Adherence Factors in Atypical Enteropathogenic Escherichia coli Strains Expressing the Localized Adherence-Like Pattern in HEp-2 Cells

Although atypical enteropathogenic Escherichia coli (aEPEC) strains are frequently implicated in childhood diarrhea in developing countries, not much is known about their adherence properties. The phenotypic and genotypic characterization of 29 aEPEC strains expressing the localized adherence-like pattern points toward the involvement of E. coli common pilus (ECP), intimins, and other known E. coli adhesins in this pattern.Atypical enteropathogenic Escherichia coli (aEPEC) strains are increasingly recognized as an emerging pathotype responsible for childhood diarrhea in many countries (2-4, 19, 23). Atypical EPEC strains together with typical EPEC (tEPEC) strains constitute two distinct groups of organisms that have in common the locus of enterocyte effacement (LEE), a pathogenicity island responsible for the development of attaching-effacing (A/E) lesions. This island encodes the type III secretion system with multiple secreted proteins and a bacterial adhesin called “intimin” encoded by the eae gene (12, 16-18). Unlike tEPEC, aEPEC strains do not possess the EPEC adherence factor (EAF) virulence plasmid that contains the bundle-forming pili (BFP) responsible for a localized adherence (LA) pattern on cultured epithelial cells (5, 8, 28), but aEPEC strains display different adherence patterns. The typical EPEC strain exhibits only the LA pattern, while aEPEC strains display LA-like (LAL), diffuse adherence (DA), or aggregative adherence (AA) patterns (1, 10, 26, 32, 33). In addition, aEPEC strains belong to serotypes other than those of tEPEC strains (1, 10, 32, 33).Despite the clear differences in adherence patterns, only the factors mediating the LA pattern have been extensively studied in tEPEC, and little is known about the factors mediating adherence of aEPEC strains. In a previous study, we identified in an aEPEC strain belonging to the O26 serogroup an adhesin gene designated as lda, for “locus for diffuse adherence,” which encodes a nonfimbrial structure conferring the DA phenotype (27).Recently, in a collection of 126 aEPEC strains isolated from Brazilian children, we found many putative E. coli adhesin-encoding genes besides lda (25), such as efa1 (enterohemorrhagic E. coli [EHEC] factor adhesion 1) (13, 20), toxB (a plasmidial locus found in EHEC O157:H7 implicated in adhesion) (31), lpfA (long polar fimbriae) (9), iha (IrgA-homologous adhesion) (30), and paa (porcine A/E-associated gene) (6). In the present study, we characterize these strains regarding intimin types, HEp-2 adherence patterns, and ability to promote actin accumulation in vitro.The eae gene was subtyped according to the restriction fragment length polymorphism assay described by Ramachandran et al. (21). This method permits detection of the intimin types α, β, γ, ɛ, ζ, θ, ι, κ, λ, ν, ξ, ο, ρ, and σ. As can be seen in Table ​Table1,1, intimins α (22 strains), β (26 strains), and γ (20 strains) were the most frequently found types. Forty-three (34%) strains had a nontypeable intimin. Intimins ξ, ι, κ, λ, and ν were not found among the entire collection of 126 aEPEC strains. In agreement with previous data (10), a close relationship between classic serotypes and intimin types was seen in this study: intimin α was found mainly among O142:H2 strains, and intimin β was detected in O26:H11, O119:H2, and O128:H2 strains, whereas intimin γ was seen in O55 strains.

TABLE 1.

Characteristics of 126 atypical enteropathogenic Escherichia coli strains^a

The EspB Protein of Enteropathogenic Escherichia coli Is Targeted to the Cytoplasm of Infected HeLa Cells

The EspB protein of enteropathogenic Escherichia coli (EPEC) is exported via a type III secretion apparatus. EspB is critical for signaling the host cell and for the development of the attaching and effacing lesion characteristic of EPEC infection. We used cellular fractionation and confocal laser scanning microscopy to determine the cellular location of EspB during infection of HeLa cells. Both methods indicated that EspB is targeted to the cytoplasm of infected cells. Using mutants, we found that EspB targeting to the host cell cytoplasm requires the type III secretion apparatus and the secreted proteins EspA and EspD, but not intimin. These results provide insights into the function of the type III secretion apparatus of EPEC and the functions of the Esp proteins.     

