesis of early divergence of this
non-motile clone is also supported by the observation that there are multiple
mutations in fliC, presumably as a
result of the long-term silencing of flagellin expression (96).
The model also stipulates that stx2 was acquired once, before the
somatic antigen transition to O157 and prior to the acquisition of the EHEC
plasmid and stx1. Recent evidence, however, indicates that
different O157:H7 strains harbor diverse Stx2-encoding phages (97). The extent to which the diversity of toxin-converting phages has
been generated in situ by
recombination or from multiple conversion events remains to be elucidated.
A second group of EHEC. An unexpected finding of the evolutionary
analysis was that the O157:H7 cluster is only distantly related to a second
group of Stx-producing strains (primarily serotypes O26:H11 and O111:H8), which
were originally classified as EHEC along with O157:H7 (93). Bacteria of these two EHEC
groups have in common a large plasmid (pO157) that encodes a variety of
putative virulence factors (73).
Much less
is known about the virulence properties of the EHEC 2 group. They
appear to be the most commonly isolated group of non-O157 Stx-producing
strains. Although they often have
serotypes O111:H8 (or its nonmotile relatives), O26:H11, or O111:H11, members
of EHEC 2 often show a diversity of O:H serotypes and many of these strains are
nonmotile or nontypeable with standard antisera (10). Because this group has the same principal virulence factors as E. coli O157:H7 (i.e. Stx and the LEE),
and are recovered from patients with hemorrhagic colitis and HUS, they have
been classified together with O157:H7 as EHEC.
However, our evolutionary genetic analysis indicates that this group is
substantially divergent from E. coli
O157:H7 (10;12). For this reason
we refer to these non-O157 EHEC strains as the EHEC 2 group (56).
EHEC 2
includes a common nonmotile O111 clone (ET 8 of reference (60)) that occurs in both North and South America. Members of this clone have eae
and produce both Stx1 and enterohemolysin (60). The EHEC 2 group includes
nonmotile O111 isolate 3007-85, an Stx-producing strain originally isolated
from a patient with hemorrhagic colitis (98), which is highly virulent in gnotobiotic pigs (99), and RDEC-1, an O15:NM isolate from a case of rabbit diarrhea (100), which has been used as a model organism for human EPEC infection.
A similar
series of steps are hypothesized in the radiation of the EHEC 2 group (Fig.
5). The emergence of this pathogenic
lineage is thought to begin with the acquisition of a LEE island into the pheU site, because this is a conserved
characteristic found in both EPEC 2 and EHEC 2 strains (12;31). This ancestral
LEE carried an ancestral b
intimin gene, which is found among the diverse serotypes in these groups. From the ancestral EPEC-like strain (A1),
one lineage led to the EPEC 2 group of strains characterized by the localized
adherence phenotype encoded on the EAF plasmid and the other lineage (A2) led
to the EHEC 2 group of strains.
The steps
in the evolution of the EHEC2 group are not clear but apparently involved
multiple gains and loss of Shiga-toxin genes and pathogenicity islands. In the top half of Figure 5, we have
assembled the information into a sequence of events that is highly speculative
and requires further study. We posit
that A2 was an ancestral O26:H11 strain that eventually acquired an stx1 phage and an EHEC plasmid to give
rise the widespread EHEC O26:H11 clone.
A2 was also the recent ancestor that experienced an antigenic shift to
O111 to produce the EHEC O111 clone.
Multilocus sequencing data as well as MLEE has shown that these two EHEC
clones are closely related genetically indicating that these events occurred
recently in evolution.
Other important genetic changes have also
occurred. For example, Karch and
colleagues (101) have recently shown that the O26:H11 clone
carries a pathogenicity island homologous to sequences from pathogenic Yersinia, and that this so-called High
Pathogenicity Island (HPI) is not found in the closely related O111
strains. From an evolutionary
perspective, this observation suggests that the HPI was either very recently
acquired in the O26 lineage or was recently lost in the O111 lineage. The divergence of the O26:H11 and O111:H8
EHEC clones also appears to have involved a recombination within the intimin
gene. EHEC O111 strains carry a mosaic
intimin allele (b/g-eae) with the sequence of the conserved
trans-membrane domain resembling the b-eae gene and the sequences encoding the
variable external domains resembling g-eae (Tarr and
Whittam, unpublished). The nature of
the recombination event and its influence on the intimin-Tir interaction has
yet to be illuminated.
The dynamic nature of clonal evolution in the
EHEC 2 groups is perhaps best seen in a recent finding that there has been a
dramatic replacement of O26 clones in Europe in the past decade (102). The
clonal replacement, detected with PFGE comparisons of O26 strains, indicates
that a new subclone with stx2 and a
distinct EHEC plasmid variant has recently spread to high frequency (102).
Presumably this new type has been recently derived from the common
O26:H11 EHEC clone (Figure 5).
Because
EHEC 2 strains share the prominent virulence factors of O157:H7 and cause
similar disease, and are also common in the bovine reservoir, it is possible
that these organisms will emerge as important foodborne pathogens in North
America.
