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Genetic Susceptibility to Enteroaggregative Escherichia coli Diarrhea: Polymorphism in the Interleukin-8 Promotor Region

  1. Zhi-Dong Jiang1,
  2. Pablo C. Okhuysen1,2,
  3. Dong-Chuan Guo2,
  4. Rumin He2,
  5. Terri M. King2,
  6. Herbert L. DuPont1,2,3,4 and
  7. Dianna M. Milewicz2,a
  1. 1University of Texas School of Public Health and
  2. 2Medical School,
  3. 3Baylor College of Medicine, and
  4. 4St. Luke’s Episcopal Hospital, Houston, Texas
  1. Reprints or correspondence: Dr. Zhi-Dong Jiang, University of Texas School of Public Health, 1200 Herman Pressler Rm. 706, Houston, TX (zjiang{at}sph.uth.tmc.edu)
 
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Abstract

Enteroaggregative Escherichia coli (EAEC) infection can be identified in 26% of travelers with diarrhea and is associated with fecal interleukin (IL)–8 production. We hypothesized that single-nucleotide polymorphisms (SNPs) in the IL-8 gene are associated with EAEC-related symptoms. Fecal IL-8 production and IL-8 SNPs at 5 loci were identified in 69 US students who remained in Mexico for 5 weeks; 23 subjects had EAEC-associated diarrhea, 7 were asymptomatic EAEC carriers, 22 had nonspecific diarrhea, and 17 were asymptomatic without an enteropathogen. The chances of having EAEC-associated diarrhea were significantly increased among those with the AA genotype at the −251 position (odds ratio [OR], 208.51; 95% confidence interval [CI], 28.5–1525.36) and among those with AT genotype (OR, 14.3; 95% CI, 1.98–105.74), compared with those with the TT genotype at the −251 position. Among subjects with EAEC-associated diarrhea, the AA genotype at the −251 position produced greater concentrations of fecal IL-8 than those with the AT or TT genotype (P=.0053). In the present study, the AA genotype at the −251 position was associated with the occurrence of EAEC-associated diarrhea and increased levels of fecal IL-8

Enteroaggregative Escherichia coli (EAEC) is an emerging foodborne infectious disease that is associated with diarrhea that occurs in international travelers [1], persistent diarrhea in children residing in developing countries [2], and chronic diarrhea in patients with AIDS [3]. Diarrhea due to EAEC is associated with markers of intestinal inflammation, such as fecal lactoferrin and fecal neutrophils, as well as with a series of proinflammatory cytokines, such as interleukin (IL)–1β, and the CXC chemokine IL-8 [4–6]. EAEC flagellin [7] and other virulence genes are involved in the production of IL-8 by the intestinal mucosa [8]. EAEC-induced IL-8 production could play a role in EAEC pathogenesis by recruiting neutrophils to the intestinal epithelium and, as has been shown for other organisms, by causing the transmigration of polymorphonuclear cells to the intestinal epithelium, leading to fluid secretion and epithelial disruption [9, 10]. EAEC diarrhea is associated with increased concentration of fecal lactoferrin but not with increased numbers of fecal neutrophils [6]

Data from our previous studies have suggested that some individuals are more susceptible to EAEC-associated diarrhea [11]. After exposure to EAEC, the clinical manifestations are variable and depend on host and pathogen factors [4, 12]. We hypothesized that single-nucleotide polymorphisms (SNPs) in the IL-8 gene are associated with EAEC-related symptoms. The present study provides evidence that a polymorphism at the IL-8 gene confers susceptibility to EAEC-associated diarrhea in those who travel to a region where EAEC is endemic

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Subjects, Materials, and Methods

Study populationStool samples were collected from US students with diarrhea that developed during short-term stays in Guadalajara, Mexico, during the summers of 1999 and 2000 [12]. Diarrhea was defined as passage of ⩾3 unformed stools within 24 h, with at least 1 additional sign or symptom within <72 h before presentation to the clinic. The signs or symptoms included abdominal pain or cramps, nausea, vomiting, fecal urgency, blood in stools, or increased intestinal gas. Stool samples also were collected from students attending the same classes as the case patients but who did not develop diarrhea. The study was approved by the University of Texas Institutional Review Board for Human Subjects Research at the University of Texas Health Science Center at Houston, and written informed consent was obtained from each subject

