Finding Regulators Associated with the Expression of the Long Polar Fimbriae in Enteropathogenic Escherichia coli.

Finding Regulators Associated with the Expression of the Long Polar Fimbriae in Enteropathogenic Escherichia coli Jia Hu,a Brittany N. Ross,a Roberto J. Cieza,a Alfredo G. Torresa,b

ABSTRACT

Enteropathogenic Escherichia coli (EPEC) is a human pathogen that requires initial adhesion to the intestine in order to cause disease. Multiple adhesion factors have been identified in E. coli strains, among them the long polar fimbriae (Lpf), a colonization factor associated with intestinal adhesion. The conditions of Lpf expression are well understood in enterohemorrhagic E. coli (EHEC); however, the expression of EPEC lpf has been found to be repressed under any in vitro condition tested. Therefore, we decided to identify those factors silencing expression of EPEC lpf. Because histone-like nucleoid structuring protein (H-NS) is a known repressor of EHEC lpf, we tested it and found that H-NS is a repressor of EPEC lpf. We also found that the adhesion of the EPEC ⌬hns strain was significantly enhanced compared to the wild-type strain. Because lpf expression was modestly increased in the hns mutant, transposon mutagenesis was performed to find a strain displaying higher lpf expression than EPEC ⌬hns. One Tn5 insertion was identified within the yhjX gene, and further in vitro characterization revealed increased lpf expression and adhesion to Caco-2 cells compared with EPEC ⌬hns. However, in a murine model of intestinal infection, the EPEC ⌬hns and EPEC ⌬hns Tn5 mutants had only a slight change in colonization pattern compared to the wild-type strain. Our data showed that EPEC Lpf is transcribed, but its role in EPEC intestinal colonization requires further analysis. IMPORTANCE

Data are presented demonstrating that the long polar fimbriae (lpf) operon in enteropathogenic E. coli (EPEC) is highly regulated; however, derepression occurs by mutagenizing two proteins associated with its control. The study demonstrates that the EPEC lpf operon can be expressed and, therefore, participates in the EPEC adherence phenotype.

E

scherichia coli bacteria are normally found in the intestines of humans and animals. Most E. coli strains rarely cause disease, except in immunocompromised hosts or in individuals whose normal gastrointestinal barrier is broken (1, 2). However, there are several E. coli strains that carry specific virulence attributes that classify them in specific pathotypes that cause distinct disease in healthy hosts (1). Two of these E. coli pathotypes, enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC), belong to the group of attaching and effacing E. coli (AEEC) strains. AEEC strains are characterized by the presence of a type III secretion system (T3SS) that injects virulence factors into the host cell, mediating the formation of the characterized histopathological attaching and effacing (A/E) lesion (1, 2). EPEC is a leading cause of human infantile diarrhea worldwide (3, 4). EHEC also causes severe human disease, including bloody diarrhea and life-threatening hemolytic uremic syndrome (HUS). Currently, EPEC is estimated to be responsible for 5 to 10% of pediatric diarrhea in developing countries (3). The Global Enteric Multicenter Study (GEMS) showed that some EPEC strains are associated with a 2.8-fold-increased risk of death among infants aged 0 to 11 months (5). In addition to the A/E lesion, EPEC and EHEC strains utilized fimbriae and nonfimbrial adhesins to mediate the initial interaction with host cells (6). Among them, the long polar fimbriae (Lpf) are adhesins that have been demonstrated to play a role in EHEC intestinal colonization in vitro and in vivo (7–10). In addition to Lpf being an important fimbria in E. coli O157:H7 adherence, it plays a significant role in pathogenesis in other EHEC strains (11, 12), as well as other pathogroups, includ-

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ing the newly described adherent and invasive E. coli (AIEC) strains (13, 14). In the case of EPEC, the prototype strain, E2348/69, carries an lpf gene cluster homologous to the lpf1 operon found in several pathogenic strains, including Salmonella enterica and EHEC O157:H7 (about 50 to 60% identical at the protein level) (15). However, when the function of Lpf was first investigated in EPEC, Tatsuno et al. found that the expression of lpf was repressed in E2348/69 under all the in vitro conditions tested, and therefore, a role for Lpf in adherence to intestinal cells or colonization of the in vitro organ culture (IVOC) model was not elucidated (15). In subsequent publications studying the regulation of the lpf operon, it was shown that the expression of Lpf in E. coli O157:H7 was controlled by a tightly regulated process (16–18). Histone-like nucleoid structuring protein (H-NS) is a global regulator controlling the expression of virulence factors in E. coli O157:H7. Ler is a master regulator of the locus for enterocyte effacement (LEE) pathogenicity island, which also regulates genes outside the EHEC and EPEC LEE. We have found that H-NS binds the regulatory

Received 23 June 2015 Accepted 2 September 2015 Accepted manuscript posted online 8 September 2015 Citation Hu J, Ross BN, Cieza RJ, Torres AG. 2015. Finding regulators associated with the expression of the long polar fimbriae in enteropathogenic Escherichia coli. J Bacteriol 197:3658 –3665. doi:10.1128/JB.00509-15. Editor: V. J. DiRita Address correspondence to Alfredo G. Torres, [email protected]. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Journal of Bacteriology

December 2015 Volume 197 Number 23

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Department of Microbiology and Immunologya and Department of Pathology,b Sealy Center for Vaccine Development, University of Texas Medical Branch, Galveston, Texas, USA

Lpf and H-NS Roles in EPEC

TABLE 1 Bacterial strains and plasmids used in this study Source or reference

Relevant characteristics

Strains EHEC 86-24 E2348/69 EPEC E2348/69 ⌬hns EPEC ⌬hns Tn5 EPEC ⌬hns ⌬yhjX DH5␣(␭pir) Sm10(␭pir) S17(␭pir)(pUTmini-Tn5-luxCDABE)

