Mapping of genetic loci that modulate differential colonization by Escherichia coli O157:H7 TUV86-2 in advanced recombinant inbred BXD mice
© Russo et al. 2015
Received: 14 July 2015
Accepted: 22 October 2015
Published: 16 November 2015
Shiga toxin (Stx)-producing E. coli (STEC) are responsible for foodborne outbreaks that can result in severe human disease. During an outbreak, differential disease outcomes are observed after infection with the same STEC strain. One question of particular interest is why some infected people resolve infection after hemorrhagic colitis whereas others progress to the hemolytic uremic syndrome (HUS). Host age and infection dose have been implicated; however, these parameters do not appear to fully account for all of the observed variation in disease severity. Therefore, we hypothesized that additional host genetic factors may play a role in progression to HUS.
Methods and Results
To mimic the genetic diversity in the human response to infection by STEC, we measured the capacity of an O157:H7 outbreak isolate to colonize mouse strains from the advanced recombinant inbred (ARI) BXD panel. We first infected the BXD parental strains C57BL/6 J (B6) and DBA/2 J (D2) with either 86–24 (Stx2a+) or TUV86-2, an Stx2a-negative isogenic mutant. Colonization levels were determined in an intact commensal flora (ICF) infection model. We found a significant difference in colonization levels between the parental B6 and D2 strains after infection with TUV86-2 but not with 86–24. This observation suggested that a host factor that may be masked by Stx2a affects O157:H7 colonization in some genetic backgrounds. We then determined the TUV86-2 colonization levels of 24 BXD strains in the ICF model. We identified several quantitative trait loci (QTL) associated with variation in colonization by correlation analyses. We found a highly significant QTL on proximal chromosome 9 (12.5–26.7 Mb) that strongly predicts variation in colonization levels and accounts for 15–20 % of variance. Linkage, polymorphism and co-citation analyses of the mapped region revealed 36 candidate genes within the QTL, and we identified five genes that are most likely responsible for the differential colonization.
The identification of the QTL on chromosome 9 supports our hypothesis that individual genetic makeup affects the level of colonization after infection with STEC O157:H7.
Shiga toxin (Stx)-producing E. coli (STEC) are Gram-negative enteric pathogens associated with foodborne outbreaks. E. coli O157:H7 accounts for the greatest incidence of disease due to a single STEC serotype in the in the United States. Indeed, E. coli O157:H7 is estimated to be responsible for 63,000 of the estimated 175,000 STEC cases per year . Stx, the primary STEC virulence factor, is an AB5 toxin that inhibits protein synthesis and leads to cell death . The A subunit is responsible for the catalytic activity of the molecule while the B pentamer binds to the host cell receptor, globotriaosylceramide (Gb3). An STEC strain may encode Stx1a and/or Stx2a, two biologically similar though antigenically distinct toxins. Some STEC strains also have the locus of enterocyte effacement (LEE) pathogenicity island , a large segment of 43 Kb DNA flanked by direct repeats, that encodes a type III secretion system and the adhesin intimin. Intimin is necessary for maximal colonization by O157:H7 in mouse, pig, and calf models [4–7].
Cattle and other ruminants are the natural carriers of STEC O157:H7, and contamination of the organism in meat most often occurs during beef processing [8–10]. Undercooked ground beef was responsible for the majority of STEC outbreaks initially [11, 12]; however, contaminated fresh produce and non-pasteurized beverages also spread STEC [13–17]. After ingestion of even low doses of E. coli O157:H7, the most common disease manifestation is bloody diarrhea or hemorrhagic colitis (HC) . A serious sequela of E. coli O157:H7 infection, the hemolytic uremic syndrome (HUS), is defined by thrombocytopenia, hemolytic anemia, and kidney failure and occurs in 10–20 % of individuals . It is not known why some individuals spontaneously clear infection while others progress to the HUS. Infectious dose and age of the patient have been implicated [19–21]; however, these factors alone do not account for the total observed disease variance [18, 22].