Stool sample analysisStool samples obtained from subjects with travelers’ diarrhea and from the asymptomatic subjects were studied in our laboratories in Guadalajara for enteric pathogens, according to published methods [13]. The following pathogenic organisms were examined: Shigella, Salmonella, Campylobacter, Aeromonas, Plesiomonas, Vibrios, Giardia, Entamoeba and Cryptosporidium species [13]. Five E. coli colonies from each subject were transported to Houston on peptone stabs to assay for enterotoxigenic E. coli (ETEC) by DNA hybridization [13] and EAEC by HEp-2 adherence assay [14]. Subjects were included in this study if they had EAEC as the only pathogen identified or had diarrhea but no specific pathogen identified. Individuals with diarrhea caused by ETEC were excluded from the study, to allow us to study the pathogenesis of EAEC diarrhea. Individuals who were asymptomatic with EAEC infection or without a pathogen identified were included

Stool samples were stored at −70°C. Aliquots of the original stool sample were diluted in PBS containing 2.5 μg/mL leupeptin, 11 μg/mL aprotinin, and 0.5 mmol/L 4-(2-aminoethyl) benzenesulfonyl fluoride (Sigma). After thorough mixing and centrifugation for 10 min at 10,000 g the supernatants were analyzed for the presence of IL-8 by use of a quantitative ELISA, as described elsewhere [4] (Quantikine; R&D systems)

DNA isolationHuman genomic DNA was extracted from each fecal and blood sample by use of the PureGene DNA isolation protocol (Gentra Systems)

Polymerase chain reaction (PCR) and pyrosequencing protocolSNP information of the human IL-8 gene was obtained from the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov). In the present study, SNPs were located in promoter, exons, and introns of the IL-8 gene. SNPs with allele frequencies >20% in the general population were analyzed (NCBI database; no. AF385628). PCR primers were designed for each of the SNPs by use of the Primer3 program (http://www.genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi). The primers for pyrosequencing assay were designed by use of the SNP Primer Design Software (Pyrosequencing AB) and were synthesized by Integrated DNA Technologies (Coralville) (table 1). PCRs were performed by use of the HotStar Taq DNA polymerase (Qiagen) with the 2-cycle amplification program (Applied Biosystems). SNP assays were performed by use of the Pyrosequencer with PSQ 96 SNP Reagent Kit, according to the conditions recommended by the manufacturer (Pyrosequencing AB)

Table 1
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Table 1

Sequence of primers used to study associations with enteroaggregative Escherichia coli infection in US students in Mexico, summers of 1999 and 2000

The presence of EAEC plasmidborne genes was assessed among E. coli derived from 23 patients with EAEC travelers’ diarrhea and 7 asymptomatic US travelers to Mexico with EAEC infection. DNA from E. coli isolates was purified by use of the ReadyAM DNA Purification System (Promega). Oligonucleotide primers for EAEC genes aggA, aggR, aafA and aspU were selected for study on the basis of previous studies, as described elsewhere [15, 16]. The positive controls used in the study for aggA, aggR, aspU and aafA were EAEC strains 17–2, JM221, 60A, and 042, respectively. The various strains were employed in each PCR assay. The final amplification mix contained the following: 98 μL of PCR mix (10 mmol/L Tris-HCl [pH 8.3], 50 mmol/L KCl, 2 mmol/L MgCl2, 100 μmol/L each dATP, dCTP, dGTP, and dTTP, and 2.5 U of AmpliTaq polymerase) (Perkin-Elmer), 25 pmol of each of the primers, and 2 μL of DNA template. The reaction mixtures were heated to 94°C for 5 min and then were subjected to 35 cycles (94°C for 30 s, 50°C for 1 min, and 72°C for 45 s), and then to a final extension at 72°C for 7 min in a DNA thermal cycler (Perkin-Elmer). Ten microliters of the amplified PCR products were added to 1% agarose electrophoresis gel with 1-kb DNA molecular weight marker (Gibco BRL). A positive reaction was defined as the presence of the PCR product of the expected size