O157:H7 serotype; Nalr Acr Smr O127:H6 serotype; Nalr Acr E2348/69 strain transformed with pLpfA-CAT; Nalr Acr Kmr Cbr E2348/69 ⌬hns strain transformed with pLpfA-CAT; Nalr Acr Kmr Cbr EPEC ⌬hns strain with a transposon insertion; Nalr Acr Kmr Cbr Tcr EPEC ⌬hns strain with deletion of yhjX; Nalr Acr Kmr Cbr Gmr DH5␣ containing the pir gene Sm10 containing the pir gene; Kmr lux Tn mutagenesis system; Tcr

Laboratory stock

Plasmids pCVD442 pLpfA-CAT

Suicide plasmid; Cbr Promoter region of lpfEPEC was amplified and cloned into the pKK232.8 plasmid

33 This study

region of the EHEC lpf1 operon, silencing (repressing) its expression, while Ler works as an antisilencer of H-NS, releasing the repression of lpf exerted by the regulator. Because the lpf operon of the EPEC prototype strain E2348/69 is intact and contains a promoter similar to the EHEC O157 lpf1 promoter region, we hypothesized that EPEC lpf should be transcribed and that the poor expression of the genes in the operon might be due to the presence of additional regulators in the EPEC genome. In the current study, we found that expression of EPEC lpf is enhanced in the EPEC ⌬hns strain, but its expression was still modest compared to that in the EHEC ⌬hns strain. Therefore, we performed transposon mutagenesis with the EPEC ⌬hns strain and identified a mutant strain (with a transposon inserted in the yhjX gene) that had further increased expression of the EPEC lpf genes. Further testing demonstrated that this transposon mutant caused increased lpf expression and that the fimbriae encoded in this operon might be responsible for the increased EPEC adhesion. Our results also showed that EPEC lpf is expressed and could act as an adhesin in vitro, but we were unable to demonstrate that Lpf plays a significant role in the initial in vivo colonization when testing the strains in the murine model of intestinal infection. MATERIALS AND METHODS Bacterial strains and plasmids. Strains and plasmids are listed in Table 1. The strains were routinely grown on Luria-Bertani (LB) broth or LB agar at 37°C. When required, growth media were supplemented with antibiotics at the following concentrations: chloramphenicol (Cm), 35 ␮g ml⫺1; carbenicillin (Cb), 100 ␮g ml⫺1; streptomycin (Sm), 100 ␮g ml⫺1; gentamicin (Gm), 3 ␮g ml⫺1; and tetracycline (Tc), 2.5 ␮g ml⫺1. ␤-Galactosidase assay. Cultures were diluted 1:10 in Z buffer (Na2HPO4 [0.06 M], NaH2PO4 [0.04 M], KCl[0.01 M], MgSO4 [0.001 M], and ␤-mercaptoethanol [0.05 M]) under the different conditions tested and assayed for activity using ␱-nitrophenyl-␤-D-galactopyranoside as the substrate. The assay was performed as described previously (19). Construction of a transposon mutant library. Random mini-Tn5 transposon mutagenesis was used to generate mutants in the EPEC ⌬hns strain carrying pLpfA-CAT. The mini-Tn5 derivative that we used carries the inner and outer transposase recognition sequences flanking a tetracycline (Tc) resistance cassette and a promoterless luxCDABE operon. This plasmid has been used to generate randomized mutants in Pseudomonas aeruginosa PAO1 (20). The mini-Tn5-lux transposon was introduced into EPEC ⌬hns(pLpfA-CAT) by conjugation with the donor strain


This study Jose Luis Puente This study This study Laboratory stock Laboratory stock 20

Sm10(␭pir). The colonies with a transposon inserted should have resistance to tetracycline (Tc) (to maintain the mini-Tn5 plasmid) and kanamycin (Km) (indicative of a recipient strain). Several colonies were randomly picked to test the insertion of the transposon using the following primers: luxF (GTG AGT GAA AGC AGC CAA CA) and luxR (GCG TTC CAT CAG GTA CAG GT). All genomic DNAs were SphI digested and circularized, followed by PCR using two outward-facing, transposon-specific primers. The PCR products were purified, sequenced, and mapped to the EPEC O127:H6 E2348/69 genome (20). Chloramphenicol acetyltransferase assay. Chloramphenicol acetyltransferase (CAT) assays were performed as described previously, with slight modifications (21). To prepare crude extracts, cell samples were collected by centrifugation (10,000 ⫻ g; 3 min). The supernatant was discarded, and the cells were washed once with solution B (10 mM TrisHCl, pH 7.8, 1 ␮M dithiothreitol). The bacterial pellet was resuspended in 400 ␮l of solution B buffer and sonicated on ice until it was clear. Intact cells and debris were eliminated by centrifugation at 10,000 ⫻ g for 15 min at 4°C, and the supernatants were transferred to clean microcentrifuge tubes. For the CAT assay, 5 ␮l of each extract was added in duplicate to a 96-well Costar plate, followed by 200 ␮l of the reaction mixture containing 1 mM DNTB [5,5=-dithio-bis(2-nitrohenzoic acid)] (Boehringer Mannheim), 0.1 mM acetyl coenzyme A (acetyl-CoA) (Pharmacia Biotech), 0.1 mM chloramphenicol (Sigma Chemical) in 0.1 M Tris-HCl, pH 7.8. Changes in absorbance at 410 nm were read and recorded every 5 s for 3 min, using a scanning Epoch Microplate Spectrophotometer. Real-time PCR. Total mRNA was extracted from E. coli O127:H6 2348/69, which was grown in LB-Dulbecco’s modified Eagle’s medium (DMEM) using an RNeasy Protect Bacteria minikit (Qiagen, Valencia, CA) and reverse transcribed using a QuantiTect reverse transcription kit (Qiagen). cDNAs were used as templates for real-time quantitative reverse transcription (qRT)-PCR analysis of selected genes with a CFX96 realtime PCR detection system (Bio-Rad, CA). SYBR green master mix (BioRad, CA) was used for all qRT-PCRs. The qlpfAO127 primers were used for gene targets: qlpfAO127F (TGC TGC GTC TAC GAT CTC AC) and qlpfAO127R (TTT TGC CTT GTC TGC ACT GG). The 16S rRNA gene was used as the housekeeping gene (q16sF [CAA GAC CAA AGA GGG GGA CC] and q16sR [TTC CAG TGT GGC TGG TCA TC]). The amplification efficiency was 0.90 to 0.99. Construction of the ⌬hns ⌬yhjX double mutant strain. The procedure for constructing the suicide plasmid pCVD442 was described previously (10). Fusion PCR was performed to amplify a Gmr cassette with about 1 kb upstream and downstream of the EPEC insertion site at both ends, using primers yhjx5=Fw (AAA GGT CTC TAG ATT ACT CGT AAG ACA GAT TGA AAT CA), yhjX5=GmFw (CAA CAT TTT CTG TTC TGA GCT CGA ATT GGC), yhjX3=GmRv (ACG ATT CGT CAG CCA GAG


