Skip to main content
Download PDF

N-Acetyl-glucosamine influences the biofilm formation of Escherichia coli

Gut Pathogens volume 10, Article number: 26 (2018) Cite this article

Abstract

Background

The intestinal mucous layer is a physical barrier that limits the contact between bacteria and host epithelial cells. There is growing evidence that microbiota-produced metabolites can also be specifically sensed by gut pathogens as signals to induce or repress virulence genes. Many E. coli, including adherent and invasive (AIEC) strains, can form biofilm. This property can promote their intestinal colonization and resistance to immune mechanisms. We sought to evaluate the impact of mucus-derived sugars on biofilm formation of E. coli.

Results

We showed that the mucin sugar N-acetyl-glucosamine (NAG) can reduce biofilm formation of AIEC strain LF82. We demonstrated that the inactivation of the regulatory protein NagC, by addition of NAG or by mutation of nagC gene, reduced the biofilm formation of LF82 in static condition. Interestingly, real-time monitoring of biofilm formation of LF82 using microfluidic system showed that the mutation of nagC impairs the early process of biofilm development of LF82. Thus, NAG sensor NagC is involved in the early steps of biofilm formation of AIEC strain LF82 under both static and dynamic conditions. Its implication is partly due to the activation of type 1 fimbriae. NAG can also influence biofilm formation of other intestinal E. coli strains.

Conclusions

This study highlights how catabolism can be involved in biofilm formation of E. coli. Mucus-derived sugars can influence virulence properties of pathogenic E. coli and this study will help us better understand the mechanisms used to prevent colonization of the intestinal mucosa by pathogens.

Background

Escherichia coli is a highly versatile bacterial species commonly found as part of the intestinal microbiota of warm-blooded animals. Most isolates are harmless but some have acquired virulence genes that allow them to cause numerous diseases within the gut (intestinal pathogenic E. coli, InPEC) or extra-intestinally (extra-intestinal pathogenic E. coli; ExPEC). Commensal E. coli colonize the large intestine in vertebrates and appear to reside inside the mucus layer without contacting the underlying epithelium [1]. In contrast, InPEC possess the ability to penetrate the mucus layer and colonize the mucosa causing disease such as diarrhea [2]. Adherent-invasive E. coli (AIEC) strains share many genetic and phenotypic features with ExPEC strains but are rather involved in inflammatory bowel disease (IBD), including Crohn’s disease (CD) [3,4,5].

The mucus layer that covers the intestinal epithelium plays a critical role in gut homeostasis. The intestinal mucus contains mucins which are highly O-glycosylated proteins. Mucins play an important role in shaping the intestinal microbiota as an alteration of the glycan availability modifies the microbiota composition [6]. By producing specific glycosidases, several species of the gut microbiota release sugars from O-glycans into the intestinal lumen [7, 8]. Released mucus-derived sugars, including N-acetylglucosamine (NAG), N-acetylneuraminic acid (NANA), galactose, fucose, mannose and N-acetylgalactosamine provide direct source of carbohydrates and promote the growth of commensal and pathogenic bacteria including E. coli [7,8,9,10]. In addition to their role as nutrients, some mucus-derived sugars can act as regulatory signals that influence bacterial colonization and adherence to intestinal cells [11,12,13,14]. Enterohemorrhagic E. coli (EHEC) uses fucose, NAG and NANA as signaling molecules to modulate its metabolism and regulate the expression of its virulence repertoire [12, 15]. We recently showed that the catabolism of NAG and NANA inhibits EHEC adhesion to epithelial cells through down-regulation of the locus of enterocytes effacement expression under NagC regulatory control [16].

Biofilms could play a key role in bacterial colonization of the healthy gut and in intestinal diseases. Mucin has been reported to be involved in biofilm formation by E. coli [17, 18], suggesting its potential role in modulating E. coli colonization in the intestinal tract. The ability of biofilm formation in vitro varies extensively among E. coli isolates [19] and many E. coli strains are believed to form biofilm in the intestinal tract [20]. As such, AIEC are known to be higher biofilm producers than non-AIEC strains [21]. The ability to form biofilm could be part of the etiology of IBD since an increased presence of biofilms formed by the Bacteroides fragilis group and the Enterobacteriaceae family has been observed in intestinal biopsy specimens of people affected with these diseases [22, 23].

