Isolimonic acid interferes with Escherichia coli O157:H7 biofilm and TTSS in QseBC and QseA dependent fashion
© Vikram et al.; licensee BioMed Central Ltd. 2012
Received: 30 March 2012
Accepted: 1 October 2012
Published: 15 November 2012
E. coli O157:H7 (EHEC) is an important human pathogen. The antibiotic treatment of EHEC reportedly results in release of Shiga toxin and is therefore discouraged. Consequently, alternative preventive or therapeutic strategies for EHEC are required. The objective of the current study was to investigate the effect of citrus limonoids on cell-cell signaling, biofilm formation and type III secretion system in EHEC.
Isolimonic acid and ichangin were the most potent inhibitors of EHEC biofilm (IC25=19.7 and 28.3 μM, respectively) and adhesion to Caco-2 cells. The qPCR analysis revealed that isolimonic acid and ichangin repressed LEE encoded genes by ≈3 to 12 fold. In addition, flhDC was repressed by the two limonoids (≈3 to 7 fold). Further studies suggested that isolimonic acid interferes with AI-3/epinephrine activated cell-cell signaling pathway. Loss of biofilm inhibitory activity of isolimonic acid in ΔqseBC mutant, which could be restored upon complementation, suggested a dependence on functional QseBC. Additionally, overexpression of qseBC in wild type EHEC abated the inhibitory effect of isolimonic acid. Furthermore, the isolimonic acid failed to differentially regulate ler in ΔqseA mutant, while plasmid borne expression of qseA in ΔqseA background restored the repressive effect of isolimonic acid.
Altogether, results of study seem to suggest that isolimonic acid and ichangin are potent inhibitors of EHEC biofilm and TTSS. Furthermore, isolimonic acid appears to interfere with AI-3/epinephrine pathway in QseBC and QseA dependent fashion.
KeywordsQuorum sensing Natural products Limonoids Citrus Escherichia coli O157:H7 LEE Epinephrine
Enterohaemorrhagic Escherichia coli (EHEC) is a major foodborne pathogen associated with frequent outbreaks of diarrheal disease. Most individuals develop watery diarrhea and recover. However, about 15–20% cases may develop life-threatening bloody diarrhea and hemolytic uremic syndrome (HUS) [1, 2]. Dissemination and contact of humans with EHEC from multiple sources such as undercooked meats, raw fruits and vegetables, physical contact with EHEC harboring animals further contribute to increased frequency of illness [2, 3].
EHEC is usually ingested through contaminated food products. Once inside the host, EHEC traverses to colon and establishes itself in the distal ileum or large bowel. Inside the colon, EHEC is thought to use guided motility, provided by flagellar motion, to reach its preferred site of attachment . Autoinducer molecules (AI-2/AI-3) and hormones (epinephrine/norepinephrine) induce various virulence factors and are speculated to help in attachment and subsequent infection process . A two-component system QseBC  induces flagellar operon in response to hormones and AI-2/AI-3, resulting in increased and guided motility  towards epithelial cell layer. Upon encountering the epithelial cell layer, the flagella and other surface structures such as type 1 pili and hemorrhagic coli pilus help EHEC to attach to the surface [7–9]. Multiple environmental and genetic factors such as pH, hormones, signaling molecules as well as quorum sensing (QS) regulate the expression of Locus of enterocyte effacement (LEE) and flagellar operons [10–13]. The hormones and AI-3 also induce type III secretion system (TTSS) in EHEC through QseEF and QseAD [14, 15]. TTSS is encoded in LEE, which is organized in five operons LEE1-LEE5. LEE1-encoded regulator (Ler) is the first gene on LEE1 operon and subject to modulation by various regulators. In turn, Ler activates the transcription of the five operons [13, 15, 16].
