Adherent-Invasive Escherichia coli Production of Cellulose InfluencesIron-Induced Bacterial Aggregation, Phagocytosis, and Induction ofColitis

Melissa Ellermann,a Eun Young Huh,b Bo Liu,b Ian M. Carroll,b Rita Tamayo,a R. Balfour Sartora,b

Department of Microbiology and Immunologya and Center for Gastrointestinal Biology and Disease,b University of North Carolina, Chapel Hill, Chapel Hill, North Carolina,USA

Adherent-invasive Escherichia coli (AIEC), a functionally distinct subset of resident intestinal E. coli associated with Crohn’sdisease, is characterized by enhanced epithelial adhesion and invasion, survival within macrophages, and biofilm formation.Environmental factors, such as iron, modulate E. coli production of extracellular structures, which in turn influence the forma-tion of multicellular communities, such as biofilms, and bacterial interactions with host cells. However, the physiological andfunctional responses of AIEC to variable iron availability have not been thoroughly investigated. We therefore characterized theimpact of iron on the physiology of AIEC strain NC101 and subsequent interactions with macrophages. Iron promoted the cellu-lose-dependent aggregation of NC101. Bacterial cells recovered from the aggregates were more susceptible to phagocytosis thanplanktonic cells, which corresponded with the decreased macrophage production of the proinflammatory cytokine interleu-kin-12 (IL-12) p40. Prevention of aggregate formation through the disruption of cellulose production reduced the phagocytosisof iron-exposed NC101. In contrast, under iron-limiting conditions, where NC101 aggregation is not induced, the disruption ofcellulose production enhanced NC101 phagocytosis and decreased macrophage secretion of IL-12 p40. Finally, abrogation ofcellulose production reduced NC101 induction of colitis when NC101 was monoassociated in inflammation-prone Il10�/� mice.Taken together, our results introduce cellulose as a novel physiological factor that impacts host-microbe-environment interac-tions and alters the proinflammatory potential of AIEC.

Inflammatory bowel diseases (IBD), including Crohn’s disease(CD) and ulcerative colitis (UC), comprise a heterogeneous col-

lection of chronic, relapsing immune-mediated disorders. Al-though the precise etiologies are incompletely understood,accumulating evidence suggests that IBD are the result of inap-propriate immune responses toward a subset of resident intestinalmicrobes and their products in genetically susceptible individuals(1, 2).

The gastrointestinal (GI) tract is home to a complex commu-nity of microbes referred to as the intestinal microbiota. The de-velopment of intestinal inflammation is associated with commu-nity-wide changes to the intestinal microbiota, including anexpansion in the relative abundance of endogenous Escherichiacoli in IBD patients (3) and in rodent models of experimentalcolitis (4–6). A functionally distinct group of resident enteric E.coli bacteria known as adherent-invasive E. coli (AIEC) are recov-ered more frequently and in larger quantities from ileal tissuebiopsy specimens from CD patients than from specimens fromnon-CD controls (7, 8). In the absence of common identifyinggenetic determinants (9), AIEC strains are characterized by theirability to adhere to and invade intestinal epithelial cells (10) and tosurvive and replicate within macrophages (11). AIEC strains arealso moderate to strong in vitro biofilm producers (12). In addi-tion, AIEC strains are capable of inducing and perpetuating intes-tinal inflammation in various rodent models of experimental coli-tis, including streptomycin-treated mice (13), dextran sodiumsulfate (DSS)-treated mice (14), Toll-like receptor 5-deficientmice (15), transgenic CEABAC10 mice (16), and gnotobiotic in-terleukin-10 (IL-10)-deficient (Il10�/�) mice (17). These func-tional attributes of AIEC, in conjunction with host factors, such asgenetic polymorphisms linked to aberrant microbial sensing and

clearance (18, 19), potentially enable enhanced mucosal associa-tion by AIEC strains (20, 21). Together, these characteristics pro-vide the physical opportunity for AIEC strains to continuouslystimulate the mucosal immune system, thus propagating a state ofchronic intestinal inflammation.

Macrophages are a key component of host innate immune de-fense in the intestines, limiting systemic microbial disseminationby destroying potential invaders through phagocytosis, while alsosensing and responding to microbial stimuli and informing con-sequent host immune responses (22). Through pattern recogni-tion receptors (PRRs), macrophages recognize the conserved mi-crobial molecular patterns synthesized by resident and pathogenicintestinal bacteria, including extracellular structures, such as fim-briae, flagella, lipopolysaccharides, and peptidoglycan. Environ-mental factors, such as iron availability, influence the microbialproduction of some of these extracellular structures, including

Received 19 July 2015 Accepted 22 July 2015

Accepted manuscript posted online 27 July 2015

Citation Ellermann M, Huh EY, Liu B, Carroll IM, Tamayo R, Sartor RB. 2015.Adherent-invasive Escherichia coli production of cellulose influences iron-inducedbacterial aggregation, phagocytosis, and induction of colitis. Infect Immun83:4068 –4080. doi:10.1128/IAI.00904-15.

Editor: A. J. Bäumler

Address correspondence to R. Balfour Sartor, ryan_balfour_sartor@med.unc.edu.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/IAI.00904-15.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

doi:10.1128/IAI.00904-15

4068 iai.asm.org October 2015 Volume 83 Number 10Infection and Immunity

on June 29, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

curli fibrils in Salmonella enterica serovar Typhimurium (23, 24)and type I fimbriae in E. coli (25), providing the opportunity forenvironmental modulation of microbial interactions with macro-phages. Indeed, iron impacts E. coli interactions with host cells,albeit in contrasting ways. Iron promotes the increased internal-ization of pathogenic E. coli by neutrophils (26) and intestinalepithelial cells (27, 28). In contrast, iron limitation promotes thephagocytosis of a nonpathogenic E. coli K-12 strain by macro-phages through the decreased expression of the outer membraneprotein OmpW (29). Extracellular microbial structures thatimpact interactions with macrophages are also produced withinmulticellular microbial communities, including biofilms and bac-terial aggregates. Curli fibrils and the exopolysaccharide celluloseare common matrix components present within multicellularstructures produced by S. Typhimurium and E. coli (30–32). Cel-lulose and/or curli production has also been implicated in modu-lating intestinal E. coli interactions with epithelial cells (33, 34)and influencing in vivo host immune responses to uropathogenicE. coli (UPEC) strains in the urinary tract (35).

Iron is a cofactor necessary for various microbial enzymes andtherefore serves as an important micronutrient for most bacteria.In E. coli, changes in cytosolic iron concentrations are directlysensed by Fur (36). When bound to Fe2�, Fur acts as a transcrip-tion factor, regulating genes involved in diverse cellular processes,such as metabolism, metal acquisition, stress responses, motility,and biofilm formation (37, 38). Changes in extracellular iron con-centrations are also sensed by the membrane-associated kinaseBasS, a member of the BasRS two-component system (39). Inresponse to Fe3�, BasR regulates genes involved in altering theouter membrane landscape of E. coli (40, 41). Given the impor-tance of iron to microbial growth and function, an integral compo-nent of the innate immune response is the secretion of iron-scaveng-ing proteins at mucosal surfaces to limit microbial iron availability, aresponse that is potentiated by inflammation (42, 43).

Studies investigating the impact of iron on E. coli physiologyand interactions with host cells have been limited to nonpatho-genic K-12 or pathogenic E. coli strains. Consequently, little isknown about the impact of iron on the functional attributes ofAIEC strains. Therefore, the goal of this study was to characterizehow iron impacts the physiology of the AIEC strain NC101 (9, 44)and subsequent interactions with macrophages. Here we showthat iron promotes the cellulose-dependent aggregation ofNC101. Bacterial cells recovered from the aggregates are moresusceptible to phagocytosis, as prevention of aggregation throughthe disruption of cellulose production reduces macrophage up-take of NC101. Conversely, under iron-limiting conditions whereaggregation is not induced, disruption of cellulose production en-hanced NC101 phagocytosis and decreased macrophage proin-flammatory responses. Abrogation of bacterial cellulose productionalso delayed the onset of colitis in inflammation-prone Il10�/�

mice monoassociated with NC101. Taken together, our resultsintroduce cellulose as a novel physiological factor that dynami-cally impacts AIEC-host interactions in the face of changing envi-ronmental conditions.

MATERIALS AND METHODSBacterial strains and growth conditions. The bacterial strains and plas-mids used in this study are listed in Table S1 in the supplemental material.E. coli NC101 was isolated from the feces of a wild-type (WT) mouse aspreviously described (17). Unless otherwise indicated, bacteria from an

overnight culture were washed prior to inoculation into M9 minimalmedium with the concentrations of iron as ferrous sulfate (catalog num-ber I146; Fisher Scientific) indicated below. Bacteria were grown at 250rpm and 37°C for all experiments. The medium was supplemented with50 �g/ml kanamycin or 100 �g/ml carbenicillin as appropriate.

Construction of isogenic mutant, chromosomally complemented,and GFP-labeled strains. All deletion mutants were created using thebacteriophage � Red recombinase system as previously described(45). Deletion mutants were chromosomally complemented using thepMCL2868 plasmid (a kind gift from M. Chelsea Lane), a mini-Tn7 vec-tor, as previously described (46). For green fluorescent protein (GFP)-labeled strains, the pEGFP plasmid was transformed into each strain byelectroporation. Transformed strains were grown with 100 �g/ml carben-icillin to maintain the plasmid.