Enterotoxigenicity of Enteropathogenic Serotypes of Escherichia coli Isolated from Infants with Epidemic Diarrhea

Enteropathogenic serotypes of Escherichia coli which have been incriminated by epidemiological evidence as responsible for epidemics of acute diarrhea in infants are often found to be nontoxigenic when tested by conventional systems such as Y1-adrenal, Chinese hamster ovary, and suckling mouse assays. Twelve such strains, representing four different enteropathogenic serotypes, were examined for their capacity to elaborate toxic materials which alter water transport. Ultrafiltration fractions prepared to contain either a high-molecular-weight, heatlabile or a low-molecular-weight, heat-stable form of toxin from each strain were perfused through rat jejuna in graded concentrations ranging from 100 mug to 0.1 ng/ml. Ten of the twelve enteropathogenic strains produced one or both toxin forms that induced water secretion at concentrations of 1 to 10 ng/ml. Values in this range are considered indicative of clinically significant enterotoxigenicity in this assay system, and toxins from well-documented toxigenic strains examined in this study were active at these same concentrations. Similar preparations from ten control strains from healthy persons were either inactive or evoked water secretion only at concentrations of 10 to 100 mug/ml. These observations suggest that enteropathogenic serotypes of E. coli isolated from epidemics of infantile diarrhea produce diarrhea by elaborating potent heat-labile and heat-stable toxin forms which alter water transport but which are inactive in conventional assay systems. The manner in which these toxins differ either quantitatively or qualitatively from those which stimulate the conventional test systems is unknown.     

Immunity to Escherichia coli in Pigs: Adhesion of Enteropathogenic Escherichia coli to Isolated Intestinal Epithelial Cells

A method was developed to test for the ability of Escherichia coli to adhere to isolated intestinal epithelial cells. Of the E. coli tested, those having either K88ac or K88ab antigens adhered to the cells, and those which did not have these antigens did not. Since some enteropathogenic E. coli did not have the ability to adhere, it is assumed that adherence is not an essential factor of pathogenesis but rather should be considered an enhancement to the pathogenicity of some E. coli. None of the E. coli enteropathogens of cattle tested adhered to either pig or cattle cells. Similarly, human strains did not adhere to pig cells. Although the test system may not have been ideal for human or bovine E. coli, the results reported here suggest that adhesiveness is a property limited to porcine enteropathogenic E. coli carrying one of the K88 antigens. Adhesiveness is associated with the K88c or K88b antigens, and their adhesive ability is only neutralizable by the homologous antisera.     

Flagellar Cap Protein FliD Mediates Adherence of Atypical Enteropathogenic Escherichia coli to Enterocyte Microvilli

The expression of flagella correlates with different aspects of bacterial pathogenicity, ranging from adherence to host cells to activation of inflammatory responses by the innate immune system. In the present study, we investigated the role of flagella in the adherence of an atypical enteropathogenic Escherichia coli (aEPEC) strain (serotype O51:H40) to human enterocytes. Accordingly, isogenic mutants deficient in flagellin (FliC), the flagellar structural subunit; the flagellar cap protein (FliD); or the MotAB proteins, involved in the control of flagellar motion, were generated and tested for binding to differentiated Caco-2 cells. Binding of the aEPEC strain to enterocytes was significantly impaired in strains with the fliC and fliD genes deleted, both of which could not form flagella on the bacterial surface. A nonmotile but flagellated MotAB mutant also showed impaired adhesion to Caco-2 cells. In accordance with these observations, adhesion of aEPEC strain 1711-4 to Caco-2 cells was drastically reduced after the treatment of Caco-2 cells with purified FliD. In addition, incubation of aEPEC bacteria with specific anti-FliD serum impaired binding to Caco-2 cells. Finally, incubation of Caco-2 cells with purified FliD, followed by immunolabeling, showed that the protein was specifically bound to the microvillus tips of differentiated Caco-2 cells. The aEPEC FliD or anti-FliD serum also reduced the adherence of prototype typical enteropathogenic, enterohemorrhagic, and enterotoxigenic E. coli strains to Caco-2 cells. In conclusion, our findings further strengthened the role of flagella in the adherence of aEPEC to human enterocytes and disclosed the relevant structural and functional involvement of FliD in the adhesion process.     