The TTSS penetrates the host cell membrane and serves as conduit for injecting effector proteins. These effector proteins manipulate the host machinery including actin cytoskeleton, resulting in attaching and effacing lesions. Some of the secreted effectors disrupt the tight junction leading to higher secretion of chloride ions and ultimately developing in diarrhea . The phage encoded Shiga toxin is the main virulence factor of EHEC and other Shiga toxin producing E. coli. The Shiga toxin disrupts the protein synthesis in host epithelial cells causing necrosis and cell death . Additionally, Shiga toxin travels to kidney through blood stream and damages renal endothelial cells inciting renal inflammation, potentially leading to HUS [2, 18]. Along with the direct injury to epithelial cells, biofilms formed by pathogenic E. coli strains can pose serious health problems such as prostatitis, biliary tract infections, and urinary catheter cystitis .
Antibiotics and antidiarrheal drug therapy of EHEC activates the stress response resulting in induction of phage lytic cycle and subsequent release of Shiga toxin. The release of Shiga toxin is directly correlated with increase in HUS incidence [2, 18]. At present, CDC recommends preventive measures such as washing hands and thorough cooking of meats etc. to control EHEC infections. However, these preventive measures need to be supported with alternative strategies for prevention and control of EHEC infections. A promising strategy is to identify anti-virulence agents, which may be used alone or in conjunction with antibiotic therapy . Anti-virulence agents target bacterial virulence determinants including toxin production, adhesion to host cells, specialized secretion systems such as TTSS . Application of anti-virulence agents is speculated to allow host immune system to prevent or clear the bacterial infection. Several synthetic and natural molecules with anti-virulence properties have been discovered [20, 21] and at least one molecule, LED209, was shown to be effective in animal models . However, none of the molecules have entered wide-scale clinical trial as of yet, owing to various concerns such as their toxicity and safety. Therefore, there is an urgent need to identify a more diverse pool of molecules with anti-virulence activities. Availability of such a pool will ensure better drug designing strategies, to combat bacterial infections like EHEC.
Secondary metabolites produced by plants present very diverse scaffolds, which have been used for designing novel drugs including antimicrobials. In nature, secondary metabolites contribute to systemic and induced plant defense system against insect, bacterial and fungal infestation . Several secondary metabolites belonging to classes such as coumarins, flavonoids, terpenoids and alkaloids demonstrate inhibitory properties against numerous microorganisms. Recently our group and others identified QS inhibitory properties of several plant secondary metabolites and extracts rich in phytochemicals [23–28].
Previously purified isolimonic acid, ichangin, isoobacunoic acid, IOAG and DNAG were used in the present study . Purity of the individual limonoids was calculated from percent peak area using high performance liquid chromatography (HPLC) analysis . A stock solution was prepared by dissolving 20 mg of each purified limonoid in 1 ml of dimethyl sulfoxide (DMSO).
Bacterial strains and plasmids
Bacterial Strains used in the study
E. coli O157:H7 EDL933
E. coli TE2680 LEE1:lacZ
E. coli TE2680 LEE2:lacZ
EHEC 86–24 ΔqseA
VS145 with plasmid pVS150
EHEC 86–24 ΔqseC
VS138 with plasmid pVS178
WT with plasmid pVS178
WT with pVS150
TEVS232 with pVS150
WT with pAV11
WT with pAV12
qseA into pACYC177
E. coli K12 qseBC in pBAD33
EHEC qseC in pBAD33
EHEC qseB in pBAD33
Growth and metabolic activity
The growth and metabolic activity of EHEC was measured as previously described . Briefly, overnight cultures of EHEC were diluted 100 fold in LB media. Two hundred microliters of diluted cultures was placed in each well of 96-well plates and grown for 16 h at 37°C in presence of 6.25, 12.5, 50, or 100 μg/ml limonoids or equivalent volume of DMSO. The plates were constantly shaken at medium speed in Synergy™ HT Multi-Mode Microplate Reader (BioTek, Instruments, Winooski, VT). OD600 was recorded every 15 min. Metabolic activity of EHEC was measured by adding AlamarBlue (25 μl/well) and absorption at 570 and 600 nm was monitored in similar fashion as growth curve.