Sedimentation assays. Sedimentation assays were performed as pre-viously described (47) with the following modifications. Briefly, bacteriawere grown in M9 minimal medium with the concentrations of iron or theiron chelator 2,2-bipyridyl (BPD; catalog number 366-18-7; Alfa Aesar)indicated below. At the specified time points, cultures were removed fromthe incubator and the aggregates were allowed to settle to the bottom of50-ml conical tubes. An aliquot from the top of the culture (planktonicphase) was taken for optical density (OD) quantification by spectropho-tometry or quantitative culture. The cultures were then vortexed thor-oughly, and the cells were pelleted, washed with phosphate-buffered sa-line (PBS), and passed through a 30-gauge needle to obtain homogeneousbacterial suspensions. An aliquot was then taken for quantitative cultureor spectrophotometry as a measure of the bacterial concentration of thewhole culture. Percent aggregation was calculated using the formula[(whole-culture OD600 � planktonic culture OD600)/whole-cultureOD600] � 100, where OD600 is the optical density at 600 nm. Whereindicated, cellulase (from Aspergillus niger [catalog number C1184;Sigma]) was added to the cultures after 2 h of growth. The cultures werethen vortexed vigorously and incubated for 10 min at 37°C.

Assessment of aggregation and CF binding by fluorescence micros-copy. GFP-labeled bacteria were grown in M9 minimal medium with theconcentrations of iron indicated below. For assessing the dispersion of thebacterial aggregates prior to the gentamicin protection assays, bacterialaggregates were physically disrupted as described above. When stainingwith calcofluor (CF), the bacteria were cultured with 1% calcofluor whitestaining solution (catalog number 18909; Sigma). The cultures were nor-malized to equivalent optical densities (600 nm). A 5-�l aliquot was spot-ted onto glass microscope slides in duplicate. The slides were visualizedusing an Olympus IX71 fluorescence microscope. ImageJ software wasutilized to quantify the aggregates, where aggregates were defined as ob-jects in the field that had a pixel intensity threshold of 5 to 215 and a pixelsize of 201 to infinity. ImageJ software was also utilized to quantify CFbinding per cell using the following formula: (mean intensity of CF � areaof CF)/(mean intensity of GFP � area of GFP). In each experiment, atleast 15 high-power fields (magnification, �200) per slide were analyzed.Representative images were taken to demonstrate the colocalization of CFwith GFP-expressing bacteria at a �400 magnification.

Gentamicin protection assays. Bacteria were grown for 2 h in M9minimal medium with the concentrations of iron indicated below. Thecultures were vortexed and washed with PBS to disrupt the aggregates asdescribed above and normalized to equivalent optical densities (600 nm).For experiments using planktonic or aggregate bacterial cells, the aggre-gates were allowed to settle to the bottom of the 50-ml conical tubes for 5min. The broth phase (top) of the culture was then collected, and cellsrecovered from this phase were defined as planktonic. The remainingbroth phase was removed by aspiration. Aggregates were recovered byresuspension in PBS and were physically disrupted as described above.Gentamicin protection assays were then performed as previously de-scribed (7). Briefly, bone marrow-derived macrophages were seeded in24-well plates, and bacteria were added at a multiplicity of infection(MOI) of 10. After centrifugation for 10 min at 228 � g, the cocultures

Impact of Iron and Cellulose on AIEC Function

October 2015 Volume 83 Number 10 iai.asm.org 4069Infection and Immunity

on June 29, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

were incubated for 30 min in RPMI 1640 medium. Gentamicin-ladenmedium (RPMI 1640 with 10% fetal bovine serum and 100 �g/ml genta-micin) was then added to eliminate any remaining extracellular bacteria.The macrophages were lysed with PBS containing 1% Triton X-100 at thetime points indicated below for enumeration of intracellular bacteria.Intracellular bacteria were quantified by serial dilution plating.

Mice. Germfree (GF) Il10�/� and WT mice of the 129S6/SvEV back-ground were originally derived under sterile conditions by hysterectomyat the Gnotobiotic Laboratory (University of Wisconsin, Madison). Micewere maintained under GF conditions at the National Gnotobiotic Ro-dent Resource Center at the University of North Carolina (UNC), ChapelHill. An overnight bacterial culture of E. coli NC101 or the bcsA mutantwas utilized to monoassociate mice via oral and rectal swabs as previouslydescribed (48). The absence of isolator contamination was confirmed byGram staining and fecal culture. Once the mice were monoassociated,fecal and cecal E. coli loads were quantified by dilution plating on LB platesas previously described (44). All animal protocols were approved by theUNC, Chapel Hill, Institutional Animal Care and Use Committee.

Histological scoring. At necropsy, proximal and distal colonic seg-ments were Swiss rolled and fixed in 10% neutral buffered formalin. Thehistological inflammation scores (range, 0 to 4) of the proximal and distalcolonic sections were blindly assessed as previously described (17).Briefly, the scoring system is as follows: 0 indicates no inflammation, 1indicates the presence of cells infiltrating the lamina propria (LP); 2 indi-cates epithelial hyperplasia, a mild loss of goblet cells, and more extensivecellular infiltration within the LP; 3 indicates marked epithelial hyperpla-sia, a loss of goblet cells, and pronounced cellular infiltration within the LPand submucosa; and 4 indicates ulceration and transmural inflammation.Data are expressed as the composite histology score (range, 0 to 8), whichwas calculated by adding the proximal and distal colonic histology scores.

Statistical analysis. P values were calculated using Student’s t testwhen two experimental groups were compared, one-way analysis of vari-ance (ANOVA) with Tukey’s multiple-comparison posttest when three ormore experimental groups were compared, or two-way ANOVA with theBonferroni multiple-comparison posttest when more than two variableswere compared. All data from the enumeration of bacteria by serial dilu-tion and plating were log transformed to normalize the data. For quanti-

fication of NC101 aggregates by microscopy, P values were calculatedusing a nonparametric Kruskal-Wallis test with Dunn’s posttest. For allanimal experiments, P values were determined using a nonparametricMann-Whitney test.

Detailed methods for the Congo red and calcofluor colony morpho-type assays, macrophage isolation, mesenteric lymph node (MLN) cul-tures, enzyme-linked immunosorbent assays, RNA isolation, and reversetranscriptase PCR are described in the Materials and Methods in the sup-plemental material.

RESULTSIron promotes aggregation of E. coli NC101. Bacterial iron avail-ability varies within the GI tract, likely decreasing toward the mu-cosal interface with the secretion of host iron-binding proteins, aphenomenon that is potentiated with inflammation (49). How-ever, the physiological responses of AIEC to changes in iron avail-ability have not been well characterized. We therefore investigatedthe impact of iron on the physiology of E. coli NC101, a murineintestinal isolate that exhibits various AIEC characteristics, in-cluding increased epithelial translocation, enhanced persistencewithin macrophages, and the ability to induce colitis in selectivelycolonized, inflammation-prone Il10�/� mice (17, 50). When cul-tured in minimal medium with 5, 10, or 50 �M iron at 37°C,NC101 formed macroscopic aggregates that sediment in culture(Fig. 1A). The proportion of bacterial cells associated with theaggregates significantly increased as the iron concentration in-creased, as assessed by quantitative plating (see Fig. S1 in the sup-plemental material) and determination of the optical density (Fig.1B). Addition of an iron chelator did not impact NC101 aggrega-tion (Fig. 1C), suggesting that further iron deprivation had noeffect on this phenotype. In contrast, the E. coli K-12 substrainMG1655 does not form aggregates in response to iron (see Fig. S2in the supplemental material), indicating that this phenomenon isnot universal to all E. coli strains.

FIG 1 Iron promotes aggregation of E. coli NC101. (A) Representative images of NC101 aggregates after 2 h of growth in minimal medium with increasingconcentrations of iron. (B and C) NC101 sedimentation assays after 2 h of growth in minimal medium with various concentrations of iron (B) or the iron chelatorBPD (C). Data represent the percent optical density associated with the aggregates relative to that for the whole culture. All data represent the mean � SEM fromat least three independent experiments. P values were determined by pairwise comparisons by one-way ANOVA. *, P � 0.05 compared to the condition with 0�M iron; **, P � 0.01 compared to the condition with 0 �M iron; ***, P � 0.001 compared to the condition with 0 �M iron.

Ellermann et al.

4070 iai.asm.org October 2015 Volume 83 Number 10Infection and Immunity

on June 29, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

Cellulose is required for iron-induced aggregation of NC101.The extracellular matrix (ECM) of multicellular structures pro-duced by other E. coli strains is composed of proteinaceous com-ponents, such as fimbriae and curli; exopolysaccharides, such ascellulose; and/or extracellular DNA (51–53). The combination ofmatrix components varies depending on the environmental con-ditions and the E. coli strain (54). Therefore, to determine theextracellular composition of the NC101 aggregates, cellulase,DNase, or proteinase was added to the cultures following 2 h ofgrowth. Addition of cellulase resulted in dispersal of the aggre-gates (see Fig. S3 in the supplemental material), while DNase andproteinase had no obvious impact (data not shown). This suggeststhat cellulose is a major extracellular component that contributesto iron-induced aggregation.

Cellulose biosynthesis and structural proteins are encoded bybcsQABZC and bcsEFG and are required for cellulose production(31, 32, 55). However, the presence of bcs genes does not neces-sarily correspond with an ability to produce cellulose. We firstassessed whether NC101 is capable of producing cellulose at 37°Cutilizing the well-established calcofluor (CF) binding and red, dry,and rough (RDAR) colony morphotype assays. In contrast to E.coli MG1655, which served as an established negative control (56),NC101 bound CF, indicating the presence of cellulose production(Fig. 2A). Similarly, on Congo red agar, NC101 produced coloniesthat had the RDAR colony morphotype (Fig. 2B), a colony mor-photype that is dependent on cellulose and curli production (23,

31). To confirm that NC101 is a cellulose producer, an isogenicmutant lacking the bcsA gene, which encodes the catalytic subunitof cellulose synthase (57), was created. Deletion of bcsA resulted inthe loss of CF binding and the production of smooth colonies onCongo red agar (Fig. 2A and B). Moreover, the bcsA mutant didnot form macroscopic aggregates (Fig. 2C and D), confirming thatcellulose production is required for iron-induced aggregation ofNC101.