Escherichia coli adherence to HEp-2 cells with prefixed cells.

We describe a new method which uses cold absolute methanol-prefixed cells for adherence of enteropathogenic Escherichia coli to HEp-2 cells. We found that a method using bacteria grown in Penassay broth to 10(6) to 10(7) CFU/ml and incubated with prefixed cells for 3 h at 37 degrees C, showed 100% sensitivity and specificity against a method using live cells.     

Interactions of Enteropathogenic Escherichia coli with Pediatric and Adult Intestinal Biopsy Specimens during Early Adherence

Enteropathogenic Escherichia coli (EPEC) strains cause watery diarrhea almost exclusively in young children. The basis for this age discrimination has never been determined, but it may be related to host cell receptors. During infection, EPEC strains express type IV bundle-forming pili composed of repeating subunits of the protein called bundlin. The very first interaction between EPEC and in vitro-cultured epithelial cells is mediated by the binding of α-bundlin to a carbohydrate receptor that contains, at a minimum, the N-acetyllactosamine (LacNAc) glycan sequence. However, bundlins expressed from the β-bundlin allele do not bind LacNAc glycan sequences. Herein, we investigated whether EPEC strains use α-bundlin to mediate early adherence to human intestinal biopsy specimens cultured in vitro by assessing the ability of isogenic EPEC mutants expressing either the α₁- or β₁-bundlin allele or a bundlin-deficient EPEC strain to bind to these specimens. Furthermore, we directly compared the abilities of a wild-type EPEC strain to bind to the epithelial surfaces of both human adult and pediatric biopsy specimens. Our results demonstrate that β-bundlin does not act as an adhesin during early EPEC adherence to adult duodenal biopsy specimens. The results also indicate that EPEC binds equally well to adult and pediatric biopsy specimens in an early adherence assay. This result is supported by the finding that the early adherence of EPEC to both adult and pediatric biopsy specimens was inhibited by LacNAc neoglycoconjugates, suggesting that organisms expressing α-bundlin-type bundle-forming pili initially bind to related glycan receptors in both age groups.Enteropathogenic Escherichia coli (EPEC) strains cause watery diarrhea in young children, an illness associated with the EPEC-mediated disruption of the small-intestinal epithelium (21, 22, 24). While EPEC strains cause clinical disease predominantly in children under the age of 2 years, disease can also be elicited in adult volunteers given a very high infectious dose of bacteria (1, 7, 9, 11). The basis for this age discrimination by EPEC has never been determined but may be related to either differences in susceptibility to EPEC colonization between adults and infants or to acquired protective immunity from repeated infections in adults.EPEC pathogenesis is dependent on a two-stage mechanism of adherence of the bacteria to host enterocytes. In the first step, EPEC cells bind to the host cell via their type IV bundle-forming pili (BFP) in a process known as localized adherence (LA). Following LA, EPEC strains inject effector proteins and their own receptor, a protein called the translocated intimin receptor (Tir), into the host cell via a type III secretion system. Tir is inserted into the host cell plasma membrane, where it acts as the receptor for the EPEC adhesin intimin and recruits cytoskeletal components, the net effect of which is the effacement of the microvilli and the formation of actin-rich pedestals at the site of EPEC