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(lpf1p::lacZ) in the wt EHEC strain 86-24, EPEC strain E2348/69, and the pEAF-cured strain JPN15. The experiments were performed in DMEM at different time points during the exponential growth phase. Shown are means and standard errors of the mean (SEM); n ⫽ 3. *, P ⬍ 0.05; **, P ⬍ 0.01. A t test was used to analyze the data.

CTC GAA TTG ACA T), yhjX3=GmFw (ATG TCA ATT CGA GCT CTG GCT GAC GAA TCG T), and yhjX3=Rw (AAA GGT CTC TAG ATG CTT TCT CTT TCG GCT TGT TAA G). The purified PCR fragments were treated with XbaI and ligated into XbaI-digested pCVD442. After transformation of DH5␣(␭pir) with the ligation mixture, Apr colonies were characterized by restriction analysis, and the right construct was transformed into SM10(␭pir). Conjugation was performed between the donor [SM10(␭pir)] and recipient (EPEC ⌬hns) strains. Overnight (O/N) colonies of the recipient and donor were cross-streaked on an LB plate and incubated at 37°C for 8 h. The double mutants were selected by isolating Kanr Gmr colonies. Correct insertion was confirmed by PCR (data not shown), using primers yhjxSeqF (ACC TGA GCA TTA CCA TAA CCA G) and yhjxSeqR (TGC TTT CTC TTT CGG CTT GTT AAG). In summary, EPEC ⌬hns is a derivative of EPEC strain E2348/69 carrying a kanamycin resistance marker in place of the deleted hns gene, while EPEC ⌬hns ⌬yhjX is derived from EPEC strain E2348/69 ⌬hns and carries a gentamicin resistance marker in place of the deleted yhjX gene. Bacterial adhesion to Caco-2 cells. (i) Cell preparation. For adhesion assays, Caco-2 cells were cultivated in 12-well plates. The cells were washed twice with minimal essential medium (MEM) without any supplement prior to infection. (ii) Bacterial suspension preparation. The strains were grown on LBDMEM to logarithmic phase (optical density at 600 nm [OD600] ⫽ 1.0) and diluted in MEM to a concentration of 1 ⫻ 108 CFU/ml. The media from the cell monolayers were then aspirated, and 500 ␮l of bacterial suspension (⬃5 ⫻ 107 CFU; multiplicity of infection [MOI] ⫽ 100) was added to each well. The bacterial suspension was serially diluted and plated to confirm the bacterial input. (iii) Measurement of adhesion. After 3 h, the monolayers were washed four times with phosphate-buffered saline (PBS) and then lysed with 200 ␮l of 0.1% Triton X-100 and plated on LB agar plates with the corresponding antibiotic. In vivo bacterial infections in mice. (i) Mice and treatment. Six- to 8-week-old female CD-1 (ICR) mice were purchased from Charles River Laboratories. The animals were housed in a specific-pathogen-free (SPF) barrier under biosafety level 2 conditions. Twenty-four hours before infection, the mice received streptomycin (5 g/liter) in their drinking water. The streptomycin-treated water also contained fructose (6.7%). Cimetidine (50 mg kg of body weight⫺1) was administered intraperitoneally to

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all the mice 1 h prior to inoculation with EPEC to reduce the effect of stomach acidity on the bacterial organisms. The animal studies were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals (34). The protocol (IACUC no. 0709042B) was approved by the Animal Care and Use Committee of the University of Texas Medical Branch. (ii) Infection dose. EPEC E2348/69, EPEC ⌬hns, EPEC ⌬hns Tn5, and EPEC ⌬hns ⌬yhjX were converted into Smr strains before the experiments. They were grown on LB medium overnight at 37°C. A bacterial suspension of 1 ⫻ 109 CFU was centrifuged and resuspended in 400 ␮l of PBS. Each animal received 1 ⫻ 109 CFU in 400 ␮l of PBS via oral gavage following the streptomycin treatment. Colonization was determined at 5 days (8 mice per group). (iii) Readout. After infection, the numbers of bacteria in the fecal pellets were monitored daily throughout the experiment. Feces were resuspended in PBS by vortexing, and bacteria were plated for enumeration. Sections of the small intestine, large intestine, and cecum were collected, homogenized, and plated on MacConkey agar containing streptomycin (50 ␮g ml⫺1) at 5 days postinfection. Colonies were counted after overnight incubation at 37°C. The colony counts were expressed as either CFU per gram of feces or CFU per gram of organ. Statistical analysis. One-way analysis of variance followed by Tukey’s posttest analysis was used to compare more than two groups, and multiplicity-adjusted P values are reported. A 95% confidence interval was used for most of the analyses. All analysis was performed using GraphPad Prism 6.0 (GraphPad Software Inc.) A P value of 0.05 or less was considered significant.