Alteration of the gut mucosal integrity and of microbiota could also change the mucus-derived sugars availability. Our hypothesis is that in healthy conditions, the integrity of the intestinal mucus and the functions of gut microbiota prevent biofilm formation of pathogenic E. coli. We evaluated the influence of mucus-derived sugars on biofilm formation of AIEC reference strain LF82. We show that NAG can reduce biofilm formation of LF82 and that the transcriptional regulator of NAG catabolism, NagC appears to be involved in the early steps of its biofilm formation. We also showed that mucus-derived sugars can influence biofilm formation of different E. coli strains from other pathotypes.

Methods

Bacterial strains and growth conditions

Bacterial strains used in this study are listed in Table 1. Bacteria were routinely cultured on lysogeny broth (LB) agar [1% (wt/vol) tryptone, 0.5% (wt/vol) yeast extract, 1% (wt/vol) NaCl, 1.5% (wt/vol) agar] at 37 °C and single colonies were transferred in liquid LB. When required, the growth medium was supplemented with kanamycin (50 μg/ml) and/or chloramphenicol (25 μg/ml). A set of commensal and pathogenic E. coli that comprises AIEC strain LF82, EHEC strain EDL933, enteroagregative E. coli (EAEC) strain 17.2, laboratory E. coli (K-12) strain MG1655 as well as commensal murine E. coli strain NC101 were included in the biofilm studies.

Table 1 List of strains and plasmids used in this study
Full size table

Mutagenesis and complementation

The LF82∆nagC mutant was constructed by allelic exchange using a suicide vector as described in our previous work [16]. Primers used for mutagenesis are listed in Additional file 1: Table S1. Briefly, the suicide vector pMEG-375 containing the kanamycin resistance cassette from pKD13 flanked with 500 pb sequences upstream and downstream of the nagC open reading frame was transformed in a diaminopimelic acid auxotrophic E. coli strain χ7213 (λpir and ∆asdA4). χ7213 was used as a donor to transfer the plasmid in E. coli strain LF82 by conjugation. Single crossover mutants were selected on LB agar without diaminopimelic acid, containing kanamycin. A second selection of double crossovers mutants was made using the sacB counter selection on LB agar without NaCl, containing 10% (wt/vol) of sucrose [24]. The mutation of nagC was confirmed by PCR and sequencing of an amplicon containing the region of interest. Complementation was performed using a derivative of the expression vector. The ORF of nagC was amplified in E. coli strain LF82 and inserted downstream of the pTrc promoter in the expression plasmid forming the pnagC. A resistance cassette of chloramphenicol was amplified from pACYC184 and inserted in pnagC, forming the pnagC-JFS.

Static biofilm assay

The assay of biofilm formation was done in 96-well microtiter plates as previously described [25]. Isolated colonies from LB agar were resuspended in fresh LB (5 ml) and incubated at 37 °C with shaking (180 rpm). Overnight cultures were diluted (1:100) in fresh medium; either LB, LB without salt (LBWS) or M9 medium with 0.4% glucose (wt/vol) and minerals (1.2 mM MgSO4, 2 µM FeCl3, 8 µM CaCl2, and 16 µM MnCl2). When required, the growth medium was supplemented with NAG, NANA or fucose (Sigma-Aldrich, St. Louis, MO, USA) at a concentration of 1 mM. A volume of 150 μl of these cultures was deposited in triplicate in the wells. Plates were incubated either at 30 or 37 °C for 24 h, without shaking. After incubation, total growth was measured at OD630 with a microplate reader (PowerWave; Bio-Tek Instruments, Winooski, VT, USA). Afterwards, the media were discarded, and each well washed three times with phosphate-buffered saline (PBS) to remove unattached cells. The biofilms were stained with crystal violet 0.1% (wt/vol) for 2 min at room temperature. The crystal violet solution was removed, and the biofilms washed six times with PBS. Finally, an 80% (vol/vol) ethanol and 20% (vol/vol) acetone solution was added to release the stain and the amount of biofilm quantified by measuring at OD595. Specific biofilm formation (SBF) index was calculated [21, 26]. Results are the ratio of biofilm measured at OD595 over total growth measured at OD630. To evaluate the effects of mucus-derived sugars addition on biofilm formation of different strains, one-way ANOVA with Dunnett’s multiple comparison test was performed to calculate P-values. To evaluate the impact of the mutation of nagC on AIEC strain LF82, two-way ANOVA with Tukey’s multiple comparison test was performed to calculate P-values *P <0.05; **P <0.01; ***P <0.001.