adherence. This is known as the attaching-and-effacing process.BFP are homopolymers of a protein called bundlin. EPEC strains express one of two bundlin types, α or β, based on sequence homology (5). There are currently three α-bundlin alleles and seven β-bundlin alleles that have been described among diverse EPEC strains (4, 5). The α-bundlin proteins are N-acetyllactosamine (LacNAc) lectins, whereas the β-bundlins are not, since they lack the carbohydrate binding domain found in the α-bundlins (13, 16). The divergence between bundlins may be the result of selective immunological pressure, since the β-bundlins are less immunogenic in rabbits and mice than are the α-bundlins (8). It appears that α₁-bundlin is the only adhesin mediating early (45-min) LA of EPEC strain E2348/69 to HEp-2 cells, since replacing the α₁-bundlin allele in this strain with β-bundlin alleles 1 to 3 abolishes early adherence despite the expression of BFP (16). The early LA of this strain, and of all the other α-bundlin allele-expressing EPEC strains tested to date, can also be inhibited by LacNAc conjugated to bovine serum albumin (BSA) or gold nanoparticles (LacNAc-Au) (14-16). We previously reported (14) that LacNAc-based neoglycoconjugates inhibit α₁-bundlin-expressing EPEC LA as a result of their ability to (i) competitively inhibit the binding of the organisms to host cell glycan receptor sequences and (ii) induce BFP retraction, resulting in the dispersal of EPEC autoaggregates. Those strains that harbor the native β-bundlin allele are either nonadherent after a 45-min incubation with HEp-2 cells or not inhibited by LacNAc neoglycoconjugates (16). The inhibition of the α-bundlin allele-carrying strains is also lost when the bacteria are incubated on HEp-2 tissue culture cells for extended periods of time (3 h) (16). These data are consistent with evidence presented previously by Cleary and colleagues (6) that demonstrates a requirement for BFP during EPEC adherence to Caco-2 cells during the first 60 min of infection. After the first 60 min, other adhesins such as intimin and EspA then contribute to EPEC adherence.Recently, significant differences were observed in the mechanism by which EPEC induces attaching and effacing lesions in tissue culture cells compared to ex vivo human intestinal biopsy specimens (3, 20). As such, we sought to determine if the roles that we have demonstrated for bundlin and LacNAc in E2348/69 early LA to intestinal cells grown in tissue culture also apply to early EPEC adherence to human intestinal explants. Of particular interest was whether the β-bundlins, represented in these studies by the β₁-bundlin allele, had completely lost the ability to act as adhesins as a result of selective pressure or whether tissue culture cells simply lack the appropriate receptor for β-bundlin. We also wished to determine if the predilection of EPEC for causing disease in the pediatric population might be related to BFP-mediated early adherence.     

Detection of enteroaggregative Escherichia coli with formalin-preserved HEp-2 cells

Formalin-stored HEp-2 cells were used to assay Escherichia coli for adherence. Cells refrigerated in formalin for up to 28 days and used in a wet assay format demonstrated an assay sensitivity ranging from 94 to 98% to detect enteroaggregative E. coli (EAEC). HEp-2 cells first fixed and stored with formalin and then stored dry in ambient conditions for 6 weeks demonstrated an assay sensitivity of 92% to detect EAEC. Using formalin-fixed HEp-2 cells will improve the efficiency of EAEC identification.     