E. coli serves as a
prime example of the role of polymorphisms within a bacterial species in human
disease. A multitude of E. coli pathotypes cause distinct
diseases. Genetic variation, both
acquired through the horizontal spread of virulence factors and present in
certain lineages that are inherently more pathogenic, is responsible for these
diverse clinical entities. Studies of
two pathotypes, EPEC and EHEC have been particularly revealing. An understanding of the molecular and
cellular basis of pathogenesis for both of these pathotypes is emerging. In addition, studies of clonal relationships
have illuminated the evolution of these pathogens. One of the important themes that has emerged from studies of
polymorphisms within virulence factor genes is the presence of increased rates of
non-synonymous substitution (amino acid altering mutations) in surface exposed
and secreted proteins, implying the influence of diversifying selection on polymorphism. This effect is seen in the divergence of the LEE genes of EPEC
and EHEC: Tir, intimin, and several of
the Esps which have levels of non-synonymous change 5 to 10 times greater than
seen in conserved products of housekeeping genes. Bundlin is also highly polymorphic and has experienced an
accelerated rate of non-synonymous substitution in the 3’ end of the gene. Presumably the increased diversity benefits
the individual organism to escape the immune response within a host or favors
spread of a variant in a population against the effects of herd immunity. Evidence for recombination within virulence
factor genes such as eae encoding
intimin also illustrates the potential for re-introduction of mobile genetic
elements containing virulence factors into established pathogens to increase
diversity. E. coli may thus be viewed as a rapidly evolving species capable of
generating new pathogenic varients that can foil host protective mechanisms and
result in new disease syndromes.
Acknowledgements
This work was supported by Public Health Service awards AI32074, AI37606, DK49720 (to MSD) and AI43291 (TSW) from the National Institutes of Health. The authors are grateful to Rick Blank for supplying the electron micrograph shown in Figure 2A.
FIGURE LEGENDS
Figure 1.
A. The dendrogram is based on analysis of polymorphism at 36 protein loci studied by multilocus enzyme electrophoresis. The number of differences between strains is converted to a genetic distance assuming that each difference results from at least one amino-acid-altering mutation at the DNA level. The diagram can be interpreted as a hypothetical phylogeny of strains that can be tested by gathering independent data. Main branches representing pathotypes are labeled. The A, B1, B2, and D groups are the clusters from the ECOR set. The triangles mark positions at which major acquisition of virulence factors are postulated to have occurred.
B.
Nucleotide
substitutions for 7 housekeeping genes against genetic distance. Nucleotide differences are analyzed
separately for synonymous sites (dS),
positions in codons where point mutations do not predict amino acid
replacements, and non-synonymous sites (dN),
where point mutations results in amino acid changes. The points are averages of the comparison of pairs of strains
(marked with circles) in panel A.
Figure 2. Pathogenesis of EPEC infection. (A) Electron micrograph of a culture of EPEC bacteria grown under conditions that lead to the production of type IV fimbria known as bundle-forming pili (BFP). BFP are required for bacterial aggregation and localized adherence to epithelial cells. (B) Electron micrograph of an EPEC bacterium engaged in attaching and effacing activity with a host intestinal epithelial cell. Note the loss of microvilli and the formation of a cup-like pedestal to which the bacterium is intimately attached. (C) A model of EPEC pathogenesis. A bacterial aggregate, connected by bundles of BFP fibers is shown near an intestinal epithelial cell (1). As infection proceeds the bacteria detach from the pilus fibers, disaggregate and become connected to the host cell through a surface appendage that contains EspA (2). It is believed that Tir, EspB and EspF travel through this appendage to the host cell. EspF is not required for attaching and effacing activity, but plays a role in disruption of intestinal barrier function and host cell death. EspB and Tir are required for attaching and effacing activity (3). The bacterial outer membrane protein intimin, composed of 3 immunoglobulin-like extracellular domains (D0 – D2, light blue) and a receptor binding lectin-like domain (D3, dark blue) binds to Tir in the host cell membrane (4). Tir forms a four helix bundle composed of two molecules each containing two anti-parallel alpha helices connected by a hairpin loop. One intimin molecule binds to each loop of the dimer. WASP is recruited to the pedestal where it activates the Arp 2/3 complex to nucleate and polymerize actin.
Figure 3. Cladograms of major evolutionary steps in the divergence of EPEC and EHEC clones. The two cladograms are based on the presence of the LEE at the selC (A) or pheU (B) loci. The diagrams are models of a branching order for the ancestry of the chromosomal backgrounds or clonal frames inferred from multilocus analysis. Branch lengths are arbitrary and not set to an evolutionary scale. Points of acquisition of principal virulence factors that define EPEC and EHEC are marked on the branches. Gains and losses of genes or phenotypes are marked below branches. The circles designate ancestral nodes referred to in the text. The EPEC (EAF) plasmid has two arrows to denote the possibility that it may have been acquired multiple times, a hypothesis to account for the a and b bundlin (bfpA) alleles occurring in both EPEC groups.
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Table 1. Clinical and epidemiological features and virulence factors of
various E. coli pathotypes.
|
Pathotype |
Clinical Features |
Epidemiological Features |
Virulence Factors |
|
Enteropathogenic |
Watery diarrhea and vomiting |
Infants in developing countries |
Bundle-forming pilus, attaching and effacing |
|
Enterohemorrhagic |
Watery diarrhea, hemorrhagic colitis, hemolytic-uremic syndrome |
Food-borne, water-borne outbreaks in developed countries |
Shiga toxins, attaching and effacing |
|
Enterotoxigenic |
Watery diarrhea |
Childhood diarrhea in developing countries, travelers’ diarrhea |
Pili, heat-labile and heat-stable enterotoxins |
|
Enteroaggregative |
Diarrhea with mucous |
Childhood diarrhea |
Pili, cytotoxins |
|
Enteroinvasive |
Dysentery, watery diarrhea |
Food-borne outbreaks |
Cellular invasion, intracellular motility |
|
Diffuse-adhering |
Poorly characterized |
Older children? |
? |
|
Uropathogenic |
Cystitis, pyelonephritis |
Sexually active women |
Type I and P fimbriae, hemolysin, pathogenicity islands |
|
Meningitis-associated |
Acute meningitis |
Neonates |
K1 capsule, S fimbriae, cellular invasion |