Dietary choice affects Shiga toxin-producing Escherichia coli (STEC) O157:H7 colonization and disease

Steven D. Zumbrun, Angela R. Melton-Celsa, Mark A. Smith, Jeremy J. Gilbreath, D. Scott Merrell, and Alison D. O’Brien1

Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814-4799

Edited by Rino Rappuoli, Novartis Vaccines and Diagnostics, Siena, Italy, and approved April 29, 2013 (received for review January 2, 2013) The likelihood that a single individual infected with the Shiga toxin One aspect of STEC pathogenesis that is not clear is why a sig- (Stx)-producing, food-borne pathogen Escherichia coli O157:H7 will nificant disparity in age and sometimes sex exists among those with develop a life-threatening sequela called the hemolytic uremic syn- STEC-related HUS. Children <10 y old are 10 times more likely to drome is unpredictable. We reasoned that conditions that enhance develop HUS following infection with STEC (12). In two recent Stx binding and uptake within the gut after E. coli O157:H7 infection large outbreaks, women developed HUS in disproportionate should result in greater disease severity. Because the receptor for numbers (9, 13). Studies that address why children are more likely Stx, globotriaosylceramide, is up-regulated in the presence of buty- than adults to develop HUS suggest that a difference in comple- rate in vitro, we asked whether a high fiber diet (HFD) that report- ment activation (14, 15), platelet activation (16), or in nitric oxide edly enhances butyrate production by normal gut flora can influence production (17) may explain the disparity. the outcome of an E. coli O157 infection in mice. To address that The Stx produced by STEC is the critical virulence factor that question, groups of BALB/c mice were fed high (10%) or low (2%) not only may contribute to intestinal epithelia damage (18, 19) but fiber diets and infected with E. coli O157:H7 strain 86-24 (Stx2+). is required for the development of HUS (20). STEC may produce Mice fed an HFD exhibited a 10- to 100-fold increase in colonization, Stx1, Stx2, or both toxins. Stx1 and Stx2 are highly related in both lost 15% more body weight, exhibited signs of morbidity, and had structure and function but cannot be neutralized by heterologous 25% greater mortality relative to the low fiber diet (LFD)-fed group. antisera. Although STEC that produce either Stx1 or Stx2 may Additionally, sections of intestinal tissue from HFD-fed mice bound cause severe disease in people, strains that make Stx2 are more more Stx1 and expressed more globotriaosylceramide than did such likely to cause HUS (21). However, it is not clear how Stx gains sections from LFD-fed mice. Furthermore, the of HFD- access to the bloodstream from the intestinal lumen. Neverthe- fed mice compared with LFD-fed mice contained reduced levels of less, the preponderance of evidence indicates that Stx targets native Escherichia , organisms that might protect the gut small vessel endothelial cells, and that toxin tropism, either di- from colonization by incoming E. coli O157:H7. Taken together, rectly or indirectly, leads to HUS (22). these results suggest that susceptibility to infection and subsequent Cell-surface expression of the Stx receptor, globotriaosylceramide E. coli disease after ingestion of O157:H7 may depend, at least in (Gb3), is required for cytotoxicity in vitro (23), and, in some fl part, on individual diet and/or the capacity of the commensal ora to models of STEC infection, Stx damages the intestinal epithelia produce butyrate. (18, 19). Historically, it was presumed that Gb3 is not present on the gut epithelia (24, 25), and, thus, the mechanism by which the tubular necrosis | HCT-8 | microbiome toxin might gain systemic access was unclear. However, contrary

higa toxin (Stx)-producing Escherichia coli (STEC) infections Significance Scontinue to be a significant health burden in the United – States. There are typically 15 20 outbreaks of STEC in the We demonstrated that dietary fiber content affects suscepti- United States per year (1) that result in 265,000 illnesses, 3,600 bility to Shiga toxin (Stx)-producing Escherichia coli (STEC) in- hospitalizations, and 30 deaths annually (2). The United States fection in mice. We showed that high fiber diet (HFD)-fed mice Department of Agriculture estimates a total cost of about $500 had elevated levels of butyrate, a beneficial gut metabolite million per year in health-related costs in the United States due that paradoxically enhances the cell-killing capacity of Stx. We to STEC infection (3). also found that the amount of gut in HFD-fed mice STEC, such as the most frequently isolated serotype in the increased whereas the percent of commensal Escherichia spe- United States, E. coli O157:H7 (1), are gut commensals of cattle. cies (spp) decreased compared with animals fed a low fiber diet E. coli However, in , E. coli O157:H7 can cause diarrhea and (LFD). These changes led to higher O157:H7 colonization hemorrhagic colitis after ingestion of as few as <50–300 organisms levels, more weight loss, and greater rates of death in HFD-fed than in LFD-fed STEC-infected animals. (4, 5). Such infections typically occur after individuals consume