Curli are commonly coexpressed with cellulose within RDARcolonies and biofilms (31, 51, 58). To determine whether curli alsocontribute to RDAR colony formation in NC101, the csgA geneencoding the major subunit of the curli fibrils was disrupted (59).The csgA mutant produced pink instead of brown-red texturedcolonies (Fig. 2B), a phenotype that has been observed in othercsgA-deficient intestinal E. coli strains (58). We also determinedwhether curli contribute to NC101 aggregation in response toiron. No differences in aggregation were observed between thecgsA mutant and NC101, suggesting that curli expression does notsignificantly contribute to iron-induced aggregate formation (Fig.2D).

Deletion of fur decreases NC101 aggregation. Given the im-pact of iron on NC101, we next sought to determine how iron-induced aggregation occurs. In E. coli, fluctuations in iron avail-ability can be sensed intracellularly by the transcription factor Fur(36) or extracellularly by the two-component system BasRS (60).To determine whether NC101 aggregation occurs in response to

FIG 2 Cellulose is required for iron-induced aggregation of NC101. (A and B) Representative colony morphologies of NC101 or the bcsA, bcsA�bcsA, orcsgA::kan mutant on calcofluor (A) or Congo red agar (B). E. coli MG1655 served as a negative control. (C) Representative images of NC101 and the bcsA mutantafter 2 h of growth in minimal medium with 10 �M iron. (D) Sedimentation assays of NC101 and the bcsA, bcsA�bcsA, or csgA::kan mutant after 2 h of growthin minimal medium with increasing concentrations of iron. Data represent the percent OD600 of the aggregates relative to that for the whole culture. Datarepresent the mean � SEM from three independent experiments. P values were determined by pairwise comparisons by one-way ANOVA. *, P � 0.05; ***, P �0.001.

Impact of Iron and Cellulose on AIEC Function

October 2015 Volume 83 Number 10 iai.asm.org 4071Infection and Immunity

on June 29, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

intracellular or extracellular iron, we tested the aggregative abili-ties of fur and basRS deletion mutants. In contrast to the findingsfor the parental strain, the fur mutant did not form visible aggre-gates after 2 h of growth (Fig. 3A and B). This was not the result ofa corresponding growth defect with the fur mutant at the sametime point (Fig. 3D). The aggregates formed by the fur mutantafter 8 h were macroscopically smaller than the aggregates formedby NC101 (Fig. 3A), with a reduced proportion of bacterial cellsrelative to the whole culture associated with the aggregates (Fig.3C). In contrast, deletion of basRS did not impact NC101 aggre-gate formation (Fig. 3B). Taken together, deletion of fur limitedNC101 aggregation, suggesting that factors promoting iron-in-duced aggregate formation may be under the control of the Furmodulon.

Decreased aggregation by the fur mutant is not the result ofan inability to produce cellulose. Given that iron-induced aggre-gation of NC101 requires the capacity to produce cellulose, wedetermined whether cellulose biosynthesis was disrupted in thefur mutant as an explanation for its reduced ability to aggregate.We first tested whether cellulose production was unperturbed inthe fur mutant. Deletion of fur did not eliminate CF binding,although the distribution of CF within the inoculum was alteredcompared to that in NC101 (Fig. 4A). As growth conditions im-pact the stimulation of E. coli cellulose production, we next deter-mined whether NC101 and the fur mutant bind CF when exposedto iron in minimal medium. NC101 microscopic aggregates colo-calized with CF (Fig. 4C), confirming that cellulose is a compo-nent of the ECM. Although binding was not uniformly observed,single NC101 cells not associated with the aggregates bound CF,

suggesting that some planktonic cells produce cellulose. The furmutant also colocalized with CF in the presence of iron, furtherdemonstrating that cellulose production was not abrogated in themutant strain. Formation of microscopic aggregates by the furmutant was also observed. However, consistent with its decreasedability to form macroscopic aggregates, the microscopic aggre-gates produced by the fur mutant were less frequent and smaller insize than those produced by NC101 (see Fig. S4 in the supplemen-tal material). Finally, to determine whether cellulose production isdecreased in the fur mutant, the extent of CF binding was assessedas a proxy for cellulose production. CF binding in the presence ofiron did not differ between NC101 and the fur mutant (Fig. 4B).CF binding was also evident for NC101 and the fur mutant whenthey were grown in minimal medium without iron (Fig. 4B; seealso Fig. S5 in the supplemental material), indicating that celluloseproduction occurs under iron-limiting conditions. Taken to-gether, these data demonstrate that decreased aggregate produc-tion by the fur mutant is not due to an inability to produce cellu-lose, suggesting the involvement of other factors, in addition tocellulose, that promote maximal NC101 aggregation.

NC101 aggregate cells are more susceptible to phagocytosisby macrophages. ECM components produced within microbialmulticellular structures potentially alter E. coli interactions withmacrophages. Given that AIEC is characterized in part by its dis-tinct interactions with macrophages (11), we investigated whetherthe physiology associated with an aggregative state alters NC101susceptibility to phagocytosis by macrophages and subsequent in-tracellular survival. To test this, aggregation of NC101 was in-duced by growth with iron, and cultures containing aggregates

FIG 3 Deletion of fur in NC101 limits iron-induced aggregation. (A) Representative images of NC101 or the fur mutant after 2 or 8 h of growth inminimal medium with 10 �M iron. (B) Sedimentation assays of NC101 and the fur, fur�fur, or basRS::kan mutant after 2 h of growth in minimalmedium with 0 or 10 �M iron. (C and D) Time course sedimentation assays (C) and growth curves (D) of NC101 or the fur or fur�fur mutant inminimal medium with 10 �M iron. Data for all sedimentation assays represent the percent OD600 of the aggregates relative to that for the whole culture.All data are represented as the mean � SEM from three independent experiments. P values were determined by pairwise comparisons by one-way ANOVA(B) and two-way ANOVA (C and D). *, P � 0.05; ***, P � 0.001.

Ellermann et al.

4072 iai.asm.org October 2015 Volume 83 Number 10Infection and Immunity

on June 29, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

were physically dispersed into single-cell suspensions and cocul-tured with macrophages. NC101 was exposed to iron prior tococulture with macrophages, as iron availability can also impactmacrophage function (61). Under aggregate-inducing conditions,NC101 phagocytosis was significantly enhanced (Fig. 5A). Al-though the quantity of intracellular NC101 was also increasedafter 4 and 8 h, the percent intracellular survival of NC101 was notsubstantially altered (Fig. 5B). These data demonstrate an associ-ation between iron-induced aggregation and increased bacterialuptake by macrophages.

To further explore this association, we physically separated theplanktonic and aggregate phases from the same culture and testedwhether NC101 cells recovered from the aggregates were moresusceptible to phagocytosis. Irrespective of the presence of iron,the extent of internalization of planktonic NC101 remained con-stant (Fig. 5C). In contrast, a significantly larger amount of NC101

aggregate cells than planktonic cells was phagocytosed by macro-phages, suggesting that NC101 aggregate cells are more suscepti-ble to phagocytosis. One explanation for the increased phagocy-tosis of aggregate cells is the incomplete disruption of theaggregates, which could result in more cells entering the macro-phage at once. To address this possibility, we investigated whetheraggregates of GFP-labeled NC101 were fully dispersed utilizingmicroscopy. Before physical disruption, the number of aggregatesper high-power field was significantly higher when NC101 wascultured with iron (see Table S3 in the supplemental material).After physical disruption, NC101 aggregates were rarely visiblemicroscopically, and importantly, there was no significant differ-ence in the number of NC101 aggregates per field with or withoutiron. These findings indicate that the increased susceptibility ofaggregate cells to phagocytosis is not likely due to more bacteriaentering the macrophage at once. Instead, these data suggest that

FIG 4 Deletion of fur does not disrupt NC101 cellulose production. (A) Colony morphologies of NC101 or the fur or bcsA mutant on calcofluor plates. (B)GFP-expressing NC101 or the fur or bcsA mutant were grown in minimal medium with 0 or 10 �M iron for 1 h and stained with calcofluor. ImageJ softwarewas utilized to calculate the mean calcofluor binding per cell per high-power field (magnification, �200). At least 15 fields were analyzed per sample. Datarepresent the mean � SEM from three independent experiments relative to the condition with NC101 and 0 �M iron. P values were determined by pairwisecomparisons by the Kruskal-Wallis test. (C) Representative images of GFP-expressing NC101 or the fur or bcsA mutant stained with calcofluor.Bacteria were grown in minimal medium with 10 �M iron for 1 h. (Insets) Magnified views of the boxed areas on the right. Magnification �400(or �2,000 for insets); bars 100 �m.

Impact of Iron and Cellulose on AIEC Function

October 2015 Volume 83 Number 10 iai.asm.org 4073Infection and Immunity

on June 29, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

the physiology of the individual cells in the aggregates promotestheir phagocytosis.