Statistical analysisAssociation of the IL-8 genotype with EAEC-associated diarrhea was analyzed by χ2 analysis. The fecal IL-8 concentrations were compared by use of the Kruskal-Wallis nonparametric method. Nonrandom distribution of SNPs in each of the groups was evaluated by χ2 analysis, as was deviation from Hardy-Weinberg equilibrium. Haplotypes were constructed with the program PHASE [17], which computed the haplotype by using the Stephens-Donnelly method of haplotype reconstruction. Logistic regression analysis was used to compare those with diarrhea and those without diarrhea as a function of the number of SNPs in each individual, as well as the derived haplotypes. Analyses were performed by use of SAS (version 8; SAS) and STATA (Stata Corp)

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Results

During the summers of 1999 and 2000, we studied the prevalence of EAEC infection among 200 US students who were in Guadalajara, Mexico, for 5-week stays [12]. These individuals lived in the same environment and had relatively the same lifestyle. A total of 87 subjects developed diarrhea associated with ⩾1 enteric pathogen: 23 experienced EAEC-associated diarrhea, and 22 subjects had nonspecific diarrhea without an identified pathogen. Twenty-four subjects who remained asymptomatic provided stool samples for analysis, 7 of which were shown to have asymptomatic EAEC infection. The remainder had diarrhea caused by ETEC or other enteropathogens and were not included in this analysis

Because genomic DNA isolated from fecal material had been used rarely for genetic analysis, we initially confirmed that genetic data obtained from fecal DNA were identical to results obtained from peripheral blood white blood cell (WBC) DNA. Genomic segments containing multiple SNPs were sequenced, and polymorphic markers were analyzed from both fecal and WBC samples obtained from 21 unrelated white subjects. Results were identical when fecal and WBC DNA were compared (data not shown)

The fecal genomic DNA obtained from 69 white subjects were analyzed for 5 SNPs in the IL-8 gene. The 5 IL-8 SNPs analyzed were located in the following positions: −251 bp in the promoter region (A or T), 386 bp (T or G), 781 bp (T or C), 1633 bp (T or C), and 2767 bp (T or A). Frequencies of these genotypes and evaluation by use of the Hardy-Weinberg equilibrium are shown in table 2. Our sample frequencies for the 386 and 781 loci significantly varied from the reported population frequency of the databases [18, 19]

Table 2
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Table 2

Evaluation of Hardy-Weinberg equilibrium in the frequency of the interleukin (IL)–8 gene and genotype in US students in Mexico with enteroaggregative Escherichia coli infection, summers of 1999 and 2000

To investigate the association between genetic polymorphisms in IL-8 and EAEC infection, we analyzed the genetic data among the study subjects. The distribution of IL-8 genotypes in symptomatic and asymptomatic subjects in each of these subgroups was determined (table 3). When the allele frequencies of the polymorphisms were analyzed, a significant difference in the frequency of the T→A polymorphism was found in the promoter region of IL-8 (located −251-bp upstream of the transcription start site). The AA homozygotes were overrepresented in subjects with symptomatic EAEC infection, compared with the AA homozygotes in subjects with asymptomatic EAEC infection (87% vs. 29%; P=.006). We also analyzed the dose-response relationship between copies of the SNP and the presence or absence of EAEC-associated diarrhea, using logistic regression. The presence of AT genotype resulted in an odds ratio (OR) of 14.43 (95% confidence interval [CI], −1.98 to 105.74), and the presence of an AA genotype resulted in an OR of 208.51 (95% CI, −28.5 to 1525.36) for EAEC-associated diarrhea when either group was compared with those without the −251 TT genotype

Table 3
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Table 3

Association between interleukin (IL)–8 polymorphisms at the −251 position and susceptibility to symptomatic enteroaggregative Escherichia coli (EAEC) infection in US students in Mexico, summers of 1999 and 2000

We compared the frequency of IL-8 SNPs from patients with nonspecific diarrhea with that of asymptomatic subjects without a pathogen identified (table 3). There were no significant differences in the proportion of IL-8 AA homozygotes at the −251 position between these 2 subgroups (P=.501). None of the other IL-8 SNPs analyzed demonstrated any association with the presence or absence of symptoms (data not shown)

The relationship between EAEC plasmidborne virulence genes and IL-8 genotypes studied is presented for the 30 subjects with EAEC infection in table 4. Of the 22 subjects with EAEC infection with the AA genotype, 14 (64%) had ⩾1 virulence factor, compared with 4 (57%) of the 7 subjects with the AT genotype. Isolates bearing the virulence factors aggA, aggR and aspU occurred with approximately equal frequency in all 3 genotypes