E. coli O157:H7-contaminated beef, fresh vegetables, water, or This work was presented in part as a poster at the 8th International Symposium on Shiga unpasteurized juice (6). On occasion, E. coli O157:H7 infections Toxin (Verocytotoxin)-Producing Escherichia coli Infections, May 2012, Amsterdam, The result from person-to-person spread of this low-infectious dose Netherlands. Abstract P-140. pathogen (6). The usual disease progression is as follows: 3 d after Author contributions: S.D.Z., A.R.M.-C., and D.S.M. designed research; S.D.Z. performed research; J.J.G. contributed new reagents/analytic tools; S.D.Z., A.R.M.-C., M.A.S., J.J.G., the ingestion of contaminated food or water, diarrhea, abdominal D.S.M., and A.D.O. analyzed data; and S.D.Z., A.R.M.-C., and A.D.O. wrote the paper. – pain, and begin; frank, bloody diarrhea follows 2 3dlater. The authors declare no conflict of interest. The bloody diarrhea generally resolves after 4 or 5 d. However, in This article is a PNAS Direct Submission. – – 4 30% (7 10) of patients, the potentially life-threatening, hemo- Freely available online through the PNAS open access option. lytic uremic syndrome (HUS) develops (6). HUS is characterized 1To whom correspondence should be addressed. E-mail: [email protected]. by acute kidney malfunction, microangiopathic hemolytic anemia, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. and thrombocytopenia (11). 1073/pnas.1222014110/-/DCSupplemental.

E2126–E2133 | PNAS | Published online May 20, 2013 www.pnas.org/cgi/doi/10.1073/pnas.1222014110 Downloaded by guest on October 2, 2021 to this perception, we recently demonstrated that small amounts butyrate treatment; however, within 4 h of butyrate removal, PNAS PLUS of Gb3 synthase mRNA and Gb3 are in fact found in cell-surface Gb3 levels returned to basal levels (Fig. 1C). The intestinal tissue (26). rapid responsiveness of cell-surface Gb3 to butyrate suggests In this study, we hypothesized that Stx uptake from the gut, as a dynamic temporal fluctuation in Gb3 that varies with local well as the degree of kidney damage (and thus susceptibility to butyrate concentration. HUS) following infection might, in part, be mediated by diet. Specifically, we theorized that a diet or an intestinal commensal Diet-Mediated Increase in Influences Gb3 Expression in population that promoted the production of butyric acid in the Vivo. We next asked whether increased butyrate levels would gut would increase susceptibility to STEC infection. Butyric acid enhance Gb3 expression in the mouse gut. To modulate the in- is normally a beneficial catabolite of intestinal fiber testinal butyrate level in mice, we fed either an HFD or an LFD that promotes cellular health (27) and acts as the primary energy to BALB/c mice. We used guar gum as the fiber source in the two source for colonic enterocytes (28, 29). Nevertheless, when cul- diets (Table S1) because it is highly fermentable in the gut (34). tured cells are treated with butyric acid, the surface expression of Mice that ate either diet appeared equally healthy (Fig. S1). We Gb3 increases, which, in turn, leads to enhanced sensitivity of collected stool and cecal contents from HFD- or LFD-fed mice those cells to the cytotoxin Stx (30–33). To test our diet hy- and quantitated the butyrate levels in the samples by gas chro- pothesis, we fed mice a high fiber diet (HFD), which in turn, matography and mass spectrometry. Stool and cecal contents increased intestinal butyrate levels, and then asked whether the from HFD-fed mice consistently contained more butyrate than response of those animals to E. coli O157:H7 infection differed similar samples from LFD-fed mice (Fig. 2A). These data are from mice fed a low fiber diet (LFD). We found that an HFD consistent with findings from human subjects (35, 36). Addi- increased Stx binding to colonocytes and renal tissues of mice, tionally, because stool from mice fed either diet contained less reduced the level of commensal E. coli, and increased the sus- butyrate than the cecal contents (Fig. 2A), we hypothesized that ceptibility of mice to E. coli O157:H7 infection and disease. some butyrate was absorbed systemically, a possibility supported by studies done in pigs (37). Furthermore, we detected more Gb3 Results on intestinal tissue from HFD-fed mice than on similar samples Influence of Butyric Acid on Stx Receptor on Human Colonic Epithelial from mice on a typical 4% fiber diet (Fig. 2B). Because butyric Cells. To confirm that butyrate enhances Gb3 levels on colonic acid enhances Gb3 expression on human proximal tubule (30) epithelial cells, we grew human colonic epithelial (HCT-8) cells and glomerular epithelial cells (31) in vitro, and it was likely that in media with or without and assayed for cell- butyrate was absorbed systemically in HFD-fed mice, we asked surface Gb3 by flow cytometry. Gb3 levels on butyrate-exposed whether those mice had greater kidney Gb3 staining than did HCT-8 cells increased 10-fold (Fig. 1A). In addition, HCT-8 cells LFD-fed animals. Although sections of kidney from HFD-mice maintained in butyrate exhibited 1,000-fold higher sensitivity to exhibited similar patterns of Gb3 staining compared with those Stx1 intoxication compared with control cells (Fig. 1B). This en- from LFD-mice (Fig. 2C), there were numerous areas of in- hancement of Gb3 levels on HCT-8 cells occurred within 24 h of creased intensity of Stx binding in tissues of HFD-fed mice (Fig. MICROBIOLOGY