Cellulose modulates NC101 susceptibility to phagocytosis.We next sought to identify the physiological factors that mediateenhanced internalization of individual NC101 aggregate cells.Given that cellulose is required for aggregation and deletion of furlimits aggregate formation in a cellulose-independent manner, wepredicted that deletion of bcsA or fur would reduce macrophageuptake of iron-exposed NC101. As hypothesized, under aggre-gate-inducing conditions, macrophage uptake of the bcsA and furmutants was significantly decreased in comparison to that ofNC101 (Fig. 6B; see also Fig. S6 in the supplemental material).However, deletion of bcsA or fur did not reduce the amount ofNC101 internalization to levels comparable to those for NC101nonaggregate cells, suggesting the involvement of other bacterialfactors that contribute to the susceptibility of aggregate cells tophagocytosis. Conversely, under iron-limiting conditions whereNC101 aggregation was not induced, the bcsA mutant was moresusceptible to phagocytosis than NC101 (Fig. 6A; see also Fig. S6 inthe supplemental material). This suggests that cellulose may act asan antiphagocytic factor for NC101 nonaggregate cells. Similarresults were also observed with the non-cellulose-producingstrain MG1655 (see Fig. S6 in the supplemental material). Impor-tantly, the bcsA mutant and MG1655 did not form microscopicaggregates (see Table S3 in the supplemental material). Moreover,deletion of bcsA did not impact the percent survival within mac-rophages (see Fig. S7 in the supplemental material). Taken to-gether, cellulose disparately modulates NC101 susceptibility tomacrophage phagocytosis by enabling the formation of an aggre-gative physiological state under iron-replete conditions and bypotentially acting as an antiphagocytic factor under iron-limitingconditions.

Cellulose alters the proinflammatory potential of NC101. Itis unclear how the differential phagocytosis of AIEC impacts mac-rophage proinflammatory responses. As deletion of bcsA altersmacrophage uptake of NC101, we investigated whether uptake ofthe bcsA mutant also alters macrophage production of p40, thecommon subunit of the proinflammatory cytokines IL-12 andIL-23. Under nonaggregating conditions (i.e., low-iron condi-tions), production of IL-12 p40 was decreased in macrophagesthat phagocytosed the bcsA mutant (Fig. 6C). Conversely, withincreased amounts of iron at levels where NC101 aggregation is

induced, macrophages that phagocytosed the bcsA mutant se-creted larger amounts of IL-12 p40 than the NC101-exposed mac-rophages (Fig. 6D). Deletion of fur did not alter IL-12 p40 secre-tion by the mutant-exposed macrophages, further demonstrating

FIG 5 NC101 aggregates are more susceptible to phagocytosis. NC101 was grown in minimal medium with 0 or 10 �M iron prior to coculture with bonemarrow-derived macrophages. (A) Following the addition of gentamicin for 30 min, intracellular NC101 was quantified after 1 h as a measure of bacterial uptakeand 4 or 8 h as a measure of intracellular survival. (B) Ratios of the amount of intracellular NC101 at 4 h to that at 1 h and of the amount of intracellular NC101at 8 h to that at 1 h as measures of percent intracellular survival. (C) The planktonic and aggregate fractions of each culture were separated prior to coculture withmacrophages. Intracellular NC101 was quantified after 1 h. Data are shown as the mean � SEM from a representative experiment of at least three independentexperiments with at least four technical replicates. P values were determined by pairwise comparisons by t test (A and B) and one-way ANOVA (C). ***, P �0.001.

FIG 6 Deletion of bcsA alters NC101 interactions with macrophages. (A andB) Intracellular NC101 (NC) or the bcsA or fur mutant after 1 h of coculturewith bone marrow-derived macrophages. (C and D) IL-12 p40 cytokine pro-duction by bone marrow-derived macrophages infected with NC101 or thebcsA or fur mutant for 8 h. All bacteria were grown in minimal mediumwith 0 �M (A and C) or 10 �M (B and D) iron prior to coculture with mac-rophages. Data are shown as the mean � SEM from a representative experi-ment of three independent experiments with at least four technical replicates.P values were determined by pairwise comparisons by one-way ANOVA. *,P � 0.05; ***, P � 0.001.

Ellermann et al.

4074 iai.asm.org October 2015 Volume 83 Number 10Infection and Immunity

on June 29, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

the importance of cellulose in mediating NC101-macrophage in-teractions. Taken together, these results demonstrate that, in ad-dition to impacting NC101 phagocytosis susceptibility, cellulosemodulates macrophage production of the proinflammatory cyto-kine IL-12 p40.

Given the contrasting effects of cellulose on macrophage pro-inflammatory responses, we next sought to determine how theability to produce cellulose contributes to the in vivo proinflam-matory potential of NC101. To investigate this, GF Il10�/� micewere monoassociated with NC101 or the bcsA mutant. AIECstrains such as NC101 uniquely induce colitis when monoassoci-ated in inflammation-susceptible Il10�/� mice (17), whereas themonoassociation of non-AIEC strains, including MG1655 (62)and Nissle (63), does not induce chronic colitis. The severity ofinflammation was assessed by histological score and proinflam-matory cytokine expression. Monoassociated WT mice served as

inflammation-resistant controls. After 10 days of colonization, nodifferences in histological inflammation were observed (Fig. 7B).After 21 days, mice colonized with the parental strain exhibitedsignificantly worse proximal and distal colonic inflammation,characterized by increased crypt hyperplasia and leukocytic infil-tration into the lamina propria (Fig. 7A and B). By 35 days,pathohistological differences were no longer apparent becausethe severity of inflammation increased in mice colonized withthe bcsA mutant. Importantly, WT animals colonized with ei-ther strain did not exhibit any pathology (Fig. 7C).

We next determined whether differences in histological in-flammation at 21 days corresponded with the differential expres-sion of proinflammatory cytokines. Il10�/� mice monoassociatedwith NC101 develop colitis that is driven by T-helper 1 (Th-1) andTh-17 responses, where the onset and exacerbation of inflamma-tion are associated with the increased production of IL-17, gamma

FIG 7 Deletion of bcsA in NC101 delays the onset of colitis in Il10�/� mice. (A) Representative histology by staining with hematoxylin and eosin of the proximalcolons of Il10�/� or WT mice monoassociated with NC101 or the bcsA mutant for 21 days. Magnification �200; bars 50 �m. (B and C) Compositeproximal and distal colon histology scores (range, 0 to 8) of Il10�/� mice (B) or WT mice (C) monoassociated with NC101 or the bcsA mutant. (D and E)Proximal colon transcript levels of Il12b (D) or Il17a (E) relative to that of Actb in Il10�/� mice monoassociated with NC101 or the bcsA mutant. Data areexpressed as the fold change relative to that for NC101-colonized mice. (F) IL-17 production by unfractionated MLN cells restimulated with the respectivebacterial lysates ex vivo. MLNs were isolated from Il10�/� mice monoassociated with NC101 or the bcsA mutant for 21 or 35 days. Each symbol represents anindividual mouse, and the data are for 5 to 8 mice per group. Open circles, mice monoassociated with NC101; closed squares, mice monoassociated with thebcsA mutant; lines at medians, P values determined by pairwise comparisons by a Mann-Whitney test. *, P � 0.05; **, P � 0.01.

Impact of Iron and Cellulose on AIEC Function

October 2015 Volume 83 Number 10 iai.asm.org 4075Infection and Immunity

on June 29, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

interferon (IFN-�), and IL-12 (17, 48). We therefore determinedwhether the earlier onset of colitis in NC101-colonized mice cor-responded with the differential expression of Il17a, Ifng, and Il12b.Prior to the onset of histological inflammation, the expression ofIl12b encoding the p40 subunit was increased in mice colonizedwith NC101 (Fig. 7D). This was consistent with the differences inmacrophage production of IL-12 p40 observed in vitro in responseto NC101 or the bcsA mutant under iron-limiting conditions. Thecolonic expression of Il17a or Ifng did not differ at 10 days (datanot shown). At 21 days, coinciding with a more severe histopa-thology, Il17a expression was increased in the proximal colon inmice colonized with NC101 (Fig. 7E). In contrast, no significantdifferences in Ifng transcript levels were observed (see Fig. S8 inthe supplemental material). Because colitis is driven by antigen-specific responses in Il10�/� mice monoassociated with NC101,we also quantified IFN-� and IL-17 production by unfractionatedMLN cells restimulated with the respective bacterial lysates. MLNcells recovered from mice colonized with NC101 produced largerquantities of IL-17 than MLN cells isolated from mice colonizedwith the bcsA mutant (Fig. 7F). The levels of IFN-� production byrestimulated MLN cells from mice colonized with NC101 or thebcsA mutant were not significantly different (see Fig. S8 in thesupplemental material). Finally, differences in the severity of coli-tis observed at 21 days also corresponded with a 2.4-fold decreasein the fecal loads of the bcsA mutant (Fig. 8). The cecal luminaldensities of the bcsA mutant were also consistently decreased rel-ative to that of NC101, although this was not uniformly observedin the feces. Decreased fecal and cecal concentrations of the bcsAmutant relative to the parental strain were likewise observed in

WT mice. Taken together, Il10�/� mice monoassociated with acellulose-deficient NC101 mutant exhibited a delayed onset ofcolitis, suggesting that the disruption of cellulose production inNC101 reduced its proinflammatory potential in an experimentalmodel of chronic immune-mediated colitis.

DISCUSSION

Environmental factors, such as iron availability, that may promoteproinflammatory interactions between AIEC, microbes clinicallyrelevant to IBD, and the host have not been well investigated.Therefore, the purpose of this study was to characterize how ironimpacts the physiology and functional attributes of the AIECstrain NC101. Our findings demonstrate that iron promotes thecellulose-dependent aggregation of NC101. Moreover, NC101 ag-gregate cells are more susceptible to phagocytosis by macro-phages. The contribution of cellulose to NC101 phagocytosis sus-ceptibility and consequent macrophage proinflammatoryresponses changes as iron availability and the physiological state ofNC101 are altered, demonstrating a dynamic role for cellulose inmodulating host-microbe interactions. Finally, abrogation of cel-lulose production in NC101 reduced its ability to induce colitis ininflammation-prone Il10�/� mice. Taken together, our resultsdemonstrate that cellulose production alters the proinflammatorypotential of NC101.