RESULTS

Expression of the EHEC lpf promoter (lpfpEHEC) is repressed in the EPEC background. To do an initial screen of lpf expression, the wild-type (wt) EPEC strain E2348/69, the pEAF-negative E2348/69 strain JPN15 (the pEAF plasmid encodes the Per regulators that are important for EPEC virulence), and EHEC strain 86-24 were transformed with the pLpfA-CAT plasmid (lpfA1p:: lacZ; the EHEC lpf1 promoter fused to the lacZ reporter gene), and ␤-galactosidase activity was assessed under in vitro conditions known to induce EHEC lpf expression. As shown in Fig. 1, ␤-ga-




FIG 1 The EHEC lpf1 promoter is repressed in the EPEC background. Shown are ␤-galactosidase assays using plasmids pRS551 (vector control) and pLpfA-CAT


lactosidase activity was enhanced in EHEC strain 86-24 during the late exponential phase of growth. No change in enzymatic activity was detected in the EPEC E2348/69 wt strain during the logarithmic phase of growth (Fig. 1), but a slight increase in expression was observed in strain JPN15. These results confirmed that additional regulators control lpf expression and that these regulatory elements can be encoded in the EPEC genome. We have previously reported the alignment of several lpf promoter regions found in EHEC and EPEC strains with the goal of identifying common regulatory elements present within these regions (16). While comparing the locations of the predicted transcriptional start site (TSS) and the ⫺10 and ⫺35 hexamers upstream of the TSS of EPEC O127:H6 and EHEC O157:H7, we found that both the TSS and hexamers were conserved. Similarly, a second predicted TSS, which we had previously identified in EHEC O157:H7, was conserved in EPEC, but the ⫺10 and ⫺35 hexamers were not (16). Although no major differences were observed in the structures of the two promoter regions that might suggest distinct regulatory mechanisms, in the current study, we found that the EHEC lpf1 promoter was under EPEC regulatory control, suggesting that perhaps an additional EPEC regulator(s) (e.g., lack of an activator or presence of a repressor) could impact the expression of EHEC lpf1. Expression of the lpf promoter from EPEC (lpfpEPEC) is significantly enhanced in the EHEC ⌬hns background. We previously showed that the expression of lpf in E. coli O157:H7 was controlled by a tightly regulated process and that H-NS is the global regulator binding to the regulatory region of the EHEC lpf1 operon, repressing its expression. Our initial testing using lacZ reporter plasmids resulted in a high background level that interfered with enzymatic activity measurements in lactose-positive EPEC and EHEC strains. Therefore, we decided to use CAT as our reporter system, and our initial measurements indicated that H-NS was a repressor of lpf in EPEC (Fig. 2A), supporting our previously published data for the EHEC strain (16). To determine whether there are other repressors of EPEC lpf, in addition to H-NS, we transformed the plasmid containing lpfpEPEC fused to the CAT reporter into both EPEC ⌬hns and EHEC ⌬hns. The results demonstrated that lpf expression is enhanced in both strains, but it is significantly lower in EPEC ⌬hns than in EHEC ⌬hns, which further confirmed the pres-


ence of another layer of regulatory control in the EPEC background (Fig. 2B). The transposon mutant has significantly enhanced CAT activity and lpf expression. To identify the regulatory factor(s), in addition to H-NS, further repressing the expression of EPEC lpf fimbrial genes, the mini-Tn5-lux transposon was conjugated into EPEC ⌬hns(pLpfA-CAT) using the donor strain SM10. After evaluating more than 1,000 colonies resulting from the transposon mutagenesis in two independent screens, we found about 50 colonies showing resistance to 35 ␮g/ml chloramphenicol. We further determined the CAT activities of all the colonies and identified one colony, initially named EPEC ⌬hns(pLpfA-CAT)::Tn5 (referred to below as EPEC ⌬hns Tn5), that had significantly higher CAT activity (Fig. 3A). Genomic analysis confirmed that the transposon mutant candidate had a single transposon insertion (data not shown). Real-time PCR was conducted to test the expression of EPEC lpf. We did two independent experiments and found that lpf expression was significantly enhanced in the isolated colony (4- to 5-fold increased compared with EPEC ⌬hns; 12- to 20-fold increased compared with EPEC) (Fig. 3B). The epigenetic effect of transposon insertion affects Lpf expression. After sequencing the transposon insertion, we found that the mutation was located immediately upstream of the EPEC lpf operon. We determined that there were 379 nucleotides between the transposon and the lpfA start codon, and the insertion was found within the 3= end of the yhjX gene, upstream of the lpfA gene. The yhjX gene is predicted to encode an inner membrane protein with 12 predicted transmembrane domains that belongs to the major facilitator superfamily (MFS) of transporters. In order to confirm that the gene product participates in the regulation of EPEC lpf, we constructed an isogenic yhjX mutant, disrupting the gene with a gentamicin resistance cassette. The mutant strain EPEC ⌬hns ⌬yhjX was evaluated by qRT-PCR analysis to define lpf expression, and it was shown that expression of lpf did not change and was similar to the levels observed in the EPEC ⌬hns strain (Fig. 4). Evaluation of the mutant strains’ adhesion properties using epithelial cells. The roles of the ⌬hns mutant strains in the interaction of EPEC with the intestinal epithelium were tested using adherence assays and comparing the wt EPEC strain with EPEC ⌬hns, EPEC ⌬hns Tn5, and EPEC ⌬hns ⌬yhjX. Reduction or in-


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FIG 2 The expression of lpfpEPEC is significantly enhanced in the EHEC ⌬hns background. (A) The overall fold change in CAT activity was examined in EPEC and EPEC ⌬hns. (B) Changes in CAT activity of EHEC ⌬hns and EPEC ⌬hns during different growth stages were also compared. Blank, empty control. *, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001; ****, P ⬍ 0.0001. Shown are means and SEM; n ⫽ 3. A t test was used to analyze the data.