Fig. 1. Butyric acid-enhanced Gb3 expression on human colonic epithelial cells. (A) Flow cytometric analysis of HCT-8 cells treated with 5 μM sodium butyrate for three passages and incubated with a monoclonal antibody against Gb3. The horizontal line indicates the gating window used to quantify Gb3-positive cells. (B) Viability of PBS- or butyrate-treated HCT-8 cells after exposure to Stx1. (C) Cell-surface Gb3 (green) detected on HCT-8 cells 2 or 4 h after cells were washed and butyrate thus removed. (Magnification: 400×.) Evan’s Blue (0.01%) was used as a counterstain (red). Arrow identifies Gb3 expression in untreated HCT-8 cells. Control cells were treated with PBS only.

Zumbrun et al. PNAS | Published online May 20, 2013 | E2127 Downloaded by guest on October 2, 2021 Fig. 2. An HFD increased intestinal butyrate and Gb3 expression in mice. (A) Quantity of butyrate in stool and cecal contents of HFD- or LFD-fed mice. (B) Gb3 expression on intestinal tissue from mice fed an HFD (detected with anti-GB3 antibody) at 100× magnification and (C) on kidney sections (detected with an Stx1 probe). Arrows indicate regions of increased signal intensity. (Magnification: 100×.) (D) Quantitation of fluorescence intensity from C by ImageJ software (65). Error bars represent the standard error of the mean (SEM).

2C, arrows). The increase in Gb3 expression on kidney tissue of luminal milieu of metabolites; these variables include age (38), mice fed an HFD was statistically significant, as determined by disease state (39), and diet (38, 40). To investigate the potential fluorescence intensity quantitation (Fig. 2D). role of the intestinal microbiota on the susceptibility of the HFD- fed mice to E. coli O157:H7, we determined the gut microbiota Diet Alters Susceptibility of Mice to Infection with E. coli O157:H7. To composition in mice fed a typical 4% fiber diet (day 0) and then determine the effect of an HFD on susceptibility to STEC in- switched to either an HFD (10% fiber) or LFD (2% fiber) for 14 d. fection, we fed two groups of 10 mice each either an HFD or LFD 11 We found that mice on an HFD had reduced numbers of com- for 2 wk and then infected them with 10 cfu E. coli O157:H7. mensal Escherichia_Shigella (hereafter Escherichia) spp compared Some of the infected mice in the HFD group consistently showed with LFD-fed mice on day 14 post diet change (Fig. 4A, Fig. S4), fl signs of greater morbidity, such as lethargy, ruf ed fur, and weight a finding consistent with studies in humans (35, 36) and piglets loss by day 7 (Fig. 3A). In most cases, weight loss that was greater (41). This reduction in Escherichia spp suggests that an HFD than 20% of body weight led to death (Fig. 3B). Over the course may create an available niche for the O157:H7 to colonize, de- of four independent infection experiments, there were 12/36 spite the overall similarity between the gut communities in the (33%) deaths in the HFD group and 4/34 (12%) in the LFD mice. LFD and HFD groups at day 14 (Fig. S5). Despite the reduction Additionally, mice that consumed an HFD maintained greater in Escherichia spp in HFD-fed mice, overall numbers of intestinal levels of E. coli O157:H7 than did the LFD-fed animals (Fig. 3C). commensals appeared to be increased compared with LFD ani- We examined sections of the intestines and kidneys from infected mals as shown by an increased quantity of bacterial DNA in the HFD-fed mice that lost >20% body weight (Fig. S2) for the B presence of lesions and compared those to samples from LFD-fed stool (Fig. 4 ) and by more bacteria in the lumen of the colon of mice that had been infected for the same amount of time (Fig. 3D, H&E-stained tissue sections from most animals (Fig. 4C). Thus, Fig. S2). Neither group of mice had intestinal lesions; however, an HFD may create an intestinal environment that provides an kidneys from the HFD-fed mice displayed moderate acute tu- advantage for STEC to colonize and cause disease. bular necrosis (Fig. 3D, multiple arrows) whereas those fed the Discussion LFD had only mild lesions (see single arrow). Here, we demonstrated in vivo a phenomenon that was previously To determine whether the greater susceptibility of the HFD fi mice to E. coli O157:H7 was specific to the presence of Stx2, we shown only in tissue culture. Speci cally, we found that butyric infected mice on an HFD or LFD with either the wild-type 86-24 acid, generated in the gut because of an HFD, increased the or an isogenic toxin minus mutant and monitored the animals for expression of the Stx receptor, Gb3, on the colonic epithelium clinical signs of disease. We found that whereas 7/8 HFD and 3/8 and in renal tissues. We further established that the butyric acid- LFD mice infected with 86-24 died, no mice lost weight (Fig. S3) mediated increase in Gb3 levels was temporally dynamic. The − or succumbed to infection with the Stx2 86-24 regardless of the amount of Gb3 was reduced to prestimulation levels within hours diet they were fed. of the withdrawal of butyric acid. Finally, we showed in a mouse model that not only did the HFD result in increased intestinal Diet Influences the Composition of the Intestinal Microbiota. Multi- butyrate levels, but it also led to a reduction in the resident ple factors influence the microbiota of the intestine and thus the Escherichia spp. The HFD-fed mice exhibited greater coloniza-