Host genetic factors are likely important determinants for O157:H7-related disease outcome. Because traditional laboratory animal models are inbred to reduce genetic heterogeneity and to minimize variably between experiments , a different animal model was required to investigate phenotypic variations and to reflect the complex genetic structure of the human population. The advanced recombinant inbred murine (ARI) BXD panel , was created by intercrossing two mouse strains, C57BL/6 J (B6) and DBA/2 J (D2). The progeny BXD strains were inbred to homozygositiy and genotyped to determine the genotypic contest of each strain and to identify the location of crossover events. The BXD panel has approximately 580,000 single nucleotide polymorphisms (SNPs) and microsatellite markers [25, 26]. In addition, the genomes of the parental parents B6  and D2 [28, 29] were sequenced and found to differ at approximately 4.8 million SNPs. Phenotypic differences among the BXD strains are primarily related to the SNPs and 500,000 insertion-deletions .
Therefore, use of the BXD panel allows for a systems genetic, genome-wide analysis to facilitate identification of a quantitative trait locus (QTL) responsible for phenotypes such as variation of severity of diabetes , forebrain weight , bone density , or addiction response to alcohol [33, 34]. Of particular relevance to this study, the BXD panel was used to explore genetic traits that underlie the response to infectious diseases such as influenza , streptococcal sepsis [36, 37], and Ebola  infections. A comparison of the human and murine genome reveals a high degree of similarity . Therefore, it is theoretically possible to translate QTL findings from BXD mice to humans (reviewed in [40, 41]). Thus, we theorized that host genetic loci that impact colonization by O157:H7 in the BXD panel may reflect human traits responsible for STEC disease.
In this study, we observed a statistically significant difference in colonization levels in the murine parental strains (B6 and D2) after infection with TUV86-2. That difference indicates the presence of a potential QTL involved in O157:H7 colonization that may be masked when Stx2a is expressed by the infecting E. coli O157:H7 strain. Analysis of colonization data from 24 BXD strains infected with TUV86-2 identified a highly significant QTL on proximal chromosome (chr) 9 between 12.5 and 26.7 Mb that strongly predicts variation in colonization levels one day post-infection, accounting for 15–20 % of variance. This QTL harbors several genes known to regulate immune responses to bacterial infections. We evaluated candidate genes within this QTL using multiple parameters that included linkage, gene ontology, variation in gene expression, co-citation networks, and biological relevance. We identified five genes of interest that may be responsible for the observed differential colonization phenotype.
All animal studies were approved by the Institutional Animal Care and Use Committee of the Uniformed Services University of the Health Sciences and were conducted in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals . Animals were housed in filter top cages with access to food and water ad libitum unless otherwise noted, in an environmentally controlled room approved by the American Association for Accreditation of Laboratory Animal Care (AAALAC).
Female mice, approximately 5–6 weeks old were used for all experiments. BXD parental strains (B6 and D2) were purchased from The Jackson Laboratory (JAX) (Bar Harbor, Maine). We obtained some BXD strains through collaboration with investigators at the University of Cincinnati (UC) who had acquired BXD breeding pairs from the University of Tennessee Health Science Center (UTHSC) (Memphis, Tennessee) . Ten BXD strains (BXD 32, 44, 49, 51, 55, 65a, 75, 86, 96, and 98) were only analyzed from UC. Additional BXD strains were from UC or JAX. Similar colonization levels between mice from UC and JAX were confirmed with four BXD strains (BXD #: 73, 73b, 83, 87) (Additional file 1: Figure S1). Ten additional BXD strains (BXD #: 45, 48, 61, 62, 66, 69, 70, 84, 100, and 101) were tested from both UC and JAX (Additional file 1: Figure S1), while five BXD strains (BXD #: 60, 71, 73a, 99, 102) were only analyzed from the JAX colony. A minimum of two biological replicates were conducted for each BXD strain. We tested total of 31 strains, 29 BXD strains and ancestral parental strains B6 and D2, with 321 mice total (each BXD strain n = 6–20; B6 and D2 n = 48).
E. coli O157:H7 strains and growth conditions
Colonization studies with the BXD parental strains (B6 and D2) were conducted with two STEC O157:H7 strains: 86–24, an Stx2a positive clinical isolate, and TUV86-2 an Stx2a-negative isogenic mutant [43–45]. BXD colonization studies were conducted only with TUV86-2. Both STEC strains are resistant to nalidixic acid (Nal) and were grown in Luria broth supplemented with 25 μg/mL Nal. To prepare the inoculum, an overnight culture (~18 h) was pelleted by centrifugation (5000 × g), the supernatant removed, and the pellet resuspended 1:100 in phosphate buffered saline (PBS) supplemented with 20 % sucrose. The inoculum was serially diluted and plated to determine the dose/mouse.