Flow-through biofilm assay (BioFlux device)

The biofilm formation assay in the BioFlux 200 device (Fluxion Biosciences, South San Francisco, CA, USA) was adapted from our previous work [25]. Biofilm formation occurs in the microfluidic channel of the device where fresh media constantly flow through causing a shear force that is similar to physiological condition [25]. E. coli strains were isolated on LB agar and resuspended in 5 ml of fresh LB medium. They were incubated overnight at 37 °C with shaking (180 rpm). A 0.5 ml volume of culture was transferred to a microtube and bacteria were collected by centrifugation (13,000g, 2 min). LB medium was used to resuspend the pellet to an OD600 ≈ 1. Prewarmed LB ± NAG (1 mM) was injected in the microfluidic system to fill the channels. Bacteria were injected from the output reservoir for 30 s at 0.5 dyne/cm2. The microfluidic plate was incubated at 30 °C for 2 h to allow the adhesion of bacteria on the surface of the channels. After adhesion, prewarmed LB ± NAG (1 mM) was added to the inlet wells and the microfluidic plate was connected to the BioFlux system. Temperature was adjusted to 30 °C and the flow was initiated at 1.0 dyne/cm2. After 4 h, the used medium was removed from the outlet well and prewarmed fresh medium was added in the inlet well. Flow was then reduced at 0.5 dyne/cm2 for the next 18 h. Images of the BioFlux biofilms were obtained with an inverted optic microscope equipped with a 40× objective (CKX41; Olympus, Markham, ON, Canada), a digital camera (Retiga EX; QImaging, Surrey, BC, Canada), and the software provided with the BioFlux 200 device. Images were treated using the software imageJ (National Institutes of Health, Bethesda, MD, USA) to quantify the amount of biofilm in the microfluidic channel. The 16-bit grayscale images were adjusted with the threshold function to include the bacterial structure before particle analysis. One-way ANOVA with Dunnett’s multiple comparison test was performed to calculate P-values.

Detection of type 1 fimbriae

The capacity of LF82 and the nagC mutant to produce type 1 fimbriae was evaluated by the ability to agglutinate to Saccharomyces cerevisiae cells in a mannose-dependant way. As previously described [27], cultures were grown at 30 °C in 20 ml of LB for 24 h without shaking to enhance expression of type 1 fimbriae. NAG was added in the medium at a final concentration of 1 mM. The concentration of an initial suspension of approximately 2 × 1011 CFU/ml in PBS was reduced by twofold serial dilutions in microtiter plate (Corning, 2797). An equal volume of a commercial yeast suspension in PBS 3% (wt/vol) (Fleischmann’s Active Dry) was added to each well. After 30 min of incubation at 4 °C, yeast agglutination was observable by precipitation of cells in the wells of the plate. The agglutination titer was recorded as the most diluted bacterial sample giving a positive aggregation reaction. Yeast agglutination was considered dependent of type 1 fimbriae if α-d-mannopyranose 5% (wt/vol) (Sigma-Aldrich) inhibited agglutination. One-way ANOVA with Dunnett’s multiple comparison test was performed to calculate P-values.

Quantitative RT-PCR (qRT-PCR)

Bacteria were precultured as described in previous section. A dilution (1:100) was done in 40 ml of LB ± NAG (1 mM) and bacteria were incubated in polystyrene petri dish overnight at 30 °C. Biofilms were washed with PBS and recovered with a cell scraper. RNA was extracted from biofilm cells using Ambion® RiboPure™-Bacteria Kit (ThermoFisher Scientific, Burlington, ON, Canada), according to the manufacturer’s recommendations. As described in [15], the absence of residual DNA in RNA samples was confirmed by PCR with primers targeting rpoA. Complementary DNA (cDNA) was synthetized from 10 μg of RNA, using a reverse transcriptase and random hexanucleotide primers. A standard curve was performed to determine the copy number of targeted transcript in 50 ng of cDNA. Primers used are listed in Additional file 1: Table S1. Results are presented as the ratios between the cDNA copy number of the gene of interest and the cDNA copy number of the housekeeping gene. One-way ANOVA with Dunnett’s multiple comparison test was performed to calculate P-values.