E2128 | www.pnas.org/cgi/doi/10.1073/pnas.1222014110 Zumbrun et al. Downloaded by guest on October 2, 2021 PNAS PLUS

Fig. 3. Mice fed an HFD are more susceptible to infection with E. coli O157:H7. (A) Average weight loss in HFD-fed (triangles) and LFD-fed (squares) mice infected with STEC (n = 10). Error bars indicate SEM. *P < 0.001, **P < 0.01, ***P < 0.05. (B) Mortality of STEC-infected HFD-fed (triangles) or LFD-fed (squares) mice (n = 10). (C) Geometric mean of colonization by O157:H7 in HFD-fed (triangles) and LFD-fed (squares) mice (n = 10). Error bars indicate 95% confidence interval. *P < 0.001. (D) Intestinal and kidney tissue sections stained with hematoxylin-eosin (H&E) from infected mice. (Magnification:100×.) Arrows indicate areas of acute tubular necrosis.

tion, morbidity, and mortality after STEC infection than did mice That butyric acid and an HFD have a negative effect on the on an LFD. Furthermore, the greater susceptibility to the HFD host with respect to STEC infection is contrary to their essential was related specifically to the presence of Stx2. In all, we dem- role in normal colonic health (44, 45). Butyrate accounts for up onstrate here that an HFD can modulate Stx receptor levels, to 70% of the energy source for colonic enterocytes (46, 47). It which, in turn, can exacerbate the outcome of STEC infection. also is important in maintaining normal physiology and mor- Our finding that Gb3 levels were temporally dynamic in re- phology of the epithelia in the gut (28) and has been proposed sponse to butyrate suggests that Gb3 expression on tissues may for use as a in cases of chronic colitis (48). Further- fluctuate with diet and within microenvironments of the tissue as more, there is a link between reduced gut butyrate a function of local butyrate exposure. This latter hypothesis may and colitis in mice (49) and humans (28, 44). Finally, at least two explain the lack of Gb3 detection on colonic tissues in previous studies show beneficial effects of butyrate during infection with studies (24, 25, 42) in which butyrate may have been incidentally Salmonella (50, 51). In contrast, this study suggests that butyrate removed during bowel preparation for surgery. Mechanical can be detrimental to the host under certain conditions. bowel preparation with one or more types of laxative up to 24 h From our results, we cannot rule out that butyric acid sensi- before surgery is the standard procedure for patients who un- tized tissues to Stx by means other than enhancement of Gb3

dergo elective colorectal procedures (43). In addition to the expression. It is clear that butyric acid has a profound effect on MICROBIOLOGY enhanced expression of Gb3 on intestinal tissues of HFD-fed cellular physiology (29) and on the cellular transcriptional pro- mice, we also noted increased Gb3-staining intensity in renal gram (27). Butyric acid also affects cellular sensitivity to another tissues from those same animals. The higher levels of Gb3 in the ribotoxin, ricin (52), which does not use Gb3 as a receptor (53). kidneys of HFD-fed mice was presumably a consequence of in- Additionally, Lingwood et al. showed that butyric acid may alter creased systemic levels of butyrate taken up from the gut. Be- the trafficking of Stx in cells to promote the interaction of toxin cause the pattern of renal Gb3 expression in mice fed an HFD with its cellular target, the endoplasmic reticulum (54). Fur- was similar to that in the kidneys of LFD-fed mice, we propose thermore, it is possible that butyrate has a direct effect on the that the butyrogenic effect on Gb3 expression occurs specifically bacterium itself. We did not explore this possibility although one in those cells that have the capacity to express Gb3. group showed that including butyrate in the growth medium of

Zumbrun et al. PNAS | Published online May 20, 2013 | E2129 Downloaded by guest on October 2, 2021 Fig. 4. An HFD alters the intestinal commensal composition. (A) Relative number of Escherichia operational taxonomic units (OTU) in stool of mice on an HFD or LFD as determined by 16S-based profiling of bacteria present in fecal pellets. (B) Total DNA (isolated to enrich specifically for bacteria DNA) present in fecal pellets of mice on different diets. (C) H&E stain (40×) of a cross-section of intestine from mice fed an LFD or an HFD. An Inset in the HFD-fed intestinal section is shown adjacent to that section at 1,000× with the luminal bacteria in blue.