Various environmental factors, including temperature and nu-trient availability, impact multicellular behaviors such as aggrega-tion in E. coli and related enteric bacteria (64, 65). Interestingly,iron has divergent effects on the multicellular behaviors of other E.coli functional subtypes, including enteroaggregative (27), uro-pathogenic (47, 66, 67), and K-12 (25) strains. Therefore, giventhe various responses of different E. coli strains to alterations iniron availability, it is likely that multiple strain-specific mecha-nisms regulate these responses. Here we show that fur-deficient,but not basRS-deficient, NC101 exhibited a reduced ability toform aggregates. This suggests that intracellular, rather than ex-tracellular, iron sensing by NC101 contributes to the induction ofthis multicellular behavior. This was not the result of an inabilityof the fur mutant to produce cellulose, suggesting that additionalfactors under the control of the Fur modulon promote maximalNC101 aggregation. Cellulose production by the fur mutant mayenable it to form smaller microscopic aggregates that, comparedto NC101 aggregates, are not macroscopically visible until later ingrowth. Additionally, the fur mutant exhibited a growth defectwhen grown in minimal medium with iron, which could contrib-ute to decreased aggregate formation. However, this growth defectwas evident only during later stages of growth, after aggregateformation had already occurred in the parental strain. Fur hasbeen linked to the regulation of additional multicellular behaviorsin UPEC, E. coli K-12, and other Gram-negative bacteria (38, 66–69), demonstrating the importance of iron as an environmentalsignal in modulating the formation of microbial communitiesacross many bacterial species.

The translocation of microbes and their products across theintestinal epithelial barrier is detected by immune cells, includingmacrophages, where engagement of pattern recognition receptorsby microbial products activates signal transduction pathways thatpromote phagocytosis (70), microbial killing, and production ofinflammatory mediators. The assumption of an aggregate physi-ological state promoted NC101 phagocytosis by macrophages,where NC101 cells recovered from the aggregates were more sus-

FIG 8 Luminal densities of NC101 or the bcsA mutant in WT or Il10�/� mice.(A and B) Quantitative bacterial culture of feces collected from Il10�/� mice(A) or WT mice (B) monoassociated with NC101 or the bcsA mutant. (C andD) Quantitative bacterial culture of cecal contents collected from Il10�/� mice(C) or WT mice (D) monoassociated with NC101 or the bcsA mutant. Eachsymbol represents an individual mouse, and the data are for 5 to 8 mice pergroup. Open circles, mice monoassociated with NC101; closed squares, micemonoassociated with the bcsA mutant; lines at medians, P values determinedby pairwise comparisons by a Mann-Whitney test. *, P � 0.05; **, P � 0.01.

Ellermann et al.

4076 iai.asm.org October 2015 Volume 83 Number 10Infection and Immunity

on June 29, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

ceptible to phagocytosis than planktonic cells. Abrogation of cel-lulose production prevented aggregation and reduced NC101 sus-ceptibility to phagocytosis under aggregate-inducing conditions(i.e., iron exposure). Coinciding with a reduced ability to aggre-gate, the fur mutant was also phagocytosed to a lesser extent, eventhough it produced cellulose. Interestingly, increased phagocyto-sis of iron-exposed (71, 72) or biofilm-associated (73, 74) bacteriahas been observed in other bacterial species and with iron-exposedextraintestinal E. coli pathogens (26). These results suggest thatalthough cellulose is required for aggregation and, presumably,the assumption of an aggregate physiological state, other factorscontribute to the phagocytosis of NC101 aggregate cells.

Under non-aggregate-inducing conditions (i.e., low-iron con-ditions) where NC101 aggregation does not occur but cellulose isexpressed, deletion of bcsA enhanced NC101 susceptibility tophagocytosis. This suggests that in a nonaggregative state, cellu-lose acts as an antiphagocytic factor, potentially masking bacterialfactors that interact with macrophage receptors to promotephagocytosis. Indeed, as no host receptor for cellulose has beenidentified, it is unlikely that microbial cellulose interacts directlywith host cells. This is consistent with the contrasting effects of thedisruption of cellulose production on NC101 phagocytosis, asboth microbial iron exposure and the resulting physiological stateof NC101 are altered. Thus, our study introduces cellulose as anovel factor that modulates interactions between AIEC and mac-rophages and highlights the complex interplay between bacterialand environmental factors in modulating host-microbe interac-tions.

Although our investigation demonstrates that cellulose is re-quired for in vitro aggregation by NC101 and modulates interac-tions with macrophages, it is unclear whether NC101 celluloseproduction and aggregation occur in vivo. In a recent study byArthur and colleagues, the impact of the inflamed and nonin-flamed colonic environments on the NC101 transcriptome wasinvestigated (75). bcs transcripts were detected at 2, 12, and 20weeks following the monoassociation of formerly GF Il10�/� andinflammation-resistant Il10�/� rag2�/� mice, indicating that bcsgenes are transcribed in vivo. However, as cellulose biosynthesis isprimarily regulated through the allosteric control of cellulose syn-thase activity (76), the presence of bcs transcripts is not conclusiveevidence of NC101 cellulose production in the colon. Moreover,current biochemical techniques for assaying the presence of bac-terial cellulose in vitro are not easily adaptable to the intestinalenvironment, given the presence of plant cellulose and other poly-saccharides consisting of glucose monomers.

The contribution of cellulose to the in vivo fitness and virulencepotential of E. coli and related bacteria has been investigated onlyin UPEC strains in the urinary tract (35, 77) and S. Typhimuriumwhen administered intraperitoneally (78). Therefore, to establishwhether cellulose contributes to the colitogenicity of AIEC in theGI tract, the severity of colitis was assessed in Il10�/� mice mono-associated with NC101 or the cellulose-deficient bcsA mutant. Theonset of colitis was delayed in mice colonized with the cellulose-deficient mutant, which corresponded with decreased Th-17-as-sociated immune responses, including the decreased expression ofIl12b. This is consistent with our in vitro observations demonstrat-ing decreased IL-12 p40 production by macrophages infected withthe bcsA mutant following exposure to non-aggregate-inducingand iron-limiting conditions. The reduced macrophage produc-tion of IL-12 p40 also corresponded with the increased phagocy-

tosis of the bcsA mutant. Therefore, cellulose may enhance theproinflammatory potential of NC101 by preventing the mucosalclearance of NC101 and, consequently, promoting increased pro-inflammatory immune responses. However, as the deletion ofbcsA did not completely prevent colitis development and its effectswere lost over time, other microbial factors likely contribute to theability of NC101 to induce colitis. Finally, these results also suggestthat iron may be limiting within the inflamed intestines, a findingthat has been reported by others (79). However, the precise bio-availability of iron remains unclear, especially as iron concentra-tions likely vary throughout the GI tract and depend on otherfactors, including host iron status, inflammation, and diet.

Coinciding with decreased inflammation, the luminal loads ofthe bcsA mutant were significantly decreased. However, it is un-clear whether a 2.4-fold decrease in fecal loads and a 1.8-fold de-crease in cecal luminal loads significantly contribute to decreasedimmune activation in mice colonized with the bcsA mutant. Thececal luminal densities of the bcsA mutant were also decreased inIl10�/� mice prior to evidence of histological inflammation and innoninflamed WT mice. However, this early difference in luminalbacterial loads was not observed in the feces. Therefore, celluloseproduction may modestly enhance AIEC colonic fitness, whichprovides a possible additional mechanism for augmenting theproinflammatory potential of NC101. Cellulose provides micro-bial resistance against a variety of stressors both in the environ-ment (55, 67, 80) and within the host (77). For example, deletionof bcsA in UPEC enhanced bacterial clearance from the kidneys ina neutrophil-dependent manner (77). Cellulose-dependent mul-ticellular behaviors can also be induced by stressors likely presentat mucosal surfaces along the normal and inflamed GI tract, in-cluding fluctuations in iron availability, peroxide stress, and mi-crobial contact with soluble IgA antibodies (47, 67, 81). Finally, asintestinal E. coli isolates demonstrate an ability to produce cellu-lose at 37°C more frequently than UPEC clinical isolates (58), it istempting to speculate that cellulose may contribute to the intesti-nal fitness of resident intestinal E. coli strains.

In the colon, under homeostatic conditions, the mucosal sur-face is home to a distinct community of bacteria. The compositionof the mucosa-associated microbial community is significantlyaltered in chronic disease states such as CD, which includes anincreased abundance of mucosally associated resident E. coli iso-lates (3). Host inflammatory responses and intrinsic host geneticdefects compromise mucosal and epithelial barrier integrity, en-abling the enhanced proximity of mucosally associated bacteria tohost cells. Consistent with this, enhanced intestinal tissue AIECloads, mucosal association, and translocation (8, 15, 82) are cor-related with more severe disease in CD and experimental modelsof colitis. Our study highlights the importance of environmentalfactors in altering AIEC physiology and subsequent host-microbeinteractions and impact on inflammation. Given the lack of iden-tifying genetic loci within AIEC, it would be interesting to inves-tigate whether iron alters the physiological state of clinical AIECisolates as a novel functional determinant. Finally, future studiesconfirming the biosynthesis of cellulose in vivo and, more broadly,assessing the in vivo physiological state of AIEC, especially withinmore defined intestinal niches, such as the normal and inflamedmucosa, are warranted. This could enable the identification ofnovel therapeutics that modulate the physiology of E. coli to limitadverse interactions with the underlying mucosa in CD patientsand individuals genetically susceptible to CD.