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lpf was significantly enhanced in EPEC ⌬hns Tn5(pLpfA-CAT) compared to EPEC ⌬hns(pLpfA-CAT) and EPEC(pLpfA-CAT). Abs, absorbance. (B) Relative lpf mRNA expression. *, P ⬍ 0.05; **, P ⬍ 0.01. Shown are means and SEM; n ⫽ 4. One-way ANOVA was used to analyze the data.

crease in adhesion was expressed in comparison to the levels observed with the wt EPEC strain, which was defined as 1-fold. The interaction of EPEC strains with nonpolarized Caco-2 cells showed that the adherence level of the EPEC ⌬hns strain was increased 9-fold compared to the wt strain levels (P ⬍ 0.0001) (Fig. 5). However, at this point, we cannot rule out the possibility that increased adhesion is due to derepression of lpf, as well as other H-NS-regulated EPEC adhesins. The EPEC ⌬hns Tn5 transposon mutant further displayed enhancement in adherence of about 10fold compared to the wt strain. In contrast, we observed that EPEC ⌬hns ⌬yhjX did not show any significant adherence difference compared to the EPEC ⌬hns strain, which confirmed that the absence of the YhjX protein did not impact adherence associated with increased expression of Lpf. Effects of the hns deletion mutants on colonization of the mouse intestine. We next determined the contributions of the EPEC hns mutants to colonization of the CD-1 mouse intestine. Groups of mice were infected intragastrically with 1.5 ⫻ 109 CFU of the EPEC wt strain, EPEC ⌬hns, EPEC ⌬hns Tn5, or EPEC ⌬hns ⌬yhjX. The fecal shedding of the mice was measured every day (Fig. 6A). During the postinfection monitoring stage, fecal shedding of EPEC ⌬hns Tn5 was significantly lower than that of the EPEC wt and the other strains early during infection, and the numbers of bacteria of the strain excreted did not drop as drastically as with the other 3 strains on the fourth and the fifth days

FIG 4 lpf expression does not further increase in EPEC ⌬hns ⌬yhjX. Real-time

RT-PCR was used to evaluate EPEC(pLpfA-CAT), EPEC ⌬hns(pLpfA-CAT), and EPEC ⌬hns ⌬yhjX(pLpfA-CAT). *, P ⬍ 0.05; ns, P ⬎ 0.05. Shown are means and SEM; n ⫽ 4. One-way ANOVA was used to analyze the data.

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postinfection. Although the mean number of EPEC ⌬hns Tn5 bacteria is a log unit lower early during infection than those of EPEC wt bacteria and the other strains of bacteria, there were no statistically significant differences between the groups. Overall, the fecal shedding indicated that 3 of the strains were eliminated in higher numbers than the EPEC ⌬hns Tn5 strain (the number of EPEC ⌬hns Tn5 bacteria was reduced 2 log units during the first 5 days postinfection, while the numbers of EPEC wt and ⌬hns ⌬yhjX bacteria dropped 4 log units and the number of ⌬hns bacteria dropped 3 log units during the same period), and this could suggest that EPEC ⌬hns Tn5 is not eliminated from the intestine because it colonizes better. Therefore, colonization in different intestinal sections was measured at day 5 postinfection (Fig. 6B). In the small intestine, the mean colonization value of EPEC ⌬hns was higher than that of the wt strain; however, there was no statistically significant difference between the two groups. In contrast, the colonization of the EPEC ⌬hns strain was statistically different from those of the other 2 mutants (P ⱕ 0.05; analysis of variance [ANOVA]; P value ⫽

FIG 5 In vitro adhesion of the EPEC wt or hns mutants to Caco-2 cells. For all the experiments, an MOI of 100 was used, and bacteria were recovered and quantified at 3 h postinfection. Shown are means and SEM; n ⫽ 6. ns, P ⬎ 0.05; *, P ⱕ 0.05; ***, P ⱕ 0.001 for comparisons between groups. One-way ANOVA followed by Tukey’s multiple-comparisons was used to analyze the data.




FIG 3 CAT activity and lpf expression were significantly enhanced in the EPEC ⌬hns Tn5 strain. (A) CAT activity showed that transcriptional expression of EPEC


gram were calculated. (B to D) Colonization of the small intestine (B), cecum (C), and large intestine (D). Intestinal segments were homogenized, and the numbers of CFU per gram were quantified at day 5 postinfection. Shown are means and SEM; n ⫽ 8. ns, P ⬎ 0.05; *, P ⱕ 0.05 for comparisons between groups. One-way ANOVA followed by Tukey’s multiple comparisons was used to analyze the data.

0.0162). In the cecum, EPEC ⌬hns and EPEC ⌬hns Tn5 strain colonization mean values were higher than that of the wt strain; however, there were no statistically significant differences between the groups. Finally, in the large intestine, there were no significant differences between the EPEC wt, EPEC ⌬hns, and EPEC ⌬hns Tn5. The only statistically significant difference observed was between the EPEC wt and EPEC ⌬hns ⌬yhjX (P ⱕ 0.05; ANOVA; P value ⫽ 0.0147). Overall, our results indicated that differences in intestinal colonization were minimal between the EPEC wt and the three mutants tested and that the potential adhesins deregulated in the EPEC ⌬hns strain might play a role in increased colonization of the small intestine. DISCUSSION

Multiple adhesins have been identified in EPEC; for example, intimin has been unequivocally found to be involved in intimate attachment in EPEC and other AEEC strains, and as such, the hallmark of EPEC pathogenesis is the formation of the A/E histopathological lesion (22). It has been reported that the EPEC E2348/69 ⌬eae ⌬espA double mutant totally lost its ability to adhere to HeLa cells (23). However, there is not a clear picture of how EPEC utilizes other adhesins to interact with the intestinal epithelium early or later during infection. Initial studies suggested that the bundle-forming pilus (Bfp) participates in the initial intestinal interaction, but later experiments demonstrated that Bfp


stabilizes microcolony formation (24). Recent studies suggested that the E. coli common pilus (ECP) and the product of the relA gene contribute to the interaction of EPEC with host epithelial cells (25, 26); however, additional experiments are required to confirm the roles of these adhesins in vivo. Here, we mainly discuss the roles of H-NS-regulated products and Lpf in adhesion and colonization of the intestinal tract. It is well established that H-NS regulates virulence factors in different pathogens, but there are a wide variety of phenotypes associated with the colonization and virulence of pathogenic bacteria lacking the hns gene product. S. enterica serovar Typhimurium strain C5 displayed a mucoid phenotype and showed attenuation when infecting mice (27). A Vibrio cholerae ⌬hns strain exhibited reduced motility, enhanced in vivo production of virulence factors, and lower colonization efficiency (28). In the plant pathogen Erwinia chrysanthemi, the ⌬hns mutant strain displayed a reduced virulence phenotype (29). In the Shiga toxin-producing E. coli O91:H21 strain B2F1, a mutation in hns reduced adherence to human colonic epithelial cells (30). However, in an EHEC O157:H7 strain, the hns mutation increased adherence to epithelial cells (17). Finally, the uropathogenic E. coli hns mutant displayed ambiguous effects (31). At a high infection dose, the hns mutant triggered more sudden death. At a lower infectious dose, the mutant was attenuated and displayed an impaired growth rate.