an STEC culture resulted in increased adherence and micro- after absorption into the blood stream from the gut, on kidney colony formation on CaCo2 cells (55). cells. Such higher colonic and renal Gb3 levels may enhance It is also possible that other unidentified factors of an HFD, in systemic transport of Stx from the gut and increase Stx sensitivity addition to butyrate production, contribute to enhanced sus- of kidney cells. Second, an HFD causes a reduction in native ceptibility to STEC infection. However, in stool samples analyzed Escherichia spp, which, in turn may promote enhanced colonization from mice on an LFD or an HFD, the other major small chain by incoming pathogenic STEC. Our model is consistent with metabolites produced in the gut, and a study in cattle in which a higher fiber grain diet in cattle led to , were at similarly low concentrations. greater colonization by STEC (57). We were surprised to find a mostly analogous microbiota In summary, our model addresses two unresolved facets of composition in the guts of HFD- and LFD-fed mice (Fig. S5). A STEC pathogenesis: (i) the mechanism of Stx uptake in the gut; significant difference in the two groups, however, was the finding and (ii) the disparity in HUS cases following STEC infection. It is of higher numbers of commensal Escherichia spp in the LFD-fed clear that intestinal ecology and diet likely result in micro- group, despite an overall reduction in gut commensal bacteria in environments with variable levels of butyrate and Gb3. Therefore, this group. A similar decrease in Escherichia spp has been ob- individual hosts likely have different Gb3 levels on and within their served in humans (36) and piglets (41) that consumed an HFD. gastrointestinal tracts, and those variations in receptor quantities However, we consistently observed higher colonization by E. coli could result in disparities in the amount of Stx delivered systemi- O157:H7 in the HFD-fed mice than the LFD-fed mice throughout cally. Higher levels of Stx in the host presumably lead to an in- the course of infection. We postulate that STEC colonize to creased risk of HUS. Besides diet, host age is a factor that a higher degree in HFD- than LFD-fed mice in three ways. First, influences microbiota composition (39). In addition, in rabbits because of lower levels of commensal Escherichia spp in the there is an age-related change in the expression of Gb3 in tissues HFD gut, there may be reduced competition against pathogenic (58). Perhaps an age-dependent change in the host microbiota Escherichia spp. Second, the overall lower quantity of commensal alters the butyrate-producing potential in the gut, which then bacteria in the LFD mice suggests that an HFD is more favorable influences Gb3 levels. Such differences in microbiota, and thus for colonization in general. Third, given that Stx enhances in- alterations in butyrate and Gb3, could explain some of the stark testinal colonization during STEC infection (56), the increase in differences in HUS susceptibility following infection in children intestinal Gb3 expression due to the HFD may promote coloni- <10 y old compared with adults. zation as well. Taken together, our results suggest that an HFD may pose Materials and Methods a two-pronged risk for those exposed to STEC such as E. coli Cell Culture. HCT-8 cells were obtained from ATCC and grown in RPMI-1640 O157:H7. First, butyrate produced under high fiber conditions media (ATCC) supplemented with 10% (vol/vol) heat-inactivated fetal bovine leads to enhanced Gb3 levels on the intestinal cell surface and, serum (FBS, Life Technologies). Vero cells (ATCC) were maintained in eagle’s