Impact of Iron and Cellulose on AIEC Function

October 2015 Volume 83 Number 10 iai.asm.org 4077Infection and Immunity

on June 29, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

ACKNOWLEDGMENTS

We acknowledge the Gnotobiotic, Histology, and ImmunotechnologyCores at the UNC Center for Gastrointestinal Biology and Disease (sup-ported by NIH P30DK34987). This work was supported by grants fromthe Crohn’s and Colitis Foundation of America (Microbiome Consor-tium), NIH P40 OD01995, NIH P30 DK34987, and NIH R01 DK053347.

REFERENCES1. Sartor RB. 2008. Microbial influences in inflammatory bowel diseases.

Gastroenterology 134:577–594. http://dx.doi.org/10.1053/j.gastro.2007.11.059.

2. Knights D, Lassen KG, Xavier RJ. 2013. Advances in inflammatory boweldisease pathogenesis: linking host genetics and the microbiome. Gut 62:1505–1510. http://dx.doi.org/10.1136/gutjnl-2012-303954.

3. Gevers D, Kugathasan S, Denson LA, Vázquez-Baeza Y, Van TreurenW, Ren B, Schwager E, Knights D, Song SJ, Yassour M, Morgan XC,Kostic AD, Luo C, González A, McDonald D, Haberman Y, Walters T,Baker S, Rosh J, Stephens M, Heyman M, Markowitz J, Baldassano R,Griffiths A, Sylvester F, Mack D, Kim S, Crandall W, Hyams J, Hut-tenhower C, Knight R, Xavier RJ. 2014. The treatment-naive micro-biome in new-onset Crohn’s disease. Cell Host Microbe 15:382–392. http://dx.doi.org/10.1016/j.chom.2014.02.005.

4. Arthur JC, Perez-Chanona E, Muhlbauer M, Tomkovich S, Uronis JM,Fan TJ, Campbell BJ, Abujamel T, Dogan B, Rogers AB, Rhodes JM,Stintzi A, Simpson KW, Hansen JJ, Keku TO, Fodor AA, Jobin C. 2012.Intestinal inflammation targets cancer-inducing activity of the microbi-ota. Science 338:120 –123. http://dx.doi.org/10.1126/science.1224820.

5. Winter SE, Winter MG, Xavier MN, Thiennimitr P, Poon V, KeestraAM, Laughlin RC, Gomez G, Wu J, Lawhon SD, Popova IE, Parikh SJ,Adams LG, Tsolis RM, Stewart VJ, Bäumler AJ. 2013. Host-derivednitrate boosts growth of E. coli in the inflamed gut. Science 339:708 –711.http://dx.doi.org/10.1126/science.1232467.

6. Maharshak N, Packey CD, Ellermann M, Manick S, Siddle JP, Huh EY,Plevy S, Sartor RB, Carroll IM. 2013. Altered enteric microbiota ecologyin interleukin 10-deficient mice during development and progression ofintestinal inflammation. Gut Microbes 4:316 –324. http://dx.doi.org/10.4161/gmic.25486.

7. Darfeuille-Michaud A, Boudeau J, Bulois P, Neut C, Glasser A-L,Barnich N, BringerM-A, Swidsinski A, Beaugerie L, Colombel J-F. 2004.High prevalence of adherent-invasive Escherichia coli associated with ilealmucosa in Crohn’s disease. Gastroenterology 127:412– 421. http://dx.doi.org/10.1053/j.gastro.2004.04.061.

8. Baumgart M, Dogan B, Rishniw M, Weitzman G, Bosworth B,Yantiss R, Orsi RH, Wiedmann M, Mcdonough P, Kim SG, Berg D,Schukken Y, Scherl E, Simpson KW. 2007. Culture independentanalysis of ileal mucosa reveals a selective increase in invasive Esche-richia coli of novel phylogeny relative to depletion of Clostridiales inCrohn’s disease involving the ileum. ISME J 1:403– 418. http://dx.doi.org/10.1038/ismej.2007.52.

9. Dogan B, Suzuki H, Herlekar D, Sartor RB, Campbell BJ, Roberts CL,Stewart K, Scherl EJ, Araz Y, Bitar PP, Lefébure T, Chandler B,Schukken YH, Stanhope MJ, Simpson KW. 2014. Inflammation-associated adherent-invasive Escherichia coli are enriched in pathways foruse of propanediol and iron and M-cell translocation. Inflamm Bowel Dis20:1919 –1932. http://dx.doi.org/10.1097/MIB.0000000000000183.

10. Boudeau J, Glasser AL, Masseret E, Joly B, Darfeuille-Michaud A. 1999.Invasive ability of an Escherichia coli strain isolated from the ileal mucosaof a patient with Crohn’s disease. Infect Immun 67:4499 – 4509.

11. Glasser AL, Boudeau J, Barnich N, Perruchot MH, Colombel JF, Dar-feuille-Michaud A. 2001. Adherent invasive Escherichia coli strains frompatients with Crohn’s disease survive and replicate within macrophageswithout inducing host cell death. Infect Immun 69:5529 –5537. http://dx.doi.org/10.1128/IAI.69.9.5529-5537.2001.

12. Martinez-Medina M, Naves P, Blanco J, Aldeguer X, Blanco JE, BlancoM, Ponte C, Soriano F, Darfeuille-Michaud A, Garcia-Gil LJ. 2009.Biofilm formation as a novel phenotypic feature of adherent-invasiveEscherichia coli (AIEC). BMC Microbiol 9:202. http://dx.doi.org/10.1186/1471-2180-9-202.

13. Small C-LN, Reid-Yu SA, McPhee JB, Coombes BK. 2013. Persistentinfection with Crohn’s disease-associated adherent-invasive Escherichiacoli leads to chronic inflammation and intestinal fibrosis. Nat Commun4:1957. http://dx.doi.org/10.1038/ncomms2957.

14. Carvalho FA, Barnich N, Sauvanet P, Darcha C, Gelot A, Darfeuille-Michaud A. 2008. Crohn’s disease-associated Escherichia coli LF82 ag-gravates colitis in injured mouse colon via signaling by flagellin. InflammBowel Dis 14:1051–1060. http://dx.doi.org/10.1002/ibd.20423.

15. Carvalho FA, Koren O, Goodrich JK, Johansson MEV, Nalbantoglu I,Aitken JD, Su Y, Chassaing B, Walters WA, González A, Clemente JC,Cullender TC, Barnich N, Darfeuille-Michaud A, Vijay-Kumar M,Knight R, Ley RE, Gewirtz AT. 2012. Transient inability to manageproteobacteria promotes chronic gut inflammation in TLR5-deficientmice. Cell Host Microbe 12:139 –152. http://dx.doi.org/10.1016/j.chom.2012.07.004.

16. Martinez-Medina M, Denizot J, Dreux N, Robin F, Billard E, Bonnet R,Darfeuille-Michaud A, Barnich N. 2014. Western diet induces dysbiosiswith increased E coli in CEABAC10 mice, alters host barrier functionfavouring AIEC colonisation. Gut 63:116 –124. http://dx.doi.org/10.1136/gutjnl-2012-304119.

17. Kim SC, Tonkonogy SL, Albright CA, Tsang J, Balish EJ, Braun J,Huycke MM, Sartor RB. 2005. Variable phenotypes of enterocolitis ininterleukin 10-deficient mice monoassociated with two different com-mensal bacteria. Gastroenterology 128:891–906. http://dx.doi.org/10.1053/j.gastro.2005.02.009.

18. Hugot JP, Chamaillard M, Zouali H, Lesage S, Cézard JP, Belaiche J,Almer S, Tysk C, O’Morain CA, Gassull M, Binder V, Finkel Y, CortotA, Modigliani R, Laurent-Puig P, Gower-Rousseau C, Macry J, Co-lombel JF, Sahbatou M, Thomas G. 2001. Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn’s disease. Nature 411:599 – 603. http://dx.doi.org/10.1038/35079107.

19. Hampe J, Franke A, Rosenstiel P, Till A, Teuber M, Huse K, AlbrechtM, Mayr G, De La Vega FM, Briggs J, Günther S, Prescott NJ, OnnieCM, Häsler R, Sipos B, Fölsch UR, Lengauer T, Platzer M, Mathew CG,Krawczak M, Schreiber S. 2007. A genome-wide association scan ofnonsynonymous SNPs identifies a susceptibility variant for Crohn diseasein ATG16L1. Nat Genet 39:207–211. http://dx.doi.org/10.1038/ng1954.

20. Lapaquette P, Glasser A-L, Huett A, Xavier RJ, Darfeuille-Michaud A.2010. Crohn’s disease-associated adherent-invasive E. coli are selectivelyfavoured by impaired autophagy to replicate intracellularly. Cell Micro-biol 12:99 –113. http://dx.doi.org/10.1111/j.1462-5822.2009.01381.x.

21. Sadaghian Sadabad M, Regeling A, de Goffau MC, Blokzijl T, WeersmaRK, Penders J, Faber KN, Harmsen HJM, Dijkstra G. 24 September2014. The ATG16L1-T300A allele impairs clearance of pathosymbionts inthe inflamed ileal mucosa of Crohn’s disease patients. Gut. http://dx.doi.org/10.1136/gutjnl-2014-307289.

22. Steinbach EC, Plevy SE. 2014. The role of macrophages and dendriticcells in the initiation of inflammation in IBD. Inflamm Bowel Dis 20:166 –175. http://dx.doi.org/10.1097/MIB.0b013e3182a69dca.