Results

NAG reduces biofilm formation of AIEC strain LF82

We investigated the impact of mucus-derived sugars, including NAG, NANA and fucose, on biofilm formation of the AIEC reference strain LF82. LB culture medium was selected for optimal growth of biofilms in static condition. Upon the addition of 1 mM of NAG, the specific biofilm formation (SBF) index of LF82 was significantly lower (P < 0.01) than the SBF index of LF82 grown in LB alone. Addition of NANA and fucose in the medium did not influence the biofilm formation of the strain (Fig. 1). The three mucus-derived sugars did not affect the growth of the strain after 24 h (data not shown). A time course measurement of NAG consumption by LF82 grown in LB supplemented with 1 mM of NAG indicated that NAG was catabolized within 4 h (Additional file 2: Figure S1).

Fig. 1
figure 1

Impact of mucus-derived sugars on specific biofilm formation of AIEC strain LF82. Biofilms of strain LF82 were formed in LB medium under static conditions at 30 °C. NAG, NANA or fucose were supplemented at a concentration of 1 mM. SBF values are the mean and standard error of at least 3 biological experiments. Statistical significance was calculated by one-way ANOVA with Dunnett’s multiple comparison test **P <0.01

Full size image

NAG reduces specific biofilm formation of LF82 by inactivating the transcriptional regulator NagC

The regulation of AIEC strain LF82 biofilm formation by NAG was further investigated. The activity of the regulator NagC is inactivated by the presence of NAG-6-P, a catabolic derivate of NAG. Therefore, a mutant strain in nagC gene was created. The SBF index of LF82ΔnagC mutant was significantly lower (P < 0.05) than the SBF index of the WT strain. This reduction of biofilm formation was similar to that observed upon the addition of NAG (Fig. 2). The wild-type phenotype was restored in the complemented strain expressing nagC. Interestingly, no additional repression was observed upon the addition of NAG in the ΔnagC mutant. This indicates that LF82 biofilm repression by NAG is NagC dependent.

Fig. 2
figure 2

NagC positively influences the biofilm formation of AIEC strain LF82. Biofilms of strain LF82, its isogenic mutant ΔnagC and complemented ΔnagC were formed in LB medium under static condition at 30 °C. NAG was supplemented at a concentration of 1 mM. SBF values are the mean and standard error of 4 biological replicates. Statistical analysis was calculated by two-way ANOVA with Tukey’s multiple comparison test *P <0.05; **P <0.01

Full size image

NagC is involved in the first steps of biofilm formation of LF82 in presence of a shear force

To further investigate the role of NagC on biofilm formation of LF82, the dynamics of biofilm formation were monitored and visualized in real time using the BioFlux device (Fig. 3a). Real time imaging with LF82 clearly revealed the apparition of microcolonies 6 h after the initiation of the flow. These microcolonies grew overtime and the production of polymeric matrix became more apparent at 10 h. After 18 h, the channel was almost entirely covered with biofilm. At 24 h, mature biofilms were formed. The kinetics of biofilm formation of LF82ΔnagC was different from that of the WT LF82. In early time points, a reduced number of microcolonies was observed in the mutant strain. Quantification results revealed a significant (P < 0.01) decrease in biofilm coverage of the mutant strain when compared to the WT strain at 6, 8 and 10 h after the activation of the flow. At 16 and 18 h, the biofilm structure of LF82ΔnagC mutant was similar to that of the WT. Again, wild-type phenotype was restored in the complemented strain (Fig. 3a, b). This suggests that the nagC mutation impairs the first steps of biofilm formation of LF82 and involves surface adhesion and/or cell to cell interactions. In contrast to what we observed in static condition, supplementation of NAG did not affect biofilm formation in the microfluidic system.