E2130 | www.pnas.org/cgi/doi/10.1073/pnas.1222014110 Zumbrun et al. Downloaded by guest on October 2, 2021 minimal essential medium (EMEM, Cell Gro) supplemented with 10% (vol/vol) Harvesting and Sectioning of Mouse Intestines and Kidneys. Intestinal sections PNAS PLUS heat-inactivated FBS and supplemented with 10 mM L-glutamine (Lonza), 10,000 were immediately removed from mice following death and either submerged units/10,000 μg/mL penicillin/streptomycin (Life Technologies), and 50 mg/mL in formalin to fix the tissue or embedded and frozen in Tissue-Tek Optimum gentamicin (ATCC). Cells were passaged at ∼80% confluency every 3–4d. Cutting Temperature (OCT) compound (Sakura Finetek) unfixed. For histo- pathological evaluation of tissue sections, hematoxylin-stained fixed sec- Antibodies. To detect Gb3 in tissue sections and on cells in culture, we used tions were deemed optimal for analysis. To minimize background staining a rat monoclonal IgM to human CD77 (Gb3), clone [38-13], (Genetex). The for detection of Gb3 on tissues, unfixed frozen tissues were found to be cognate secondary antibody used to detect bound Gb3 antibody was goat superior to fixed tissue. anti-rat IgM Alexa Fluor 488 (Life Technologies). To detect Stx1 on tissues and on cells, we used a rabbit polyclonal antibody developed in our laboratory Immunofluorescence of Stained Tissues and Cells. Immunostaining of in- (59). The cognate secondary antibody used to detect antibody against Stx1 testinal tissue sections or HCT-8 cells for Gb3 was done as follows. An ∼2-cm or Stx2 was goat anti-rabbit IgG Alexa Fluor 488 (Life Technologies). Sodium length of intestine distal to the cecum was removed from a mouse that had butyrate was purchased from Sigma-Aldrich. Guar gum used in custom fiber been killed. The luminal contents of that tissue piece were gently flushed diets was purchased from Sigma-Aldrich. out with PBS contained in a syringe fitted with a pipet tip. The intestinal segment was cut longitudinally with scissors to expose the lumen. Tissue was Flow Cytometric Analysis. HCT-8 cells maintained in RPMI-1640 (ATCC) and positioned on semisolid OCT medium (Sakura Finetek) in a sectioning boat 5 μM butyrate (Sigma-Aldrich) and grown in six-well dishes to ∼80% con- and covered in liquid OCT, then frozen on dry ice. From the tissue block, 6- fluency were washed with PBS and then detached from the plate with 0.5 mL μm sections of tissue were sliced onto positively charged Superfrost Plus of 0.05% trypsin/EDTA (Cell Gro) for 5 min at 37 °C. Cell clumps were then slides (Fisher Scientific), and slides were kept frozen on dry ice. Slides that removed gently with a 25G needle/syringe followed by transfer through were to be stained were thawed and fixed for 5–10 min in cold a 70-μm nylon filter. Cells were transferred to a 96-well v-bottom poly- (−20 °C). Excess acetone was removed, and the specimen was encircled in propylene plate (Becton Dickinson), and the plate was subjected to centri- a hydrophobic barrier with a Papanicolaou (PAP) pen (Polysciences). To fugation at 400 × g at 4 °C for 4 min. Primary anti-Gb3 rat monoclonal prepare HCT-8 cells for staining, cells were grown in wells of eight-chamber antibody (1:50 in 3% BSA/PBS; Matreya) was added to the cells, and the plate slides. The slides were then washed with PBS and fixed in acetone (−20 °C) was incubated at 4 °C for 1 h. The cells were then washed with PBS, and for 10 min. The slides were then allowed to air dry. Slides with fixed in- secondary goat-anti-rat AF488 (1:1,000 in 3% BSA/PBS; Life Technologies) was testinal sections or HCT-8 cells were then incubated for 30 min with 3% BSA added. The plate was then incubated at 4 °C in the dark. Following a final PBS in PBS as a blocking reagent, washed in PBS, incubated with an anti-Gb3 rat wash of the cells, they were resuspended in 400 μL of PBS and immediately monoclonal antibody (Matreya), diluted 1:50 in 3% BSA/PBS for 1 h, and analyzed with an LSR II Flow Cytometer (Becton Dickinson). then washed again with PBS. Goat anti-rat AF488 (Life Technologies) sec- ondary antibody diluted in 3% BSA/PBS to 1:1,000 was then added to the Purification of Stx. Stx1 was purified as previously described (60) by a single- slides for 1 h, and then slides were washed in PBS. To counterstain HCT-8 step immunoaffinity procedure. cells on the slides, 0.01% Evan’s Blue dye in PBS was added to the slides for 30 s followed by a final wash with PBS. Intestinal tissue sections on slides Stx Cytotoxicity Assay. A cytotoxicity assay modified from Gentry and were mounted with anti-fade glycerol solution (Life Technologies) and ob- Dalrymple (61) was carried out on HCT-8 cells in the presence or absence served with an Olympus BX60 fluorescence microscope. of sodium butyrate. HCT-8 cells were seeded at 1 × 104 per well of a 96-well For immunostaining of mouse kidney sections, the presence of Gb3 was fi plate (Corning) and grown for 48 h at 37 °C in 5% CO2. The cells were then detected indirectly with Stx1 as a probe of un xed frozen tissue sections by exposed to 10-fold serial dilutions of a stock of Stx1 at 25 ng/mL, prepared in a previously described method (64). Briefly, kidneys were removed from mice RPMI-1640 tissue culture medium that contained 10% FBS for an additional and immediately frozen in liquid OCT. Sections (6 μm) prepared on positively 48 h of incubation. Control cells were given medium alone. Cells were then charged slides were air dried overnight at room temperature to fix the tis- fixed in Formaldefresh (Fisher Scientific) and stained with 1.3% crystal violet sue. Tissue sections were then blocked for 30 min in 1% normal goat serum (Sigma-Aldrich) in 5% (Fisher Scientific). The optical density (OD) at (NGS) in PBS. Excess NGS was removed, and 200 μL of Stx1 (200 ng/mL) was 630 nm of fixed, stained cells in each well on a plate was determined in added, followed by incubation of the slide at room temperature for 1 h. The μ a microplate reader. The average OD630 of three replicates for each sample slide was then washed with PBS, and 200 L of rabbit polyclonal anti-Stx1 was calculated. The butyrate-treated group and the control group were each (59) diluted to 1:50 in 1% NGS/PBS was added for 1 h. The kidney section on