23. Römling U, Sierralta WD, Eriksson K, Normark S. 1998. Multicellularand aggregative behaviour of Salmonella typhimurium strains is con-trolled by mutations in the agfD promoter. Mol Microbiol 28:249 –264.http://dx.doi.org/10.1046/j.1365-2958.1998.00791.x.

24. White AP, Gibson DL, Grassl GA, Kay WW, Finlay BB, Vallance BA,Surette MG. 2008. Aggregation via the red, dry, and rough morphotype isnot a virulence adaptation in Salmonella enterica serovar Typhimurium.Infect Immun 76:1048 –1058. http://dx.doi.org/10.1128/IAI.01383-07.

25. Wu Y, Outten FW. 2009. IscR controls iron-dependent biofilm forma-tion in Escherichia coli by regulating type I fimbria expression. J Bacteriol191:1248 –1257. http://dx.doi.org/10.1128/JB.01086-08.

26. Wise AJ, Hogan JS, Cannon VB, Smith KL. 2002. Phagocytosis andserum susceptibility of Escherichia coil cultured in iron-deplete and iron-replete media. J Dairy Sci 85:1454 –1459. http://dx.doi.org/10.3168/jds.S0022-0302(02)74213-6.

27. Alves JR, Pereira ACM, Souza MC, Costa SB, Pinto AS, Mattos-Guaraldi AL, Hirata-Júnior R, Rosa ACP, Asad LMBO. 2010. Iron-limited condition modulates biofilm formation and interaction withhuman epithelial cells of enteroaggregative Escherichia coli (EAEC). JAppl Microbiol 108:246 –255. http://dx.doi.org/10.1111/j.1365-2672.2009.04417.x.

28. Kortman GAM, Boleij A, Swinkels DW, Tjalsma H. 2012. Iron avail-ability increases the pathogenic potential of Salmonella typhimurium andother enteric pathogens at the intestinal epithelial interface. PLoS One7:e29968. http://dx.doi.org/10.1371/journal.pone.0029968.

29. Wu X-B, Tian L-H, Zou H-J, Wang C-Y, Yu Z-Q, Tang C-H, Zhao F-K,Pan J-Y. 2013. Outer membrane protein OmpW of Escherichia coli is

Ellermann et al.

4078 iai.asm.org October 2015 Volume 83 Number 10Infection and Immunity

on June 29, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

required for resistance to phagocytosis. Res Microbiol 164:848 – 855. http://dx.doi.org/10.1016/j.resmic.2013.06.008.

30. Römling U, Rohde M, Olsen A, Normark S, Reinköster J. 2000. AgfD,the checkpoint of multicellular and aggregative behaviour in Salmonellatyphimurium regulates at least two independent pathways. Mol Microbiol36:10 –23. http://dx.doi.org/10.1046/j.1365-2958.2000.01822.x.

31. Zogaj X, Nimtz M, Rohde M, Bokranz W, Römling U. 2001. Themulticellular morphotypes of Salmonella typhimurium and Escherichiacoli produce cellulose as the second component of the extracellular ma-trix. Mol Microbiol 39:1452–1463. http://dx.doi.org/10.1046/j.1365-2958.2001.02337.x.

32. Serra DO, Richter AM, Hengge R. 2013. Cellulose as an architecturalelement in spatially structured Escherichia coli biofilms. J Bacteriol 195:5540 –5554. http://dx.doi.org/10.1128/JB.00946-13.

33. Monteiro C, Saxena I, Wang X, Kader A, Bokranz W, Simm R, NoblesD, Chromek M, Brauner A, Brown RM, Jr, Römling U. 2009. Charac-terization of cellulose production in Escherichia coli Nissle 1917 and itsbiological consequences. Environ Microbiol 11:1105–1116. http://dx.doi.org/10.1111/j.1462-2920.2008.01840.x.

34. Wang X, Rochon M, Lamprokostopoulou A, Lünsdorf H, Nimtz M,Römling U. 2006. Impact of biofilm matrix components on interac-tion of commensal Escherichia coli with the gastrointestinal cell lineHT-29. Cell Mol Life Sci 63:2352–2363. http://dx.doi.org/10.1007/s00018-006-6222-4.

35. Raterman EL, Shapiro DD, Stevens DJ, Schwartz KJ, Welch RA.2013. Genetic analysis of the role of yfiR in the ability of Escherichiacoli CFT073 to control cellular cyclic dimeric GMP levels and to persistin the urinary tract. Infect Immun 81:3089 –3098. http://dx.doi.org/10.1128/IAI.01396-12.

36. Hantke K. 2001. Iron and metal regulation in bacteria. Curr Opin Micro-biol 4:172–177. http://dx.doi.org/10.1016/S1369-5274(00)00184-3.

37. McHugh JP, Rodríguez-Quinoñes F, Abdul-Tehrani H, SvistunenkoDA, Poole RK, Cooper CE, Andrews SC. 2003. Global iron-dependentgene regulation in Escherichia coli. A new mechanism for iron homeosta-sis. J Biol Chem 278:29478 –29486.

38. Seo SW, Kim D, Latif H, O’Brien EJ, Szubin R, Palsson BO. 2014.Deciphering Fur transcriptional regulatory network highlights its com-plex role beyond iron metabolism in Escherichia coli. Nat Commun5:4910. http://dx.doi.org/10.1038/ncomms5910.

39. Nagasawa S, Ishige K, Mizuno T. 1993. Novel members of the two-component signal transduction genes in Escherichia coli. J Biochem 114:350 –357.

40. Wösten MM, Kox LF, Chamnongpol S, Soncini FC, Groisman EA.2000. A signal transduction system that responds to extracellular iron. Cell103:113–125. http://dx.doi.org/10.1016/S0092-8674(00)00092-1.

41. Ogasawara H, Shinohara S, Yamamoto K, Ishihama A. 2012. Novelregulation targets of the metal-response BasS-BasR two-component sys-tem of Escherichia coli. Microbiology 158:1482–1492. http://dx.doi.org/10.1099/mic.0.057745-0.

42. Raffatellu M, George MD, Akiyama Y, Hornsby MJ, Nuccio S-P, PaixaoTA, Butler BP, Chu H, Santos RL, Berger T, Mak TW, Tsolis RM,Bevins CL, Solnick JV, Dandekar S, Bäumler AJ. 2009. Lipocalin-2resistance confers an advantage to Salmonella enterica serotype Typhimu-rium for growth and survival in the inflamed intestine. Cell Host Microbe5:476 – 486. http://dx.doi.org/10.1016/j.chom.2009.03.011.

43. Chassaing B, Srinivasan G, Delgado MA, Young AN, Gewirtz AT,Vijay-Kumar M. 2012. Fecal lipocalin 2, a sensitive and broadly dynamicnon-invasive biomarker for intestinal inflammation. PLoS One 7:e44328.http://dx.doi.org/10.1371/journal.pone.0044328.

44. Patwa LG, Fan T-J, Tchaptchet S, Liu Y, Lussier YA, Sartor RB, HansenJJ. 2011. Chronic intestinal inflammation induces stress-response genes incommensal Escherichia coli. Gastroenterology 141:1842–1851.e1–10.http://dx.doi.org/10.1053/j.gastro.2011.06.064.

45. Datsenko KA, Wanner BL. 2000. One-step inactivation of chromosomalgenes in Escherichia coli K-12 using PCR products. Proc Natl Acad SciU S A 97:6640 – 6645. http://dx.doi.org/10.1073/pnas.120163297.

46. Choi K-H, Gaynor JB, White KG, Lopez C, Bosio CM, Karkhoff-Schweizer RR, Schweizer HP. 2005. A Tn7-based broad-range bacterialcloning and expression system. Nat Methods 2:443– 448. http://dx.doi.org/10.1038/nmeth765.

47. Rowe MC, Withers HL, Swift S. 2010. Uropathogenic Escherichia coliforms biofilm aggregates under iron restriction that disperse upon the

supply of iron. FEMS Microbiol Lett 307:102–109. http://dx.doi.org/10.1111/j.1574-6968.2010.01968.x.

48. Kim SC, Tonkonogy SL, Karrasch T, Jobin C, Sartor RB. 2007. Dual-association of gnotobiotic IL-10�/� mice with 2 nonpathogenic commen-sal bacteria induces aggressive pancolitis. Inflamm Bowel Dis 13:1457–1466. http://dx.doi.org/10.1002/ibd.20246.

49. Kortman GAM, Raffatellu M, Swinkels DW, Tjalsma H. 2014. Nutri-tional iron turned inside out: intestinal stress from a gut microbial per-spective. FEMS Microbiol Rev 38:1202–1234. http://dx.doi.org/10.1111/1574-6976.12086.

50. Liu B, Schmitz JM, Holt LC, Jarvis W, Bringer M-AS, Kim SC, Dar-feuille-Michaud A, Sartor RB. 2009. Increased intracellular survival of E.coli NC101 within macrophages may contribute to its ability to inducecolitis. Gastroenterology 136:A-704.

51. Saldaña Z, Xicohtencatl-Cortes J, Avelino F, Phillips AD, Kaper JB,Puente JL, Girón JA. 2009. Synergistic role of curli and cellulose in celladherence and biofilm formation of attaching and effacing Escherichiacoli and identification of Fis as a negative regulator of curli. EnvironMicrobiol 11:992–1006. http://dx.doi.org/10.1111/j.1462-2920.2008.01824.x.

52. Hung C, Zhou Y, Pinkner JS, Dodson KW, Crowley JR, Heuser J,Chapman MR, Hadjifrangiskou M, Henderson JP, Hultgren SJ. 2013.Escherichia coli biofilms have an organized and complex extracellularmatrix structure. mBio 4(5):e00645-13. http://dx.doi.org/10.1128/mBio.00645-13.