Intact commensal flora (ICF) infection model
Colonization levels were determined in the ICF infection model as previously reported . Briefly, food and water were removed from the mice for 20 or 2 h, respectively, prior to infection. Mice were fed a high inoculum, approximately 1010 colony forming units (CFU) in 100 μL by pipette tip. Each experiment included three mice per strain, with six to seven strains total. The parental B6 and D2 strains were included in all experiments as an internal control for colonization levels. Mice were weighed daily and colonization levels were reported as CFU per g feces on day one through day four post-infection.
To determine the CFU per g feces, mice were placed in individual cages with no bedding for 30–40 min. After this time, mice were returned to their original cage and fecal pellets were collected, weighed, and resuspended 1:10 w/v in PBS. The fecal slurry was further diluted 1:10 in PBS and plated on sorbitol MacConkey (SMAC) agar supplemented with Nal to select for the inoculating strain. The dilution that contained between 30 and 300 colonies was counted to determine CFU per g feces. The limit of detection for this model is 100 CFU per g.
Data analysis and QTL mapping
We dedicated a considerable amount of time to data error checking and filtration after each experiment. Data from individual mice were flagged in the database and excluded from the final analysis if there were factors that affected colonization levels other than infection. For the final analysis we used data from 24 BXD strains and the parental mice. We performed general linear model (GLM) analysis of covariates using ordinary least-squares analysis of variance (OLS ANOVA) to determine the relative effect and interactions of covariates on the genetic factor, represented as mouse strain. GLM analysis revealed that there were no differences associated with mouse age, source, or the seasonality of the experiment (P > 0.15), Additional file 2: Table S1. Mouse strain was the most significant predictor of colonization (P <0.0001), followed by inoculum (P = 0.02). Although the differences in inoculum were statistically significant, we do not believe them to be biologically significantly, as they were within the same log for all experiments and did not result in any difference in colonization levels of the parental strains. We used the open-access web-based interval analysis on the GeneNetwork (GN) platform for complex trait analysis to identify QTLs. The primary data has been entered under trait IDs 17467, 16607, 16608, 18071, and 18072. The genome-wide interval mapping module allowed us to analyze phenotypes in the context of mouse genotypic differences and estimate the significance at each location using 5000 permutation tests . We did ten sets of analyses of the log CFU means, log CFU medians or corrected coefficient of log CFU/g feces for the following variables: 1) colonization one day post-infection; 2) colonization two days post-infection; 3) colonization 3 days post-infection; 4) colonization 4 days post-infection; 5) difference in colonization between day four and one post-infection; 6) difference in colonization between day four and two after infection; 7) difference in colonization between day four and three after infection; 8) difference in colonization between day three and one after infection; 9) difference in colonization between day three and two after infection; and, 10) difference in colonization between day two and one after infection, for 30 traits analyzed. In addition, we determined the overall variation in median colonization across the BXD panel over time from the linear and polynomial slopes of colonization change per strain. Therefore 32 traits overall were mapped for QTLs.
Bioinformatic analyses of mapped QTLs
Haplotype analyses of significant QTL on Chr 9
We analyzed haplotypes of the BXD strains used in this study at the significant mapped QTL on Chr 9 between 12.6 and 25.6 Mb. We downloaded the BXD genotype data set as a Microsoft excel file from http://www.genenetwork.org/genotypes/BXD.geno and selected for the Chr 9 mapped QTL region and examined the genotypes of the BXD strains from that database. The different BXD strains were rank-ordered according to colonization levels on day one post-infection.