independently normalized to the OD630 value of the Stx1-negative samples. the slide was washed again with PBS, and secondary goat anti-rabbit AF488 diluted to 1:500 in 1% NGS/PBS was added to the slide for 1 h. The tissue Animal Studies. All animal work was approved by the Institutional Animal Care section was then subjected to a final PBS wash and mounted and observed and Use Committee at the Uniformed Services University. An intact-commensal as described above. flora model (62) in female BALB/c mice (Charles River) was adapted for these studies. Mice were weighed and then introduced to one of two custom diets: Quantitation of Immunofluorescence. Image J software (65) was used to an HFD (10% guar gum) or an LFD (2% guar gum) (Harlan Laboratories). quantitate fluorescence intensity. Ten random positively stained kidney Control animals were maintained on the standard diet (Teklad Global Protein tubules (Fig. 2C) from each tissue were quantitated by calculating the ratio 18% with 4% soluble fiber; Harlan Laboratories). The specifications for each of fluorescence intensity to the area detected. custom diet are listed in Table S1. Mice were acclimated to each diet for 2 wk and then infected by gavage with 109 to 1011 cfu of E. coli strain O157:H7, Butyrate Extraction and Gas Chromatography/Mass Spectroscopy. The butyrate isolate 86-24 (Stx2+) that was originally obtained from Phil Tarr (Washington extraction procedure was adapted from the method of Richardson et al. (66). University, St. Louis, MO). Alternatively, some mice were infected with an Fecal pellets or cecal contents were collected from mice, weighed, and sus- fi isogenic stx2 mutant of 86-24 (TUV86-2) provided to us by A. Donahue-Rolfe pended in 2 mL of PBS in glass vials (Fisher Scienti c) with screw caps. The (63). To prepare the inoculum, the bacteria were cultured in LB media over- vials were immediately stored at −20 °C. To extract butyrate from the night at 37 °C with shaking at 225 rpm and then pelleted by centrifugation for samples, the vials were thawed at room temperature, and 5 mM ethyl- 10 min at 1,000 × g. The organisms were then resuspended in 1/40th volume of butyric acid (Sigma-Aldrich) in dimethyl sulfoxide (Sigma-Aldrich) was added PBS. One hundred microliters of culture was gently gavaged into each mouse. to each vial as an internal reference. The vial contents were then mixed by The day of infection was designated as day 0 in each study. After mice were shaking on a vortex for at least 2 min until the samples within them were MICROBIOLOGY infected, they were weighed daily for 2 wk. They were also monitored for homogenous. The largest solid particles were then allowed to settle in the other signs of morbidity such as ruffled fur and lethargy. To determine the vial, and a 1-mL homogenate was then transferred from each vial to a 5-mL level of colonization in the gut of infected mice, the amount of E. coli O157:H7 glass screw-cap tube (Fisher Scientific). To acidify the samples, 50 μLof5M in fecal pellets was assessed as follows. Fecal pellets from individual mice were HCl (Sigma-Aldrich) was added to each tube. Next, 2 mL of collected, weighed, and suspended 1:10 (wt/vol) in PBS. The fecal pellet/PBS (Sigma-Aldrich) was added to each tube. The tubes were then shaken on mixtures were mixed by shaking on a vortex at room temperature for 1 min, a vortex for 2 min, and then they were subjected to centrifugation for 10 min every 10 min, for 30 min. Ten-fold dilutions of the mixtures were made in at 3,000 × g. Aliquots of each resultant supernatant (1 μL) were separated PBS, and 100-μL aliquots of the homogenates were plated onto Sorbitol– on a Nukol Supelco capillary GC column; 30 m × 0.25 mm × 0.25 μm (Sigma- MacConkey agar plates. Plates were incubated overnight at 37 °C. Colony Aldrich). Helium was used as the carrier gas at 0.80 mL/min from 100 °C– counts were determined the next day. 200 °C at 8 °C/min. A mixture of standard free fatty acids (Sigma-Aldrich)