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FIG 6 EPEC levels in feces and intestinal segments during 5 days postinfection. (A) Feces were collected for 5 days postinfection, and the numbers of CFU per

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In contrast to the EHEC lpf1 locus, the EPEC lpf operon and regulatory regions are intact, and there is no apparent reason for the fimbriae not to be expressed in the EPEC background (15). As such, we found that the EPEC ⌬hns and ⌬hns Tn5 strains showed significant increases in lpf expression with slightly increased colonization in the cecum. Surprisingly, EPEC ⌬hns ⌬yhjX colonization was decreased in both the small and large intestine. Initial analysis of the function of YhjX, a predicted membrane transporter and an acid-resistant protein, did not provide any clues about its participation in colonization. With respect to the EPEC ⌬hns and ⌬hns Tn5 strains, further analysis needs to be performed to define the contributions of these mutations to increased lpf expression. For example, we need to determine whether changes in the structure of the regulatory region due to the transposon insertion prevent binding of repressors or enhanced binding of activators, mediating lpf expression. Unfortunately, at this moment, we are unable to provide satisfying answers to this conundrum. Enteropathogenic E. coli causes acute infection in humans, and the pathogen utilizes a plethora of adhesins to guarantee intestinal colonization (2, 24). We have demonstrated that lpf in EPEC strain 2348/69 is a highly regulated operon that might function as an adhesin when expressed, and as such, our study found that lpf expression is enhanced in the EPEC ⌬hns strain. Because Lpf does not seems to play a significant role in initial colonization, we speculate that EPEC E2348/69 or other lpf-containing EPEC strains possess other H-NS-regulated adhesion mechanisms mediating initial adhesion while repressing lpf, and therefore, expression of this fimbria might confer an advantage later during the process of infection or during colonization of other niches or environmental reservoirs. ACKNOWLEDGMENTS We thank Jose Luis Puente for sharing the EPEC ⌬hns mutant strain with us. This work was partially supported by NIH/NIAID grant AI079154 to A.G.T. The contents are solely our responsibility and do not necessarily represent the official views of the NIAID or NIH.

REFERENCES 1. Kaper JB, Nataro JP, Mobley HL. 2004. Pathogenic Escherichia coli. Nat Rev Microbiol 2:123–140. http://dx.doi.org/10.1038/nrmicro818. 2. Hu J, Torres AG. 28 January 2015. Enteropathogenic Escherichia coli: foe or innocent bystander? Clin Microbiol Infect http://dx.doi.org/10.1016/j .cmi.2015.01.015. 3. Ochoa TJ, Barletta F, Contreras C, Mercado E. 2008. New insights into the epidemiology of enteropathogenic Escherichia coli infection. Trans R Soc Trop Med Hyg 102:852– 856. http://dx.doi.org/10.1016/j.trstmh.2008 .03.017. 4. Ochoa TJ, Contreras CA. 2011. Enteropathogenic Escherichia coli infection in children. Curr Opin Infect Dis 24:478 – 483. http://dx.doi.org/10 .1097/QCO.0b013e32834a8b8b. 5. Kotloff KL, Nataro JP, Blackwelder WC, Nasrin D, Farag TH, Panchalingam S, Wu Y, Sow SO, Sur D, Breiman RF, Faruque AS, Zaidi AK, Saha D, Alonso PL, Tamboura B, Sanogo D, Onwuchekwa U, Manna B, Ramamurthy T, Kanungo S, Ochieng JB, Omore R, Oundo JO, Hossain A, Das SK, Ahmed S, Qureshi S, Quadri F, Adegbola RA, Antonio M, Hossain MJ, Akinsola A, Mandomando I, Nhampossa T, Acácio S, Biswas K, O’Reilly CE, Mintz ED, Berkeley LY, Muhsen K, Sommerfelt H, Robins-Browne RM, Levine MM. 2013. Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-control study. Lancet 382:209 –222. http://dx.doi.org/10.1016/S0140 -6736(13)60844-2.