Polymorphism analysis (SNP analysis)
We did SNP analysis on genes and transcripts in the mapped QTLs associated with STEC O157:H7 differential colonization. The analysis was done on the 24 BXD strains and the parental strains B6 and D2. SNP analysis data were retrieved on March 22, 2015 from the mouse phenome database (MPD) with the SNP wizard online tool at http://phenome.jax.org/SNP. The retrieved data originated from several databases: national center for biotechnology information (NCBI) Mouse Build 37 (known as mm9), NCBI SNP database (dbSNP) build 128 and the joint project of the European Molecular Biology Laboratory (EMBL), European Bioinformatics Institute (EBI) and the Wellcome Trust Sanger Institute, (Ensembl) build 48. SNPs among BXD strains were based on Wellcome-CTC Mouse Strain SNP Genotype Set .
We did correlation analyses of all traits associated with the differential colonization parameters described in the QTL analysis section above (12 traits) with QTL heatmap. QTL heatmap is a bioinformatics tool offered by the GeneNetwork (GN) platform in which a hierarchical cluster tree of the mapped set of traits is computed and distances between pairs of traits is calculated with the formula1-r, where r is the Pearson product–moment correlation.
Candidate gene co-citation analysis
We did co-citation analyses using open-source Chilibot platform (www.chilibot.net), a literature search engine that identifies all relevant relationships among search terms . We cross-referenced the 36 candidate polymorphic genes using key words associated with STEC O157:H7 colonization to identify genes most likely responsible for the QTL on Chr 9. The initial key words were: STEC; colonization; normal flora; colonization resistance; epithelial cell; tight junction; cell polarity; adhesion; mucus; mucin; nucleolin. The key words for the final interaction diagram, chosen because of the high degree of interconnectivity to the top genes and for clarity of the figure were: STEC; colon; mucus; colonization.
All primary calculations and sorting operations were done with features and functions of Microsoft Excel. Statistical analyses were executed with Data Desk (version 6.3) software (Data Description, Inc., Ithaca, NY; www.datadesk.com) and included correlation, regression, and general linear model (GLM) analyses by ordinary least squares analysis of variance (OLS ANOVA). Mouse age, weight, inoculum, and log CFU/g feces for each day post-infection were analyzed to determine the possible effect of those variables on the final results.
Our data sets were stored as part of the BXD published phenotypes on Gene Network Platform and can be found at (www.genenetwork.org) under BXD published phenotypes record trait IDs 17467, 16607, 16608, 18071, and 18072.
Infection with TUV86-2, but not 86–24, results in significant colonization differences between the parental murine strains
Colonization differences between parental and BXD strains infected with TUV86-2
QTL identified on proximal Chr 9 associated with TUV86-2 colonization in BXD mice
Summary of TUV86-2 colonization QTLs in ARI BXD micea
Name of mapped trait (Phenotype)
Genetic marker(s) associated with peak locus
Location of genetic markers (Mb)
Colonization one day after infection
gnf09.010.169, rs13480073, and mCV24962297
rs13482392, gnf14.114.290, rs13482396
Colonization two days after infection
rs13482396, gnf14.085.610 and rs3707842
Colonization four days after infection
gnf09.010.169, rs13480073, mCV24962297
Difference in colonization between day two and one after infection
rs6209128 and rs3023086
Difference in colonization between day three and one after infection
rs3718618 and rs3669949
rs13483513, gnf19.005.316, rs4232041 and rs4232042
Difference in colonization between day four and one after infection
rs13478413 and rs3688859
Difference in colonization between day three and two after infection
Difference in colonization between day four and two after infection
rs13475818 and UT_1_38.719268
rs3717268 and rs13482709
rs13483746, rs13483748 and rs13483736
Difference in colonization between day four and three after infection
Overall strain specific variation in pattern of colonization across BXD strains (Linear slopes of medians)
Overall strain specific variation in pattern of colonization across BXD strains (Polynomial slopes of medians)
rs13483770, gnfX.044.260, rs13483785, rs13483786
We also identified suggestive QTLs that overlapped on Chr 14 for colonization levels on days one or two post-infection with a peak LRS of 13.84 and 12.09, respectively (Table 1). We further identified multiple suggestive QTLs for the following traits: difference in colonization between two independent days post-infection [such as colonization day two minus colonization day 1 (QTL on Chr 13)], and the linear (Chr 17) and polynomial slopes of colonization change (Chr X) (Table 1).