Fig. 3
figure 3

The biofilm formation is delayed in nagC mutant of LF82 in a microfluidic system. a Biofilms of strain LF82, its isogenic mutant ΔnagC and complemented ΔnagC were formed in LB medium at 30 °C in the BioFlux 200 microfluidic system. NAG was supplemented at a concentration of 1 mM. A field of view representative of 3 independent replicates is shown for each test. Images were captured by phase contrast microcopy using a digital camera. b Quantification of biofilm formation was made using the “Analyze Particles” function of ImageJ and results show the percentage of the area covered by biofilm structures in images pictures. Statistical significance was calculated by one-way ANOVA with Dunnett’s multiple comparison test **P 0.01; ***P 0.001

Full size image

The mutation of nagC impaired the production of type 1 fimbriae of LF82

To determine the role of type 1 pili in the NagC-dependent reduction of biofilm formation of LF82, the expression and the production of type 1 fimbriae were evaluated in the mutant strain. The expression of fimA, encoding the major structural subunit of type 1 fimbriae, was measured in grown biofilms (Fig. 4a). Results showed a significant (P < 0.01) reduction of 3.5-fold of fimA transcription in the LF82ΔnagC strain when compared to WT strain (Fig. 4a). The expression of fimA was restored in the complemented strain. However, the effect of supplementation of NAG in medium on the expression of fimA was not statistically significant but slightly increased (Fig. 4a). The production of type 1 fimbriae was measured by mannose-sensitive agglutination to S. cerevisiae and was significantly lower (P < 0.01) by 2 Log2 in LF82ΔnagC mutant in comparison to WT strain (Fig. 4b). Type 1 pili production was restored in the complemented strain. Addition of NAG during growth did not affect the production of type 1 fimbriae. Interestingly, NAG is rapidly consumed within 4 h of bacterial growth in LB medium supplemented with NAG, as measured by mass spectrometry (Additional file 2). Thus, the repressive effect of NAG on type 1 fimbriae production might be transient. Addition of mannose was previously reported to reduce the biofilm of LF82 by interfering with type 1 fimbriae adhesion [40]. In presence of mannose, biofilm formation of LF82 was decreased in either LB or NAG supplemented medium indicating that type 1 fimbriae is involved in biofilm in these conditions (Additional file 3: Figure S2).Taken together these results show that expression and production of type 1 fimbriae are regulated by NagC in LF82. Type 1 fimbriae or other structures participating to LF82 biofilm might be influenced by NAG supplementation.

Fig. 4
figure 4

NagC influences the expression and the production of type 1 fimbriae of LF82. a The expression of type 1 fimbriae subunit fimA was measured by qRT-PCR in LF82 WT strain, its ΔnagC derivative and the complemented mutant grown as biofilm in LB media. NAG was also supplemented at a concentration of 1 mM in media used for WT strain. Results are shown as the ratio of copy number of fimA transcript/copy numbers of tufA transcript. b Production of type 1 fimbriae was also evaluated for each strain by mannose specific agglutination to the yeast S. cerevisiae. LB medium was used in the experiments and NAG was supplemented at a concentration of 1 mM when needed. Results are the mean values and standard error of 3 biological experiments. Statistical analyzes were made using one-way ANOVA with Dunnett’s multiple comparison test *P 0.05; **P 0.01

Full size image

The impact of mucus-derived sugars on biofilm formation varies among E. coli strains

Biofilm formation of a set of distinct E. coli strains was tested in different culture media to determine optimal conditions. LB medium was used for strains 17.2 and MG1655, whereas LBWS and M9 with glucose were used as the optimized culture media for biofilm formation of strains NC101 and EDL933, respectively. The effect of addition of NAG, NANA or fucose on biofilm formation was evaluated in static condition. The influence of mucus-derived sugars was variable, strain-dependent and did not affect the growth of bacteria as measured with optical density (Additional file 4: Figure S3). Our data showed that the mean OD value of biofilm formed by murine strain NC101 was significantly decreased in the presence of the all three sugars (P < 0.01). Supplementation of the medium with NAG (P < 0.01) or NANA (P < 0.05), significantly reduced biofilm formation of EHEC strain EDL933. A slight reduction was also observed upon the addition of NAG in EAEC strain 17.2 and K-12 strain MG1655 (Fig. 5).