53. Hadjifrangiskou M, Gu AP, Pinkner JS, Kostakioti M, Zhang EW,Greene SE, Hultgren SJ. 2012. Transposon mutagenesis identifies uro-pathogenic Escherichia coli biofilm factors. J Bacteriol 194:6195– 6205.http://dx.doi.org/10.1128/JB.01012-12.

54. Hancock V, Witsø IL, Klemm P. 2011. Biofilm formation as a function ofadhesin, growth medium, substratum and strain type. Int J Med Microbiol301:570 –576. http://dx.doi.org/10.1016/j.ijmm.2011.04.018.

55. Solano C, García B, Valle J, Berasain C, Ghigo J-M, Gamazo C, Lasa I.2002. Genetic analysis of Salmonella enteritidis biofilm formation: criticalrole of cellulose. Mol Microbiol 43:793– 808. http://dx.doi.org/10.1046/j.1365-2958.2002.02802.x.

56. Da Re S, Ghigo JM. 2006. A CsgD-independent pathway for celluloseproduction and biofilm formation in Escherichia coli. J Bacteriol 188:3073–3087. http://dx.doi.org/10.1128/JB.188.8.3073-3087.2006.

57. Omadjela O, Narahari A, Strumillo J, Mélida H, Mazur O, Bulone V,Zimmer J. 2013. BcsA and BcsB form the catalytically active core ofbacterial cellulose synthase sufficient for in vitro cellulose synthesis.Proc Natl Acad Sci U S A 110:17856 –17861. http://dx.doi.org/10.1073/pnas.1314063110.

58. Bokranz W. 2005. Expression of cellulose and curli fimbriae by Esche-richia coli isolated from the gastrointestinal tract. J Med Microbiol 54:1171–1182. http://dx.doi.org/10.1099/jmm.0.46064-0.

59. Olsen A, Arnqvist A, Hammar M, Sukupolvi S, Normark S. 1993. TheRpoS sigma factor relieves H-NS-mediated transcriptional repression ofcsgA, the subunit gene of fibronectin-binding curli in Escherichia coli.Mol Microbiol 7:523–536. http://dx.doi.org/10.1111/j.1365-2958.1993.tb01143.x.

60. Hagiwara M, Yamashino T, Mizuno T. 2004. A genome-wide view of theEscherichia coli BasS-BasR two-component system implicated in iron-responses. Biosci Biotechnol Biochem 68:1758 –1767. http://dx.doi.org/10.1271/bbb.68.1758.

61. Nairz M, Schroll A, Sonnweber T, Weiss G. 2010. The struggle foriron—a metal at the host-pathogen interface. Cell Microbiol 12:1691–1702. http://dx.doi.org/10.1111/j.1462-5822.2010.01529.x.

62. Kim SC, Tonkonogy SL, Jarvis HW, Darfeuille-Michaud A, Sartor RB.2008. Escherichia coli strains differentially induce colitis in IL-10 genedeficient mice. Gastroenterology 134:A-23.

63. Schumann S, Alpert C, Engst W, Klopfleisch R, Loh G, Bleich A, BlautM. 2014. Mild gut inflammation modulates the proteome of intestinalEscherichia coli. Environ Microbiol 16:2966 –2979. http://dx.doi.org/10.1111/1462-2920.12192.

64. Gerstel U, Römling U. 2001. Oxygen tension and nutrient starvation aremajor signals that regulate agfD promoter activity and expression of themulticellular morphotype in Salmonella typhimurium. Environ Micro-biol 3:638 – 648. http://dx.doi.org/10.1046/j.1462-2920.2001.00235.x.

65. Spurbeck RR, Tarrien RJ, Mobley HLT. 2012. Enzymatically active andinactive phosphodiesterases and diguanylate cyclases are involved in reg-

Impact of Iron and Cellulose on AIEC Function

October 2015 Volume 83 Number 10 iai.asm.org 4079Infection and Immunity

on June 29, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

ulation of motility or sessility in Escherichia coli CFT073. mBio 3(5):e00307-12. http://dx.doi.org/10.1128/mBio.00307-12.

66. Hancock V, Dahl M, Klemm P. 2010. Abolition of biofilm formation inurinary tract Escherichia coli and Klebsiella isolates by metal interferencethrough competition for Fur. Appl Environ Microbiol 76:3836 –3841.http://dx.doi.org/10.1128/AEM.00241-10.

67. DePas WH, Hufnagel DA, Lee JS, Blanco LP, Bernstein HC, Fisher ST,James GA, Stewart PS, Chapman MR. 2013. Iron induces bimodal pop-ulation development by Escherichia coli. Proc Natl Acad Sci U S A 110:2629 –2634. http://dx.doi.org/10.1073/pnas.1218703110.

68. Banin E, Brady KM, Greenberg EP. 2006. Chelator-induced dispersaland killing of Pseudomonas aeruginosa cells in a biofilm. Appl EnvironMicrobiol 72:2064 –2069. http://dx.doi.org/10.1128/AEM.72.3.2064-2069.2006.

69. Wu CC, Lin CT, Cheng WY, Huang CJ, Wang ZC, Peng HL. 2012.Fur-dependent MrkHI regulation of type 3 fimbriae in Klebsiella pneu-moniae CG43. Microbiology 158:1045–1056. http://dx.doi.org/10.1099/mic.0.053801-0.

70. Doyle SE, O’Connell RM, Miranda GA, Vaidya SA, Chow EK, Liu PT,Suzuki S, Suzuki N, Modlin RL, Yeh WC, Lane TF, Cheng G. 2004.Toll-like receptors induce a phagocytic gene program through p38. J ExpMed 199:81–90.

71. James BW, Mauchline WS, Fitzgeorge RB, Dennis PJ, Keevil CW. 1995.Influence of iron-limited continuous culture on physiology and virulenceof Legionella pneumophila. Infect Immun 63:4224 – 4230.

72. Domingue PA, Lambert PA, Brown MR. 1989. Iron depletion alterssurface-associated properties of Staphylococcus aureus and its associationto human neutrophils in chemiluminescence. FEMS Microbiol Lett 50:265–268.

73. Spiliopoulou AI, Kolonitsiou F, Krevvata MI, Leontsinidis M,Wilkinson TS, Mack D, Anastassiou ED. 2012. Bacterial adhesion,intracellular survival and cytokine induction upon stimulation ofmononuclear cells with planktonic or biofilm phase Staphylococcusepidermidis. FEMS Microbiol Lett 330:56 – 65. http://dx.doi.org/10.1111/j.1574-6968.2012.02533.x.

74. Daw K, Baghdayan AS, Awasthi S, Shankar N. 2012. Biofilm and plank-tonic Enterococcus faecalis elicit different responses from host phagocytesin vitro. FEMS Immunol Med Microbiol 65:270 –282. http://dx.doi.org/10.1111/j.1574-695X.2012.00944.x.

75. Arthur JC, Gharaibeh RZ, Mühlbauer M, Perez-Chanona E, Uronis JM,McCafferty J, Fodor AA, Jobin C. 2014. Microbial genomic analysisreveals the essential role of inflammation in bacteria-induced colorectalcancer. Nat Commun 5:4724. http://dx.doi.org/10.1038/ncomms5724.

76. Römling U. 2005. Characterization of the rdar morphotype, a multicel-lular behaviour in Enterobacteriaceae. Cell Mol Life Sci 62:1234 –1246.http://dx.doi.org/10.1007/s00018-005-4557-x.

77. Larsen S, Bendtzen K, Nielsen OH. 2010. Extraintestinal manifestations ofinflammatory bowel disease: epidemiology, diagnosis, and management. AnnMed 42:97–114. http://dx.doi.org/10.3109/07853890903559724.

78. Pontes MH, Lee E-J, Choi J, Groisman EA. 2015. Salmonella promotesvirulence by repressing cellulose production. Proc Natl Acad Sci U S A112:5183–5188. http://dx.doi.org/10.1073/pnas.1500989112.

79. Deriu E, Liu JZ, Pezeshki M, Edwards RA, Ochoa RJ, Contreras H,Libby SJ, Fang FC, Raffatellu M. 2013. Probiotic bacteria reduce Salmo-nella typhimurium intestinal colonization by competing for iron. CellHost Microbe 14:26 –37. http://dx.doi.org/10.1016/j.chom.2013.06.007.

80. Gualdi L, Tagliabue L, Bertagnoli S, Ierano T, De Castro C, Landini P.2008. Cellulose modulates biofilm formation by counteracting curli-mediated colonization of solid surfaces in Escherichia coli. Microbiology154:2017–2024. http://dx.doi.org/10.1099/mic.0.2008/018093-0.

81. Amarasinghe JJ, D’Hondt RE, Waters CM, Mantis NJ. 2013. Exposureof Salmonella enterica serovar Typhimurium to a protective monoclonalIgA triggers exopolysaccharide production via a diguanylate cyclase-dependent pathway. Infect Immun 81:653– 664. http://dx.doi.org/10.1128/IAI.00813-12.

82. Martin HM, Campbell BJ, Hart CA, Mpofu C, Nayar M, Singh R,Englyst H, Williams HF, Rhodes JM. 2004. Enhanced Escherichia coliadherence and invasion in Crohn’s disease and colon cancer. Gastroenter-ology 127:80 –93. http://dx.doi.org/10.1053/j.gastro.2004.03.054.

Ellermann et al.

4080 iai.asm.org October 2015 Volume 83 Number 10Infection and Immunity

on June 29, 2020 by guesthttp://iai.asm

.org/D

ownloaded from