Association of Diarrheagenic Escherichia coli Pathotypes with Infection and Diarrhea among Mexican Children and Association of Atypical Enteropathogenic E. coli with Acute Diarrhea

Seventy-six children ≤2 years old were prospectively followed for 1 year in a peri-urban community of Mexico City to determine asymptomatic infection and acute diarrhea associated with diarrheagenic Escherichia coli pathotypes (DEPs). By use of a pathogen-specific multiplex PCR, DEPs were sought in 795 stool samples, of which 125 (16%) were positive for DEP; of these, 4 represented shedding episodes and 4 parasite coinfections. Most single-DEP infections (85/117) were asymptomatic (P < 0.001), and of the 32 DEP diarrhea episodes, 41% were associated with atypical enteropathogenic E. coli (aEPEC), 37.5% with enterotoxigenic E. coli, 9% with typical EPEC, 9% with enteroinvasive E. coli, and 3% with Shiga toxin-producing E. coli strains. Among the 76 children, 54 had at least one stool positive for DEP, of which 23 experienced a DEP-associated diarrhea episode. In the last group of children, DEP infection was significantly associated with a diarrhea episode (relative risk [RR] = 2.5; 95% confidence interval [CI], 1.79 to 3.57; P < 0.001), with ETEC (RR = 2.30; 95% CI, 1.49 to 3.54; P = 0.003) and aEPEC (RR = 1.92; 95% CI, 1.23 to 3.0; P = 0.019) being the pathotypes associated with diarrhea. aEPEC-associated diarrhea episodes were frequently in the <12-month age group (RR = 2.57; 95% CI, 1.05 to 6.27; P = 0.04). aEPEC infections were distributed all year round, but associated diarrheal episodes were identified from April to October, with a May-June peak (rainy season). Most ETEC infections and diarrhea episodes characteristically occurred during the summer (rainy season), with a diarrhea peak in August. Of all DEPs, only aEPEC was associated with acute diarrhea episodes lasting 7 to 12 days (P = 0.019). DEPs are important causes of community-acquired enteric infection and diarrhea in Mexican children.     

Expression of Enteropathogenic Escherichia coli Map Is Significantly Different than That of Other Type III Secreted Effectors In Vivo

The enteropathogenic Escherichia coli (EPEC) locus of enterocyte effacement (LEE)-encoded effectors EspF and Map are multifunctional and have an impact on the tight junction barrier while the non-LEE-encoded proteins NleH1 and NleH2 possess significant anti-inflammatory activity. In order to address the temporal expression of these important genes in vivo, their promoters were cloned upstream of the luxCDABE operon, and luciferase expression was measured in EPEC-infected mice by bioluminescence using an in vivo imaging system (IVIS). Bioluminescent images of living mice, of excised whole intestines, and of whole intestines longitudinally opened and washed were assessed. The majority of bioluminescent bacteria localized in the cecum by 3 h postinfection, indicating that the cecum is not only a major colonization site of EPEC but also a site of EPEC effector gene expression in mice. espF, nleH1, and nleH2 were abundantly expressed over the course of infection. In contrast, map expression was suppressed at 2 days postinfection, and at 4 days postinfection it was totally abolished. After 2 to 4 days postinfection, when map is suppressed, EPEC colonization is significantly reduced, indicating that map may be one of the factors required to maintain EPEC colonization. This was confirmed in a competitive colonization study and in two models of chronic infection, repeated exposure to ketamine and Citrobacter rodentium infection. Our data suggest that map expression contributes to the maintenance of EPEC colonization.     

HEp-2 Cell-Adherent Escherichia coli and Intestinal Secretory Immune Response to Human Immunodeficiency Virus (HIV) in Outpatients with HIV-Associated Diarrhea