In our current in vitro experiments, the adhesion of the EPEC ⌬hns mutant increased compared to the wt EPEC strain. However, the in vivo experiment showed that there is no significant difference between wt EPEC and EPEC ⌬hns strains. It is plausible to propose that in vivo regulation of adhesins in the hns mutant is different from conditions in vitro, and therefore, the expression level of the adhesins in the hns mutant may be further regulated in vivo. Lpf constitutes an important colonization factor of EHEC O157:H7 strains (6, 32). Our previous publications showed that E. coli O157:H7 harboring a mutation in hns caused derepression of the lpf1 locus and that Ler acts as an antisilencer, removing H-NS from the lpf promoter region (16–18). In the current study, we also found that H-NS is a silencer of lpf in EPEC. We did not study the role of Ler in EPEC lpf regulation because in the absence of the H-NS protein, Ler does not have an effect on Lpf. Because hns is a global regulator modulating the expression of several adhesins and virulence factors and we did not observe complete derepression of EPEC lpf, it was a logical approach to try to identify the second repressor of lpf. After screening a transposon mutant library, we found one candidate that had significantly enhanced expression of EPEC lpf. After identification of the disrupted gene and further construction of an in-frame deletion, RT-PCR showed that insertion due to the transposon instead of clean disruption of the yhjX gene was associated with enhanced expression of EPEC lpf. We propose that the transposon insertion might be causing some epigenetic effects that changed the expression of Lpf. We speculate that the transposon insertion caused structural changes in the lpf regulatory region, either allowing the binding of an activator or preventing the access of a repressor; however, further experiments are required to confirm this hypothesis. Regardless of this unsolved mechanistic issue, we showed that overexpression of EPEC lpf significantly increased bacterial adhesion to Caco-2 cells, which suggested that Lpf might participate in the adhesion process. In contrast, the in vivo results were quite different. The initial fecal shedding of EPEC ⌬hns Tn5 was significantly lower than that of the other strains, which might suggest that the strain is not cleared from the intestine as fast as the other strains and that Lpf fimbriae might contribute to the initial in vivo colonization. The fecal shedding of EPEC ⌬hns Tn5 was reduced by 2 log units during the first 5 days postinfection, while the EPEC wt and ⌬hns ⌬yhjX dropped 4 log units and ⌬hns dropped 3 log units during the same period, indicating that adhesins derepressed in the transposon mutant strain might contribute to further persistence in the intestine. When the intestinal segments were analyzed, we found that the mean values of EPEC ⌬hns and ⌬hns Tn5 mutants’ colonization of the cecum were slightly enhanced (no statistical differences), and there was no difference in the large intestine compared to the EPEC wt strain. In contrast, the EPEC ⌬hns mutant colonized the small intestine better than the other 3 strains. Our interpretation of the results is that the expression levels of lpf or other H-NS-regulated adhesins might not play a significant role during colonization of the murine intestine; however, the H-NS-regulated colonization factors might confer an advantage on the EPEC ⌬hns mutant in colonizing the small intestine, which is the intestinal site colonized by EPEC strains. We cannot rule out the possibility that EPEC Lpf plays a role later during colonization of the intestinal tract, and therefore, we were unable to establish meaningful differences during intestinal infection.



20.

21.

22.

23.

24. 25.

26. 27.

28.

29.

30.

31.

32.

33.

34.

Escherichia coli O157:H7. Int J Med Microbiol 297:177–185. http://dx.doi .org/10.1016/j.ijmm.2007.01.005. Lewenza S, Falsafi RK, Winsor G, Gooderham WJ, McPhee JB, Brinkman FS, Hancock RE. 2005. Construction of a mini-Tn5-luxCDABE mutant library in Pseudomonas aeruginosa PAO1: a tool for identifying differentially regulated genes. Genome Res 15:583–589. http://dx.doi.org /10.1101/gr.3513905. Martinez-Laguna Y, Calva E, Puente JL. 1999. Autoactivation and environmental regulation of bfpT expression, the gene coding for the transcriptional activator of bfpA in enteropathogenic Escherichia coli. Mol Microbiol 33:153–166. http://dx.doi.org/10.1046/j.1365-2958.1999.01460.x. Bueris V, Huerta-Cantillo J, Navarro-Garcia F, Ruiz RM, Cianciarullo AM, Elias WP. 2015. Late establishment of the attaching and effacing lesion caused by atypical enteropathogenic Escherichia coli depends on protein expression regulated by Per. Infect Immun 83:379 –388. http://dx .doi.org/10.1128/IAI.02563-14. Hernandes RT, De la Cruz MA, Yamamoto D, Giron JA, Gomes TA. 2013. Dissection of the role of pili and type 2 and 3 secretion systems in adherence and biofilm formation of an atypical enteropathogenic Escherichia coli strain. Infect Immun 81:3793–3802. http://dx.doi.org/10.1128 /IAI.00620-13. Torres AG, Zhou X, Kaper JB. 2005. Adherence of diarrheagenic Escherichia coli strains to epithelial cells. Infect Immun 73:18 –29. http://dx.doi .org/10.1128/IAI.73.1.18-29.2005. Saldana Z, Erdem AL, Schuller S, Okeke IN, Lucas M, Sivananthan A, Phillips AD, Kaper JB, Puente JL, Girón JA. 2009. The Escherichia coli common pilus and the bundle-forming pilus act in concert during the formation of localized adherence by enteropathogenic E. coli. J Bacteriol 191:3451–3461. http://dx.doi.org/10.1128/JB.01539-08. Spira B, Ferreira GM, de Almeida LG. 2014. relA enhances the adherence of enteropathogenic Escherichia coli. PLoS One 9:e91703. http://dx.doi .org/10.1371/journal.pone.0091703. Harrison JA, Pickard D, Higgins CF, Khan A, Chatfield SN, Ali T, Dorman CJ, Hormaeche CE, Dougan G. 1994. Role of hns in the virulence phenotype of pathogenic salmonellae. Mol Microbiol 13:133–140. http://dx.doi.org/10.1111/j.1365-2958.1994.tb00408.x. Ghosh A, Paul K, Chowdhury R. 2006. Role of the histone-like nucleoid structuring protein in colonization, motility, and bile-dependent repression of virulence gene expression in Vibrio cholerae. Infect Immun 74: 3060 –3064. http://dx.doi.org/10.1128/IAI.74.5.3060-3064.2006. Nasser W, Faelen M, Hugouvieux-Cotte-Pattat N, Reverchon S. 2001. Role of the nucleoid-associated protein H-NS in the synthesis of virulence factors in the phytopathogenic bacterium Erwinia chrysanthemi. Mol Plant Microbe Interact 14:10 –20. http://dx.doi.org/10.1094/MPMI.2001 .14.1.10. Scott ME, Melton-Celsa AR, O’Brien AD. 2003. Mutations in hns reduce the adherence of Shiga toxin-producing E. coli 091:H21 strain B2F1 to human colonic epithelial cells and increase the production of hemolysin. Microb Pathog 34:155–159. http://dx.doi.org/10.1016/S0882-4010(03)00002-0. Muller CM, Dobrindt U, Nagy G, Emody L, Uhlin BE, Hacker J. 2006. Role of histone-like proteins H-NS and StpA in expression of virulence determinants of uropathogenic Escherichia coli. J Bacteriol 188:5428 – 5438. http://dx.doi.org/10.1128/JB.01956-05. Lloyd SJ, Ritchie JM, Torres AG. 2012. Fimbriation and curliation in Escherichia coli O157:H7: a paradigm of intestinal and environmental colonization. Gut Microbes 3:272–276. http://dx.doi.org/10.4161/gmic .20661. Philippe N, Alcaraz JP, Coursange E, Geiselmann J, Schneider D. 2004. Improvement of pCVD442, a suicide plasmid for gene allele exchange in bacteria. Plasmid 51:246 –255. http://dx.doi.org/10.1016/j.plasmid.2004 .02.003. National Research Council. 2011. Guide for the care and use of laboratory animals, 8th ed. National Academies Press, Washington, DC.