Table 4
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Table 4

Defined enteroaggregative Escherichia coli (EAEC) virulence factors in HEp-2 cell–adherent E. coli and interleukin (IL)–8 polymorphisms at the −251 position in US students with EAEC infection acquired in Mexico, summers of 1999 and 2000

The derived IL-8 haplotypes were analyzed to determine whether there was an association between the IL-8 haplotypes and symptomatic EAEC infection. The haplotypes were stratified by the first SNP (A vs. T), which resulted in findings that were similar to the single SNP analysis. To determine whether the haplotype was significant, we took only those haplotypes with the “A” allele at the −251 position and stratified them by their second allele. In that analysis, we found that there was no significant association between the second allele, given the first allele, and the development of EAEC-associated diarrhea (P=.17). This signifies that there is no relationship between particular haplotypes, but, rather, that the increased risk for acquiring EAEC diarrhea is related only to the “A” SNP at the −251 position

The T→A polymorphism at the −251 position previously had been shown to affect IL-8 blood levels [19]. Fecal IL-8 levels were determined to verify that this IL-8 SNP has an effect on fecal IL-8 levels. Fecal IL-8 levels ranged from 0 to 4074.74 pg/mL and correlated with the A/T SNP at the −251 position of the IL-8 gene. The subjects with the AA genotype (n=42) produced greater concentrations of fecal IL-8 (median, 132.6 pg/mL) than subjects with the AT genotype (n=21; median 20.3 pg/mL) or the TT genotype (n=6; median, 3.8 pg/mL; P=.0053). No relationship was noted between the T→A polymorphism at the −251 position and the presence of fecal leukocytes

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Discussion

EAEC is rapidly becoming recognized as an important enteropathogen in subjects traveling from industrialized countries to developing regions of the world. EAEC is defined by its ability to form a characteristic stacked-brick aggregative pattern of adherence to HEp-2 cells in culture [20]. Several virulence factors have been described, including the production of a thick biofilm, AAF/I, AAF/II, the heat-stable enterotoxin EAST-1 [21], the Shigella-derived enterotoxin ShET-1 [22], and a plasmid-encoded toxin (Pet) [23]. In addition, EAEC are motile by virtue of possessing flagella, a multimer structure composed of the subunit component flagellin. Recently, EAEC has been noted to elicit the production of IL- 8 in vivo and in vitro in a process that involves flagellin [7] and other nonflagellar virulence genes [8]. The production of IL-8 by the epithelial cells probably contributes to EAEC pathogenicity by recruiting neutrophils to the epithelial mucosa, which results in epithelial disruption and fluid secretion. Of interest, despite inducing the production of IL-8, EAEC-associated diarrhea is frequently not associated with fecal leukocytes in stools. However, lactoferrin, a product of polymorphomuclear cells, is frequently identified. Potential reasons for this discrepancy are individual host response to infection or lysis of neutrophils in the intestinal lumen

SNPs in regulatory and coding regions of cytokines have been associated with susceptibility to a number of complex disorders. Polymorphisms in a number of cytokine genes, such as tumor necrosis factor–α, IL-1β, IL-1ra, IL-6, and IL-4, have an impact on the type of immune response that occurs after infectious and noninfectious stimuli have been identified [24, 25]. Previous studies have suggested that the production of IL-8 is also genetically determined and that neutrophils from individuals who are homozygous for the AA genotype at the −251 position demonstrated a trend toward higher levels of IL-8 in response to lipopolysaccharide than those without the allele [18]. Studies also have indicated the severity of respiratory syncytial virus (RSV) bronchiolitis is associated with the A variant at this location [18], and a family linkage study that evaluated 9 SNPs in the IL-8 gene demonstrated that the -251A/781T haplotype also had a significant association with RSV bronchiolotis severity [19]

In the present study, we used a novel approach for the identification of human SNPs in fecal material. There is a growing body of evidence that validates the use of fecal-derived DNA for genotyping purposes; this approach has been used to identify mutations associated with gastrointestinal malignancies [26, 27]. We successfully obtained DNA of sufficient quality to perform genotyping. Furthermore, the genotypes obtained from fecal material were confirmed with the genotypes seen in DNA derived from whole blood. The advantage for using fecal-derived DNA for genotype studies is that it is a noninvasive method that can be collected easily in the field and can be performed in stored samples. The disadvantage is that, if samples are not adequately preserved, only short DNA segments can be amplified