HEp-2 cell-adherent Escherichia coli and the human immunodeficiency virus (HIV) itself have recently been incriminated as causes of chronic HIV-associated diarrhea. This study sought to determine the prevalence of these two agents among HIV-infected patients with diarrhea in an outpatient setting in the United States and to compare their prevalence to that of other commonly recognized enteropathogens known to be present in this population. HEp-2 cell-adherent E. coli was found in 20 of 83 (24.1%) patients with diarrhea. A diffuse pattern of adherence was the most common, found in 14 of 20 (70%) patients, followed by a localized adherence pattern (6 of 20; 30%). An intestinal secretory immune response against the p24 antigen of HIV was found in 9 of 34 (27.5%) patients with HIV-associated diarrhea. The following pathogens or products were also detected in lower frequencies: Cryptosporidium spp. (10.8%), Clostridium difficile toxin (8.8%), microsporidia (6%), Isospora belli (3.6%), Blastocystis hominis (2.4%), Giardia spp. (1.2%), Salmonella spp. (1.2%), and Mycobacterium spp. (1.2%). The role of HEp-2 cell-adherent E. coli and HIV enteric infections in patients with HIV-associated diarrhea deserves further study.     

Contribution of the pst-phoU Operon to Cell Adherence by Atypical Enteropathogenic Escherichia coli and Virulence of Citrobacter rodentium

Strains of enteropathogenic Escherichia coli (EPEC) generally employ the adhesins bundle-forming pili (Bfp) and intimin to colonize the intestine. Atypical EPEC strains possess intimin but are negative for Bfp and, yet, are able to cause disease. To identify alternative adhesins to Bfp in atypical EPEC, we constructed a transposon mutant library of atypical EPEC strain E128012 (serotype O114:H2) using TnphoA. Six mutants that had lost the ability to adhere to HEp-2 cells were identified, and in all six mutants TnphoA had inserted into the pstSCAB-phoU (Pst) operon. To determine if the Pst operon is required for adherence, we used site-directed mutagenesis to construct a pstCA mutant of E128012. The resultant mutant showed a reduced ability to adhere to HEp-2 cells and T84 intestinal epithelial cells, which was restored by trans-complementation with intact pstCA. To determine if pst contributes to bacterial colonization in vivo, a pstCA mutation was made in the EPEC-like murine pathogen, Citrobacter rodentium. C57BL/6 mice infected perorally with the pstCA mutant of C. rodentium excreted significantly lower numbers of C. rodentium than those given the wild-type strain. Moreover, colonic hyperplasia and diarrhea, which are features of infections with C. rodentium, were not observed in mice infected with the pstCA mutant but did occur in mice given the trans-complemented mutant. As mutations in pst genes generally lead to constitutive expression of the Pho regulon, our findings suggested that the Pho regulon may contribute to the reduced virulence of the pstCA mutants. To investigate this, we inactivated phoB in the pstCA mutants of EPEC E128012 and C. rodentium and found that the phoB mutation restored the adherent phenotype of both mutant strains. These results demonstrate that Pst contributes to the virulence of atypical EPEC and C. rodentium, probably by causing increased expression of an unidentified, Pho-regulated adhesin.Enteropathogenic Escherichia coli (EPEC) is a prominent cause of diarrhea worldwide, especially among young children (28, 32, 41). In developing countries, EPEC is responsible for endemic infantile diarrhea and is estimated to cause the deaths of several hundred thousand children each year (32, 41). EPEC employs a large number of determinants to colonize the intestine and produces characteristic attaching and effacing (A/E) lesions in the intestinal mucosa (8, 20). The genetic determinants required for the production of A/E lesions are located on a pathogenicity island called the locus of enterocyte effacement (LEE), which encodes a type III protein secretion system, an outer-membrane protein adhesin (called intimin and encoded by the eae gene), and a translocated intimin receptor (Tir), as well as other type III secreted proteins (8, 14). Many EPEC strains also carry an adherence factor plasmid (pEAF) that encodes bundle-forming pili (Bfp), which promote bacterial adherence to epithelial cells and are essential for virulence (7, 25, 39).Carriage of the bfpA gene, which encodes the major structural pilin subunit, is used to classify EPEC into two major subgroups, known as typical (Bfp positive) and atypical (Bfp negative) EPEC (19, 41). Typical EPEC bacteria adhere to HEp-2 cells in a localized pattern, whereas atypical EPEC, if they adhere to HEp-2 cells at all, do so in a variety of patterns, termed localized-like adherence, diffuse adherence, and aggregative adherence (33, 41). Despite their lack of Bfp, the results of epidemiological, clinical, and volunteer studies indicate that atypical EPEC are able to cause diarrhea (25, 33, 41).Given that, as a group, atypical EPEC lack Bfp and display variable patterns of adherence to HEp-2 cells, we hypothesized that atypical EPEC strains carry novel adhesin(s) responsible for these phenotypes. Other than intimin, however, only one adhesin has so far been described in an atypical EPEC strain. This is a novel afimbrial adhesin called the locus for diffuse adherence (LDA), which was present in an atypical EPEC strain (O26:H11) isolated from an infant with diarrhea (36). However, the prevalence of LDA in other atypical EPEC strains is low (36). The aim of this study was to identify the determinants of atypical EPEC strain E128012 (O114:H2) which allow this strain to adhere to HEp-2 cells. Originally isolated from an infant with sporadic diarrhea in Bangladesh, E128012 shows localized-like adherence to HEp-2 cells and, when fed to volunteers, caused diarrhea of severity similar to that caused by a typical EPEC strain, E2348/69 (25). Our results indicated that atypical EPEC strain E128012 requires an intact pst-phoU operon to adhere to HEp-2 cells and, moreover, that Citrobacter rodentium strain ICC169, an A/E pathogen of mice that is used as a model of infections with A/E strains of E. coli, requires the same operon for virulence.     