Fig. 5
figure 5

Mucus-derived sugars can influence the specific biofilm formation of different strains of E. coli. Strain specific optimized conditions for biofilm formation were identified. LB was used for EAEC strain 17.2 and K-12 strain MG1655. LBWS was used for AIEC strain NC101 and M9 with glucose was used for EHEC strain EDL933. Biofilms were formed under static conditions at 30 °C except for strain EDL933 that was grown at 37 °C. NAG, NANA and fucose were supplemented at a concentration of 1 mM. SBF values are the mean and standard error of at least 3 biological experiments. Statistical analysis was made using one-way ANOVA with Dunnett’s multiple comparison test *P 0.05; **P 0.01; ***P 0.001

Full size image

Discussion

There is growing evidence that microbiota-produced metabolites can also be specifically sensed by pathogens as signals to induce or repress virulence genes [28]. We show that the mucin sugars NAG, NANA and fucose can reduce the biofilm formation of AIEC strain LF82 and other pathogenic E. coli. A recent study showed that fucose modulated biofilm formation of Campylobacter jejuni [29]. We observed that the influence of mucus-derived sugars on biofilm formation was strain-dependent, reflecting the high genetic diversity and the variability of metabolic patterns between E. coli strains [30, 31]. It is known that E. coli preference for mucus-derived sugars varies from one strain to another [10, 32, 33]. Interestingly, the repressor effect of NAG was common among the tested E. coli strains as NAG supplementation reduced biofilm formation of AIEC strain LF82, murine strain NC101, EHEC strain EDL933 and to a lower extent EAEC strain 17.2 and K-12 strain MG1655.

We demonstrated that the effect of NAG on biofilm formation of AIEC strain LF82 is NagC-dependent. NagC is a repressor of nag operon involved in catabolism of NAG in E. coli [34, 35]. The uptake of this sugar leads to the production of intracellular NAG-6-P that will inactivate the regulator NagC [36]. Our study shows that the inactivation of NagC, whether it is caused by the catabolism of NAG or by a mutation of nagC, is responsible for the decreased biofilm formation. Thus, NagC is a positive regulator of biofilm formation in LF82. Interestingly, NagC is also involved in the expression of the locus of enterocytes effacement virulence genes of EHEC as well as type 1 fimbriae of E. coli K-12 [13, 16, 36].

The real-time monitoring of LF82 biofilm formation using microfluidic system showed that early steps are impaired by the mutation of nagC. Biofilm formation initially required the attachment of the bacteria to a surface and the cell-to-cell adhesion that leads to the formation of microcolonies. Thus, during these steps, NagC might influence the expression of structures involved in early adhesion of bacteria. In contrast to the situation in static conditions, the biofilm in dynamic conditions was insensitive to the presence of NAG. It is possible that constant renewal of media in dynamic conditions influences NAG catabolism and thus NagC activity.

Type 1 fimbriae are key factors that facilitate adhesion to a surface and cell-to-cell aggregation during establishment of biofilm on abiotic surfaces [37, 38]. They also participate in biofilm formation and in the adhesion-and-invasion process in AIEC strains such as LF82 [21, 39, 40]. In the present study, we show that NagC activates the gene expression and the production of type 1 fimbriae of AIEC strain LF82. This is similar to the work showing NagC regulation on type 1 fimbriae in E. coli K-12 MG1655 [13]. Based on NagC consensus DNA binding site generated from known NagC binding sequences [13, 16, 41, 42], two different binding sites were found upstream of the promoter of fimB recombinase in LF82. FimB is involved in the OFF-to-ON switching of type 1 fimbriation and sequences found in LF82 were identical and in the same distance to those of fimB promoter in K-12 strain MG1655 [13] (Additional file 5: Figure S4). This indicates that NagC control on type 1 fimbriae could influence at least in part the biofilm formation of LF82. In contrast, NAG supplementation did not influence type 1 fimbriae production of LF82 strain. It is possible that NAG-dependant repression on type 1 fimbriae was transient because NAG is rapidly consumed by the strain). NAG might also influence other factors contributing to biofilm formation of LF82 as NAG was also shown to influence the production of curli in E. coli K-12 [11].