Candidate genes analyses
Candidate genes on Chr 9
Name or description
Chr location (GRCm38)
Mastermind like 2 (Drosophila)
Meiotic recombination 11 homolog A (S. cerevisiae)
RIKEN cDNA 4931406C07 gene
TATA box binding protein (Tbp)-associated factor, RNA polymerase I, D
coiled-coil domain containing 67
RIKEN cDNA 4930540 M03 gene
FAT tumor suppressor homolog 3 (Drosophila)
N-acetylated alpha-linked acidic dipeptidase 2
Olfactory receptor 39
Collagen, type V, alpha 3
DNA methyltransferase (cytosine-5) 1
Phosphodiesterase 4A, cAMP specific
Interleukin enhancer binding factor 3
Coactivator-associated arginine methyltransferase 1
Dedicator of cytokinesis 6
Predicted gene 6484
Zinc finger protein 599
Zinc finger protein 810
Bardet-Biedl syndrome 9 (human)
BMP-binding endothelial regulator
Neuropeptide S receptor 1
dpy-19-like 1 (C. elegans)
dpy-19-like 2 (C. elegans)
Endonuclease/exonuclease/phosphatase family domain containing 1
Predicted gene 1110
Acyl-Coenzyme A dehydrogenase family, member 8
Non-SMC condensin II complex, subunit D3
Opioid binding protein/cell adhesion molecule-like
The major finding from this study was the identification of a significant QTL on proximal Chr 9 associated with TUV86-2 colonization levels in BXD mice one day post-infection. The identification of this QTL supported our hypothesis that host genetics affect STEC O157:H7 colonization levels in mice. Since establishment of infection is critical for comparison of colonization levels across multiple experiments, we included the BXD parental strains in every experiment as an internal control. Because the B6 and D2 day one colonization levels were consistently within the expected range (106–108), we are confident that the variation in BXD colonization levels is due to genotypic differences among the strains. The variation in colonization levels across BXD strains is consistent with the mosaic genotypes of the panel. We predict that such genetic predispositions to low or high initial colonization levels could influence the severity of disease from an STEC infection. The mosaic-like genetic complexity of the ARI BXD panel provided the diversity required to map the QTL and would allow us to predict if an individual animal would be susceptible or relatively resistant to O157:H7 colonization.
We initially determined the colonization profiles of the parental murine strains after infection with both 86–24, an Stx2a-positive strain, and TUV86-2, an Stx2a-negative isogenic strain. However, since there was no difference in colonization levels of the parental strains after 86–24 infection, we decided to only infect the BXD strains with TUV86-2 to have the greatest chance of identifying a QTL associated with colonization. The reason that we did not observe a difference in colonization levels after infection by 86–24 is most likely due to the fact that Stx2a enhances STEC colonization levels in a traditional mouse model . The decision to proceed with the toxin-negative strain enabled us to identify host genetic markers that may be associated with O157:H7 colonization. Future studies could determine whether the toxin positive strain would overcome the low colonization phenotype associated with the “D” allele at the Chr 9 QTL.
Suggestive QTLs linked to colonization levels on days two and four post-infection were also mapped to Chr 9 in the same region as the significant day one colonization QTL. In addition, an LRS that approached a suggestive QTL in the same Chr 9 region was mapped for colonization levels on day three post infection (data not shown), and the QTL heatmap for day three colonization also indicates B haplotype (blue color) dominance (Additional file 1: Figure S1). It is possible that the addition of more strains to the panel would strengthen the suggestive QTLs for colonization on days 3 and 4 post infection, time points for which days the parental mice also showed a difference in colonization. We believe that the overlap of multiple QTLs in one location bolsters the likelihood that this region of Chr 9 is tightly linked to colonization capacity. However, it is possible that different host factors are responsible for variations in colonization on day one compared to colonization persistence as measured on days 2 and 4. The additional suggestive QTLs identified in Table 1 linked to multiple traits illustrate the complexity of host genetic factors that respond to the presence of a bacterial pathogen. The QTLs associated with the differences in colonization between the days after infection are likely related to colonization persistence. That multiple QTLs on different Chrs are implicated in the persistence phenotype suggests that genes located across QTLs may be connected through similar pathways. This is especially evident by the three suggestive QTLs linked to the difference in colonization between day 3 and day 1 on Chrs 7, 18, 19.