Variations in the csgD Promoter of Escherichia coli O157:H7 Associated with Increased Virulence in Mice and Increased Invasion of HEp-2 Cells

Promoter alterations in the csgD gene of Escherichia coli O157:H7 strains ATCC 43894 and ATCC 43895 are associated with variations in curli expression and the ability to bind Congo red dye. Red variants of each strain were more invasive for cultured HEp-2 cells than were white variants. An ATCC 43895 red variant was more virulent than a white variant in a mouse model. However, there were no differences in Shiga toxin production between red and white variants.     

Localization of a determinant for HEp-2 adherence by enteropathogenic Escherichia coli.

pMAR2, a 60-megadalton plasmid encoding localized HEp-2 adherence in enteropathogenic Escherichia coli, was mapped with BamHI, HindIII, and SalI. Deletion and insertion mutants were constructed and used to define a potential DNA probe. Preliminary results indicate that this probe is sensitive and specific for the genes encoding the enteropathogenic E. coli adherence factor.     

Agents That Inhibit Rho, Rac, and Cdc42 Do Not Block Formation of Actin Pedestals in HeLa Cells Infected with Enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) induces formation of actin pedestals in infected host cells. Agents that inhibit the activity of Rho, Rac, and Cdc42, including Clostridium difficile toxin B (ToxB), compactin, and dominant negative Rho, Rac, and Cdc42, did not inhibit formation of actin pedestals. In contrast, treatment of HeLa cells with ToxB inhibited EPEC invasion. Thus, Rho, Rac, and Cdc42 are not required for assembly of actin pedestals; however, they may be involved in EPEC uptake by HeLa cells.     

The AIDA Autotransporter System Is Associated with F18 and Stx2e in Escherichia coli Isolates from Pigs Diagnosed with Edema Disease and Postweaning Diarrhea