In the present study, we investigated the association between polymorphisms in the IL-8 gene and EAEC infection. Our results suggest that the intestinal inflammatory response also is partially determined by IL-8 gene polymorphisms and indicate the association of the AA genotype at the −251 position with symptomatic illness after exposure to EAEC. Although the high incidence of AA genotype in subjects with EAEC-associated diarrhea suggests that individuals with this genotype are more likely to develop symptomatic disease when exposed to EAEC, the number of subjects studied was too small to determine whether having the AT or TT genotype is protective of symptomatic diarrhea. EAEC isolates bearing virulence factors aagR, aggR and aspU were isolated with similar proportions among the different host genotypes, which suggests that host-determined IL-8 response contributes significantly to disease manifestation

We were unable to identify the −251A/781T haplotype association with EAEC infection, as has been described elsewhere for RSV infection in a family linkage analysis conducted in England [18, 19]. Potential explanations for the lack of haplotype association are as follows: our study population consisted of a heterogeneous group of white subjects with diverse genetic background and did not include their family members or was a result that was related to the small sample size. In addition, we focused on subjects with EAEC-associated diarrhea, asymptomatic EAEC infection, and nonspecific diarrhea without a pathogen identified, as well as asymptomatic subjects without a pathogen identified. We excluded subjects with other causes of infectious inflammatory and noninflammatory EAEC-associated diarrhea. Larger ongoing studies will assist in determining whether specific haplotypes are associated with EAEC and other agents of infectious diarrhea

We also measured fecal IL-8 levels in all subjects. IL-8 levels were higher in subjects with the AA genotype than in those with the AT or TT genotype. As indicated in our previous studies, subjects with EAEC infection excreted higher levels of fecal IL-8 in their stool samples, compared with those in whom other agents could not be identified or in whom diarrhea did not develop [4]. Steiner et al. [5] previously demonstrated that fecal IL-8, IL-1β, and lactoferrin could be identified in Brazilian children infected with EAEC. In the study of travelers’ diarrhea [4], patients with diarrhea in whom a pathogen could not be identified also were negative for these inflammatory products, which indicates an important relationship between cytokine-mediated intestinal inflammation and diarrhea caused by defined bacterial pathogens in travelers. The IL-8 −251 SNP also is likely to impact the fecal IL-8 levels and outcome of infection due to other agents of inflammatory diarrhea, such as Shigella, Salmonella, Campylobacter, C. difficile and Cryptosporididium species, which are also known to lead to increased levels of fecal IL-8 [4, 28–30]

The above data demonstrate that fecal IL-8 production is associated with EAEC-associated diarrhea in subjects who possess specific virulence genes. However, the number of subjects studied was too small to determine whether having the AT or TT genotype was protective from symptomatic EAEC diarrhea. We are now studying the interaction of human IL-8 gene with susceptibility to other forms of inflammatory diarrhea. These studies should help us determine whether the −251 SNP controls susceptibility to symptomatic EAEC and to help characterize its relationship to specific EAEC virulence genes

The present study was designed to test the hypothesis that the presence of SNPs in the genes coding for IL-8 may influence the pathogenesis of EAEC-associated diarrhea. The association of the −251 AA SNP with symptomatic EAEC infection identifies a group of individuals who are particularly susceptible to infection with this agent and may explain the heterogenous clinical presentations seen with EAEC. Determining the host IL-8 genotype will be important in studying the epidemiology of EAEC, as well as for risk assessment, pathogenicitiy, and vaccines studies in volunteers and in the field. In the future, determining IL-8 genotypes will assist in defining populations that are likely to benefit from therapeutic interventions, such as prophylactic antibiotic therapy or vaccination, or, ultimately, to advise against foreign travel in selected cases

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Footnotes

  • ↵Financial support: Public Health Service (grant DK56338), which funds the Texas Gulf Coast Digestive Diseases Center; National Institute of Allergy and Infectious Diseases (grant NO1-AI-25465); University of Texas Medical School at Houston Clinical Research Center (grant M01-RR-02558)

  • ↵D.M.M. is a Doris Duke Distinguished Clinical Scientist

  • Received December 13, 2002.
  • Accepted March 21, 2003.
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  1. J Infect Dis. (2003) 188 (4): 506-511. doi: 10.1086/377102
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