Zumbrun et al. PNAS | Published online May 20, 2013 | E2131 Downloaded by guest on October 2, 2021 was used to generate a standard curve and to identify the appropriate bp or greater than 72 bp in length as well as any sequences that contained peak by mass spectroscopy that corresponded to butyric acid. homopolymer runs of >4 bp. The resulting alignment was then filtered to remove any columns that contained missing information. Reads were pre- Immunohistochemistry. Tissues were removed, washed in PBS, and immedi- clustered so that any sequences that contained a single base pair difference ately submerged in 10% neutral buffered formalin, pH 6.8–7.2 at 25 °C were considered to be the same. Preclustered reads were classified with the (Fisher Scientific). Tissues were embedded in paraffin and sectioned at 6 μm mothur implementation of the RDP Bayesian classifier (73) using a cutoff of onto glass slides. For hematoxylin and eosin (H&E) staining, sections were 60% bootstrap support over 100 iterations. Sequences from mitochondria, deparaffinized in ethanol and stained by standard protocol. Tissues were cyanobacteria, chloroplasts, and sequences that were classified as “un- sectioned and stained by the Biomedical Instrumentation Center, Uniformed known” or “unclassified” at the phylum level were removed from the data- Services University of the Health Sciences, Bethesda, MD and by Histoserv, set. The remaining aligned sequences were used to generate a pairwise Inc., Gaithersburg, MD. distance matrix and clustered into operational taxonomic units (OTUs) using average-linkage clustering at an identity of 97%. OTUs were classified with Histopathological Analysis of Tissue Sections. To assess tissues for the presence the mothur implementation of the RDP classifier (73) as described above. To of lesions, a veterinary pathologist evaluated sections in a blinded manner. compare the intrasample diversity of each of the murine gut communities, we normalized the sequences from each sample based on the lowest number of fl Mouse Gut Microbiota Sequencing Experiment. The mice used for the gut sequences found in any of the samples. This work ow provided adequate > ’ microbiota sequencing experiment were housed individually to prevent sample coverage ( 0.97) as determined by Good s estimate (74). Rarefaction mouse-to-mouse transmission of endogenous microbiota. Ten mice fed either curves were generated by plotting the mean number of OTUs from all ani- an HFD or an LFD were included in this study. Fecal pellets were collected from mals in each experimental group (Fig. S6). Coordinates for nonmetric multi- mice in cages that contained a wire grid to limit the contamination of fecal dimensional scaling plots were generated using mothur and plotted in fi samples with microbiota present on skin and fur. Fecal pellets were collected Microsoft Excel. A complete list of OTUs identi ed in this study is given in before starting the HFD or LFD, and then 7 and 14 d after the start of each diet. Dataset S1. On day 15, mice were infected by gavage with 1 × 1011 cfu of E. coli O157:H7. Fecal pellets from each mouse were weighed, and bacterial DNA was isolated Statistical Analyses. Prism v4 (Graphpad Software) was used to analyze data from the pellets by the QiaAmp DNA Stool extraction system (Qiagen). throughout the manuscript. SEM was calculated for replicate samples in Figs. 2 A and D,3A, and 4B, and Fig. S3. A Student t test was used to determine fi 16S V6 Hypervariable Region PCR, Illumina Sequencing, and Subsequent statistical signi cance between groups in Figs. 2 A and D and 4B. A Two-way Microbial Community Profiling. The V6 region of the bacterial 16S rRNA ANOVA analysis with two-way repeated measures and the Bonferroni gene was amplified by the PCR from fecal pellet DNA with a combination of posttest correction was performed on data shown in Fig. 3A and Fig. S3 to fi five forward (CNACGCGAAGAACCTTANC, CAACGCGAAAAACCTTACC, CAA- determine statistical signi cance between time-matched samples. To de- fi CGCGCAGAACCTTACC, ATACGCGARGAACCTTACC, and CTAACCGANGAA- termine statistical signi cance in Fig. 3C, a two-way ANOVA analysis with CCTYACC) and four reverse primers (CGACAGCCATGCANCACCT, CGACA- two-way repeated measures and the Bonferroni posttest correction was ACCATGCANCACCT, CGACGGCCATGCANCACCT, and CGACGACCATGCAN- performed on the log-transformed individual values. Because the geometric CACCT) (67–69). V6 PCRs were performed in triplicate for each sample, and mean was plotted in Fig. 3C, the error represented among replicate samples fi the replicate reactions were pooled before sequencing. V6 amplicons were indicates the 95% con dence interval of time-matched values between each sequenced on an illumina Hi-sEq (100-bp single-end run) at the Tufts Uni- group. Analysis of molecular variance (AMOVA) (75, 76) was used to com- versity Core Facility. Raw reads were processed in Galaxy (70) as follows: pare the intestinal bacterial community structures of day 0, day 14 LFD, and illumina adapters, primer sequences, and low quality bases at the ends of day 14 HFD mice, shown in Fig. S5. each read were removed; any sequences with an “N” base were removed from the dataset; sequences were filtered to remove any reads with a quality ACKNOWLEDGMENTS. We thank the following individuals at the Uniformed score of less than 15 for 95% of the read. All subsequent sequence analyses Services University: Kenneth Gable for gas chromatography-mass spectrom- were done with mothur (71). To simplify the dataset and reduce computa- etry support; Joseph Royal, Farhang Alem, Alyssa Flora, and Lisa Russo for aid with animal work; Kateryna Lund for flow cytometry assistance; Dr. Cara tional time, the sequences from each sample that remained after initial Olsen for statistics support; Dr. James Vergis for assistance with figures; and processing were randomly subsampled to a depth of 115,000 reads per Dr. Christy Ventura for laboratory support. We also thank Dr. Petra Louis fi sample. To increase ef ciency and accuracy, sequences were aligned to a V6- (Rowland Institute, United Kingdom) for suggestions in butyrate extraction specific, curated database (72) derived from the full-length SILVA alignment. methods. This work was supported by National Institutes of Health Grant The alignment was screened to remove sequences that were shorter than 56 R37 AI020148 (to A.D.O.).

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