Pathogenic Escherichia coli strains are known to cause edema disease (ED) and postweaning diarrhea (PWD) in piglets. Although the exact mechanisms of pathogenicity that lead to ED-PWD remain to be elucidated, E. coli-borne Shiga-like toxin and adhesion-mediating virulence factors such as F18 adhesin or F4 fimbriae are believed to play a central role in ED-PWD. In light of these observations we investigated whether another E. coli adhesin, the plasmid-encoded AIDA (adhesin involved in diffuse adherence) might also be present in ED-PWD-causing E. coli isolates. For rapid screening for the AIDA system in large numbers of isolates, a multiplex PCR method along with a duplex Western blot procedure was developed. When screening 104 strains obtained from pigs with or without ED-PWD, we observed a high prevalence of the AIDA operon in porcine E. coli isolates, with over 25% of all strains being AIDA positive, and we could demonstrate a significant association of the intact AIDA gene (orfB) with ED-PWD, while defects in orfB were associated with the absence of disease. Although our data hint toward a contribution of AIDA to ED-PWD, further studies will be necessary since the presence of the AIDA genes was also associated with the presence of the Shiga-like toxin and F18 adhesin genes, two reported virulence factors for ED-PWD.     

Mannose-Sensitive and Mannose-Resistant Adherence to Human Uroepithelial Cells and Urinary Virulence of Escherichia coli

The adherence to human uroepithelial cells of 23 Escherichia coli strains belonging to three groups with different levels of virulence was investigated, and the mechanism of adherence was studied. It was found that strains belonging to the most virulent group adhered better to human uroepithelial cells than did avirulent strains. Adherence of loss virulent but supposedly nephropathogenic strains was more variable. These results suggest that adherence is an important virulence factor, especially in the group of strains with the highest but a more general virulence. Piliated strains adhered better than did nonpiliated strains. We found strong evidence for the existence of at least two different mechanisms of adherence: (i) mannose-sensitive adherence by piliated strains, very likely mediated by type I pili because this mannose-sensitive adherence was associated with mannose-sensitive hemagglutination of guinea pig erythrocytes by broth cultures of the strains; (ii) mannose-resistant adherence by piliated strains, very likely mediated by non-type I pili because this mannose-resistant adherence was invariably associated with mannose-resistant hemagglutination of human group A erythrocytes by the strains, whether grown in broth or on plates. Additionally, one strain without pili and without hemagglutinating activity adhered well. Thus in most cases adherence seemed to be mediated by bacterial pili, although different types might be involved.     

Intestinal Secretory Immunoglobulin A Response to Enteroaggregative Escherichia coli in Travelers with Diarrhea

We examined stool samples from travelers for secretory immunoglobulin A (sIgA) to enteroaggregative Escherichia coli (EAEC) during episodes of acute diarrhea. Ten paired samples from 10 patients with diarrhea caused by EAEC were examined for the presence of specific sIgA by dot blot and Western blot immunoassays. Five samples were positive by dot blotting, and two samples were positive by Western blotting.     

Elevation of intracellular free calcium levels in HEp-2 cells infected with enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) are a class of diarrheagenic organisms that induce a characteristic attaching and effacing lesion in enterocytes and various cultured cell lines. Infection of cultured HEp-2 cells by EPEC isolates 2036-80 (serotype O119) and E2348-69 (serotype O127) resulted in significant elevation of intracellular free calcium levels, determined quantitatively with the fluorescent calcium indicator dye 2-([2-bis(carboxymethyl)amino-5-methylphenoxy]methyl)-6-methoxy-8- bis(carboxymethyl)aminoquinoline. This effect, which was not observed on infection with non-lesion-forming E. coli strains, was inhibited by dantrolene, a drug that prevents calcium mobilization from intracellular stores. Moreover, activated protein kinase C in infected cells was dissociated from cell membranes by a process that was inhibited by cyclosporin A, suggesting involvement of the calcium-dependent protease calpain. A qualitative method for observing intracellular calcium fluxes by fluorescence microscopy with the recently described fluorescein-based indicator fluo-3 was used to screen a collection of well-characterized E. coli isolates from patients with infantile enteritis. Increased localized calcium-dependent fluo-3 fluorescence was observed only in HEp-2 cells infected with known lesion-forming EPEC strains. We propose that enhancement of intracellular free calcium levels in enterocytes infected with EPEC would result in formation of the characteristic lesion by calcium-dependent activation of actin-depolymerizing proteins, with eventual loss of absorptive capacity.