EHEC biofilms were grown in polystyrene 96-well plates by plating 200 μl/well of 100 fold diluted overnight cultures in presence of 6.25, 12.5, 50, or 100 μg/ml of limonoids at 26°C for 24 h without shaking [23, 39]. For VS138 (ΔqseC) and VS179 (VS138 + qseBC) biofilms were quantified after 48 h growth in 96-well plates. The biofilms were quantified by staining with 0.3% crystal violet (Fisher, Hanover Park, IL) for 20 min. Extra stain was washed with phosphate buffer (0.1 M, pH 7.4) and dye associated with attached biofilm was dissolved with DMSO. An absorbance at 570 nm was used to quantify the total biofilm mass.
In vitro adhesion assay
Human epithelial Caco-2 cells were purchased from ATCC (Manassas, VA) and maintained in Dulbecco’s Minimal Essential Medium (DMEM) with nonessential amino acids and 10% fetal bovine serum without antibiotics. Caco-2 cells were seeded at 1 × 105 cells/well in 6-well plates and infected with approximately 5 × 106 cells/well of freshly grown EHEC ATCC 43895 in presence or absence of 100 μg/ml isolimonic acid, ichangin, isoobacunoic acid, IOAG and DNAG. The plates were incubated for 3 h at 37°C in 5% CO2 environment. After completion of incubation, plates were washed 3× with sterile PBS to remove any loosely attached cells. Caco-2 cells were lysed with 0.1% Triton-X in PBS to release the bacteria and serial dilutions were plated on LB-agar and incubated at 37°C for 24 h. Colonies were counted after incubation period and presented as log10CFU/ml.
Caco-2 cell survival assay
Caco-2 cells (1 × 104/well) were seeded in 96-well plate and exposed to 100 μg/ml of isolimonic acid, ichangin, isoobacunoic acid, IOAG and DNAG for 6 h in humidified incubator at 5% CO2, 37°C. Cell survival was determined by measuring lactate dehydrogenase using CytoTox-ONE™ Homogeneous Membrane Integrity Assay (Promega Corp., Madison, WI).
Sequences of the Primers used in this study
AI-3 reporter assay
Preconditioned media (PM) was prepared as described . Overnight cultures of TEVS232, TEVS21 and AV45 (EHEC ATCC 43895 harboring pVS150) were diluted 100 fold in LB medium and grown till OD600 ≈0.2. The cells were collected by centrifugation at 2500 × g for 10 min and resuspended in either fresh LB media supplemented with 50 μM epinephrine or PM and treated with 100 μg/ml isolimonic acid or equivalent amount of DMSO. The β-galactosidase activity was measured after 30 min incubation at 37°C using o-nitrophenyl β-D-galactopyranoside as previously described  and reported as mean ± SD of three replicates.
Percent inhibition of biofilm formation was calculated from three experiments consisting of three replicate wells using the formula 100- [(OD570 of sample well/ OD570 of positive control) × 100]. Effects of different limonoids for each activity were analyzed using analysis of variance (ANOVA) followed by Tukey’s pairwise multiple comparison test on SPSS 16.0 (SPSS Inc., Chicago, IL, USA). The effect was considered significant at p <0.05. The data for EHEC biofilm was fitted to a 3-parameter sigmoid models y= a/(1+exp(−(x-x0)/b)) using SIGMAPLOT 11.0 (Systat Software, Inc.). In order to conduct the analysis, concentration of each limonoids was converted to Log10 μM and plotted against percent inhibition values.