jb.asm.org

3665


6. Farfan MJ, Torres AG. 2012. Molecular mechanisms that mediate colonization of Shiga toxin-producing Escherichia coli strains. Infect Immun 80:903–913. http://dx.doi.org/10.1128/IAI.05907-11. 7. Lloyd SJ, Ritchie JM, Rojas-Lopez M, Blumentritt CA, Popov VL, Greenwich JL, Waldor MK, Torres AG. 2012. A double, long polar fimbria mutant of Escherichia coli O157:H7 expresses Curli and exhibits reduced in vivo colonization. Infect Immun 80:914 –920. http://dx.doi .org/10.1128/IAI.05945-11. 8. Fitzhenry R, Dahan S, Torres AG, Chong Y, Heuschkel R, Murch SH, Thomson M, Kaper JB, Frankel G, Phillips AD. 2006. Long polar fimbriae and tissue tropism in Escherichia coli O157:H7. Microbes Infect 8:1741–1749. http://dx.doi.org/10.1016/j.micinf.2006.02.012. 9. Jordan DM, Cornick N, Torres AG, Dean-Nystrom EA, Kaper JB, Moon HW. 2004. Long polar fimbriae contribute to colonization by Escherichia coli O157:H7 in vivo. Infect Immun 72:6168 – 6171. http://dx.doi .org/10.1128/IAI.72.10.6168-6171.2004. 10. Torres AG, Giron JA, Perna NT, Burland V, Blattner FR, AvelinoFlores F, Kaper JB. 2002. Identification and characterization of lpfABCC=DE, a fimbrial operon of enterohemorrhagic Escherichia coli O157:H7. Infect Immun 70:5416 –5427. http://dx.doi.org/10.1128/IAI.70.10.5416 -5427.2002. 11. Svab D, Galli L, Horvath B, Maroti G, Dobrindt U, Torres AG, Rivas M, Tóth I. 2013. The long polar fimbriae operon and its flanking regions in bovine Escherichia coli O157:H43 and STEC O136:H12 strains. Pathog Dis 68:1–7. http://dx.doi.org/10.1111/2049-632X.12038. 12. Galli L, Torres AG, Rivas M. 2010. Identification of the long polar fimbriae gene variants in the locus of enterocyte effacement-negative Shiga toxin-producing Escherichia coli strains isolated from humans and cattle in Argentina. FEMS Microbiol Lett 308:123–129. http://dx.doi.org /10.1111/j.1574-6968.2010.01996.x. 13. Chassaing B, Rolhion N, de Vallee A, Salim SY, Prorok-Hamon M, Neut C, Campbell BJ, Söderholm JD, Hugot JP, Colombel JF, Darfeuille-Michaud A. 2011. Crohn disease-associated adherent-invasive E. coli bacteria target mouse and human Peyer’s patches via long polar fimbriae. J Clin Invest 121:966 –975. http://dx.doi.org/10.1172/JCI44632. 14. Chassaing B, Etienne-Mesmin L, Bonnet R, Darfeuille-Michaud A. 2013. Bile salts induce long polar fimbriae expression favouring Crohn’s disease-associated adherent-invasive Escherichia coli interaction with Peyer’s patches. Environ Microbiol 15:355–371. http://dx.doi.org/10.1111/j .1462-2920.2012.02824.x. 15. Tatsuno I, Mundy R, Frankel G, Chong Y, Phillips AD, Torres AG, Kaper JB. 2006. The lpf gene cluster for long polar fimbriae is not involved in adherence of enteropathogenic Escherichia coli or virulence of Citrobacter rodentium. Infect Immun 74:265–272. http://dx.doi.org/10.1128/IAI .74.1.265-272.2006. 16. Torres AG, Lopez-Sanchez GN, Milflores-Flores L, Patel SD, RojasLopez M, Martinez de la Pena CF, Arenas-Hernández MM, MartínezLaguna Y. 2007. Ler and H-NS, regulators controlling expression of the long polar fimbriae of Escherichia coli O157:H7. J Bacteriol 189:5916 – 5928. http://dx.doi.org/10.1128/JB.00245-07. 17. Torres AG, Slater TM, Patel SD, Popov VL, Arenas-Hernandez MM. 2008. Contribution of the Ler- and H-NS-regulated long polar fimbriae of Escherichia coli O157:H7 during binding to tissue-cultured cells. Infect Immun 76:5062–5071. http://dx.doi.org/10.1128/IAI.00654-08. 18. Rojas-López M, Arenas-Hernández MM, Medrano-López A, Martínez de la Peña CF, Puente JL, Martínez-Laguna Y, Torres AG. 2011. Regulatory control of the Escherichia coli O157:H7 lpf1 operon by H-NS and Ler. J Bacteriol 193:1622–1632. http://dx.doi.org/10.1128 /JB.01082-10. 19. Torres AG, Milflores-Flores L, Garcia-Gallegos JG, Patel SD, Best A, La Ragione RM, Martinez-Laguna Y, Woodward MJ. 2007. Environmental regulation and colonization attributes of the long polar fimbriae (LPF) of

The Role of Long Polar Fimbriae in Escherichia coli O104:H4 Adhesion and Colonization.

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Enterohemorrhagic Escherichia coli pathogenesis: role of Long polar fimbriae in Peyer's patches interactions.

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