Haplotype analysis in the region of Chr 9 that contains the significant QTL revealed that the D allele is associated with low colonization levels of BXD strains, a finding that contrasts with the colonization data from the parental strains in which the D allele was associated with high, sustained colonization. When there is such a reversal of SDP the QTL is referred to as a “cryptic” QTL, and is likely a reflection of the fact that the BXD panel was derived from the F2 progeny of the initial D2 x B6 cross . It may be that the D2 have other factors that allow high colonization levels that mask the cryptic QTL on Chr 9. Future studies to test that latter hypothesis would determine the colonization levels of additional BXD strains that are “D” at the Chr 9 QTL to try to map a host factor(s) that allows high colonization in DBA but not the progeny.
We focused our QTL analysis on the significant QTL on proximal Chr 9. The QTL is located in a chromosomal region with limited SNPs. We chose the 36 candidate genes based on the highest number of polymorphisms, because such polymorphisms are more likely to be responsible for the QTL. We used Chilibot to compare scientific literature that included one or more of the 36 genes and key words associated with STEC colonization in the colon. Fig. 6 represents the final interconnectivity plot of the five genes most likely to be linked to the QTL: Acad8; Bmper; Pdea; Panx1; Dnmt1. All five of the genes selected are expressed in the colon of humans and mice. Acad8 is a member of the acyl-Coenzyme A dehydrogenase family and necessary for butyrate oxidation . Butyrate, a short chain fatty acid, is an important source of energy for colonic enterocytes [50, 51] and helps to maintain intestinal epithelial cell physiology . Defects in butyrate oxidation are linked to mucosal inflammation and ulcerative colitis . Additionally, butyrate increases STEC adherence to CaCo2 cells  and increases the concentration of the Stx receptor, Gb3, on intestinal epithelial cells . Polymorphisms of Acad8 may drive the QTL on Chr 9 by affecting the health of colonic enterocytes, which in turn promote or inhibit colonization.
Bmper, Pde4a, Panx1, and Dnmt1 are four genes that modulate inflammation of colonic enterocytes. Bmper is a BMP-binding endothelial regulator . It limits endothelial inflammation by inhibiting expression of intercellular adhesion molecule 1 (ICAM1) [56, 57] and by regulating leukocyte extravasation and adhesion . Pde4a is a cAMP-specific phosphodiesterase 4A that participates in multiple signal transduction pathways, such as platelet aggregation and immune cell activation . Use of Pde4a-specific inhibitors has anti-inflammatory effects, such as reduced neutrophil adhesion  and inhibition of cellular trafficking and microvascular leakage . Additionally, Pde4 inhibitors have been proposed to prevent STEC-mediated brain damage . Panx1 is part of the innexin family, and as such a structural component of gap junctions  Panx1 is responsible for the release of ATP to the extracellular space, which can initiate cellular migration and inflammation . Additionally, ATP release can modulate mucus secretion  which may impact colonization. Dnmt1 is a DNA methyltransferase responsible for the establishment and regulation of tissue specific methylated cytosine residues . Since Dnmt1 activity affects global methylation patterns, variation in expression can change epithelial cell morphology . Finally, Dnmt1 levels are elevated in response to UPEC infection . Variation of inflammation levels could affect initial colonization, while the effect on leukocyte transit could impact colonization persistence. Further studies are needed to confirm or refute the actual involvement of one or more of the identified genes. The long term goal of this project is to correlate the genes identified within the murine QTL(s) with human host factor(s) responsible for the variable disease states observed during STEC O157:H7 outbreaks.
We identified a QTL associated with colonization 1 day post-infection by O157:H7 on murine Chr 9. The identification of this QTL suggests that host genetics affect STEC O157:H7 colonization levels in mice
We thank Stephen Darnell for assistance with animal work.
The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Department of Defense, the Uniformed Services University of the Health Sciences, or the National Institutes of Health.
This work was supported by Uniformed Services University Intramural Grant R073RV to ADO, National Institutes of Health grant R37 AI020148 to ADO, and University of Cincinnati College of Medicine & The Cincinnati VA Medical Center Merit Award to MK.
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