Effect of citrus limonoids on EHEC growth and biofilm formation
Generation time (in minutes) of E. coli O157:H7 upon exposure of different concentrations of limonoids
23.56 ± 0.71
23.11 ± 0.76
22.97 ± 0.96
23.65 ± 0.95
23.58 ± 1.06
22.96 ± 1.06
24.90 ± 1.82
22.97 ± 0.97
23.12 ± 0.92
23.16 ± 0.93
23.27 ± 1.09
23.64 ± 1.08
23.62 ± 2.47
23.58 ± 1.19
23.26 ± 1.23
22.58 ± 1.26
23.68 ± 0.91
23.51 ± 1.26
23.68 ± 1.84
23.54 ± 1.01
22.69 ± 1.09
23.12 ± 1.08
23.97 ± 1.31
23.69 ± 1.32
23.91 ± 0.63
23.70 ± 1.09
23.90 ± 1.02
23.55 ± 1.05
23.61 ± 1.05
23.76 ± 1.01
Effect of limonoids on adhesion of EHEC to Caco-2 cells
Citrus limonoids repress the LEE, flagellar and stx2 genes
Expression of LEE encoded, flagellar and stx2 genes in presence of 100 μg/ml limonoids
Shiga toxin produced by EHEC is responsible for HUS . We were further interested in learning if any of the limonoids modulate expression of stx2. Isolimonic acid and ichangin (100 μg/ml) repressed the stx2 by 4.9 and 2.5 fold, respectively (Table 4), while IOAG, isoobacunoic acid and DNAG did not seem to affect the expression of stx2.
Effect of isolimonic acid on AI-3/epinephrine induced LEE expression
QseBC dependent inhibition of biofilm by isolimonic acid
QseA dependent inhibition of ler by isolimonic acid
EHEC is an important gastrointestinal pathogen, prolific biofilm former and demonstrates resistance to various antimicrobials in biofilm mode of growth . For successful colonization of gastrointestinal tract and initiation of infection, adhesion of EHEC to intestinal epithelium is an essential early event [47, 48]. Additionally, several E. coli pathovars were reported to produce and live in biofilms inside the human body . In order to counteract these maladies, an antivirulence molecule with anti-adhesion and/or anti-biofilm properties may be highly desirable. Research in our laboratory has identified several molecules with differing anti-virulence effects [23, 28, 36, 37, 52, 53]. The current work examined the potential of five citrus limonoids- isolimonic acid, ichangin, isoobacunoic acid, IOAG and DNAG, to inhibit EHEC biofilm and TTSS. All the tested limonoids seem to interfere with the EHEC biofilm formation in a dose dependent fashion (Figure 2). Isolimonic acid was the most potent inhibitor of the EHEC biofilm and adhesion to Caco-2 cells. Moreover, the limonoids do not seem to affect growth of EHEC, suggesting that limonoids, especially isolimonic acid inhibits EHEC biofilm and adhesion without adversely affecting the growth or metabolic activity (Table 1, Additional file 1: Figure S1).
In EHEC, the initial attachment to various surfaces such as epithelial cells and plastic surface is regulated by several factors including TTSS, flagella and fimbriae [47, 48, 54]. LEE encoded TTSS, effector proteins as well as flagella and intimin [47, 48] play an important role in adhesion of EHEC to gastrointestinal tract surface, while flagella and fimbriae also contribute in biofilm formation. Results of the adhesion and biofilm assay indicated that one or more of above-mentioned factors may be affected by limonoids particularly by isolimonic acid. To investigate this hypothesis, expression of LEE encoded genes and flagellar master regulators flhDC was determined by qRT-PCR. Isolimonic acid and ichangin appear to exert their antivirulence and biofilm inhibitory effect by repressing TTSS carried on LEE, stx2, which encodes for Shiga toxin and flagellar master regulon flhDC (Table 4).
In EHEC, expression of LEE and flagellar operons are regulated by multiple environmental and genetic factors including QS [10–13]. In particular AI-2/AI-3/epinephrine mediated cell-cell signaling regulates the expression of both flagellar operon and LEE, which contribute to adhesion and biofilm formation. Furthermore, expression of stx2 is also regulated by QS [2, 12, 55, 56]. Therefore, repression of TTSS, flagella and stx2 indicated a possibility that limonoids, especially isolimonic acid may interfere with EHEC QS. Isolimonic acid was chosen for further studies, as it demonstrated the most potent inhibition of biofilm formation, adhesion, LEE, flhDC and stx2. For determination of AI-3/epinephrine mediated QS in EHEC, reporter strains TEVS 232 and TEVS21 containing chromosomal fusions LEE1:LacZ and LEE2:LacZ were used. The analysis was confined to LEE1 and LEE2, because these two operons have been reported to be directly activated by AI-3/epinephrine mediated QS [15, 41]. To test if the isolimonic acid acts as an QS inhibitor, PM/epinephrine stimulated activation of LEE1 and LEE2 in reporter strains was measured . The PM, described earlier , was used as a source of AI-3 molecules as the purified AI-3 was not available. Repression of AI-3/epinephrine-induced ler, LEE1 and LEE2 (Figure 5) indicated that isolimonic acid interferes with EHEC QS system.
The autoinducers and hormones reportedly increase the autophosphorylation levels of histidine kinase QseC, which then activates QseB to regulate motility and biofilm formation . Furthermore, interaction of AI-3/epinephrine with QseA activates LEE encoded genes [15, 57]. It was possible that isolimonic acid interferes with EHEC QS in a mechanism involving QseBC and QseA. If activity of isolimonic acid depends upon functional QseBC, deletion of qseBC will eliminate the inhibitory effect. On the other hand, complementation of ΔqseBC with plasmid borne QseBC is likely to restore the inhibitory effect of isolimonic acid. Furthermore, overexpression of qseBC in wild type background (EHEC ATCC 43895) will result in higher levels of QseBC proteins in the cell and consequently will have a higher activity. This higher level of activity may compensate and relieve the inhibitory effect of isolimonic acid on biofilm formation. In order to verify QseBC dependent inhibition, biofilm formation in ΔqseBC strain (VS138) and complemented strain (VS179)  in presence of 100 μg/ml of isolimonic acid was measured. As expected, isolimonic acid did not reduce the biofilm formation in VS138. In contrast, isolimonic acid exposure resulted in a significant decrease in VS179 (qseBC complemented strain) biofilm as measured by crystal violet (Figure 6A), indicating involvement of QseBC. Additionally, overexpression of qseBC, qseB and qseC in EHEC ATCC 43895, under the control of arabinose operon restored the inhibitory effect of isolimonic acid on EHEC biofilm formation (Figure 6B). Taken together these results suggest that effect of isolimonic acid is dependent upon QseBC. Furthermore, the effects of isolimonic acid did not seem to arise from modulation of qseBC expression. However, based on the current data it was not possible to differentiate, if the effect is dependent solely upon qseB or qseC, as supplementation of EHEC by both qseB and qseC relieved the inhibitory effect. Further studies are required to precisely determine if the target of isolimonic acid is qseB or qseC.
The present study demonstrates that the citrus limonoids, particularly isolimonic acid and ichangin are strong inhibitors of biofilm formation and attachment of EHEC to Caco-2 cells. Furthermore, isolimonic acid and ichangin seems to affect biofilm formation and TTSS by repressing LEE and flagellar operon. Isolimonic acid seems to exert its effect by inhibiting AI-3/epinephrine mediated cell-cell signaling in QseBC and QseA dependent manner. However, the mechanism by which isolimonic acid affects the QseBC and QseA remains to be elucidated. One possibility is that the isolimonic acid may interfere with the DNA binding activities of QseB and QseA. Another possible scenario will be that isolimonic acid interferes with phosphorylation events. However, further study is required to determine the target of isolimonic acid for the modulation of flhDC and ler. In addition, determination of the structure-activity relationship will provide further insights into the inhibitory action of isolimonic acid. In nutshell, isolimonic acid acts as an antivirulence agent in EHEC and may serve as lead compound for development of novel agents. Furthermore, the fact that isolimonic acid is present in citrus juices and do not demonstrate cytotoxic effect on normal human cell line , increases the desirability to develop it as antivirulence agent.
We would like to thank Dr. V. Sperandio (University of Texas Southwestern Medical Center, Dallas, TX) for generously providing AI-3 reporter strains harboring chromosomal LEE1:lacZ (TEVS232), LEE2:lacZ (TEVS21) and EHEC mutants VS145, VS151, VS138, VS179.
This project is based upon the work supported by the USDA-NIFA No. 2010-34402-20875, “Designing Foods for Health” through the Vegetable & Fruit Improvement Center. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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