Dysbiosis that occurs during IBD can favor AIEC growth and probably biofilm formation [43]. As glycosylation of the mucin is defective in CD [44] and microbiota activity is modified, it is possible that the availability of mucin sugars will influence not only the metabolic activity but also the virulence behavior including the pathogens’ ability of biofilm formation. Thus, factors that regulate biofilm formation could signal to repress expression of the type 1 fimbriae and other factors contributing to biofilm of LF82. By affecting the concentration of free NAG available in the digestive tract, gut bacterial species expressing N-acetylglucosaminidase [6] might therefore influence E. coli biofilm formation through a modulation of NagC activity. Interestingly, administration of glucosamine can reduce production of pro-inflammatory cytokines and therefore intestinal inflammation in murine model of IBD could influence the activity of the flora including AIEC and reduce their biofilm formation and colonization ability [45, 46].

Conclusion

In conclusion, the presence of mucin-derived sugars can influence biofilm formation of different E. coli strains. This study highlights that the diminution of biofilm formation of AIEC strain LF82 in the presence of NAG is NagC-dependent. Indeed, the NAG sensor NagC is involved in the early steps of biofilm formation of strain LF82 and its implication could be partly due to the control of type 1 fimbriae.

Abbreviations

AIEC:

adherent and invasive E. coli

NAG:

N-acetyl-glucosamine

InPEC:

intestinal pathogenic E. coli

ExPEC:

extra-intestinal pathogenic E. coli

IBD:

inflammatory bowel disease

CD:

Crohn’s disease

NANA:

N-acetylneuraminic acid

EHEC:

enterohemorrhagic E. coli

LB:

lysogeny broth

EAEC:

enteroagregative E. coli

LBWS:

LB without salt

PBS:

phosphate-buffered saline

OD:

optical density

SBF:

specific biofilm formation

cDNA:

complementary DNA

References

  1. Poulsen LK, Lan F, Kristensen CS, Hobolth P, Molin S, Krogfelt KA. Spatial distribution of Escherichia coli in the mouse large intestine inferred from rRNA in situ hybridization. Infect Immun. 1994;62(11):5191–4.

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Torres AG, Zhou X, Kaper JB. Adherence of diarrheagenic Escherichia coli strains to epithelial cells. Infect Immun. 2005;73(1):18–29.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Darfeuille-Michaud A. Adherent-invasive Escherichia coli: a putative new E. coli pathotype associated with Crohn’s disease. Int J Med Microbiol. 2002;292(3–4):185–93.

    Article  CAS  PubMed  Google Scholar 

  4. Darfeuille-Michaud A, Boudeau J, Bulois P, Neut C, Glasser AL, Barnich N, Bringer MA, Swidsinski A, Beaugerie L, Colombel JF. High prevalence of adherent-invasive Escherichia coli associated with ileal mucosa in Crohn’s disease. Gastroenterology. 2004;127(2):412–21.

    Article  PubMed  Google Scholar 

  5. Martinez-Medina M, Garcia-Gil LJ. Escherichia coli in chronic inflammatory bowel diseases: an update on adherent invasive Escherichia coli pathogenicity. World J Gastrointest Pathophysiol. 2014;5(3):213–27.

    Article  PubMed  PubMed Central  Google Scholar 

  6. Martens EC, Chiang HC, Gordon JI. Mucosal glycan foraging enhances fitness and transmission of a saccharolytic human gut bacterial symbiont. Cell Host Microbe. 2008;4(5):447–57.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Bertin Y, Chaucheyras-Durand F, Robbe-Masselot C, Durand A, de la Foye A, Harel J, Cohen PS, Conway T, Forano E, Martin C. Carbohydrate utilization by enterohaemorrhagic Escherichia coli O157:H7 in bovine intestinal content. Environ Microbiol. 2013;15(2):610–22.

    Article  CAS  PubMed  Google Scholar 

  8. Ng KM, Ferreyra JA, Higginbottom SK, Lynch JB, Kashyap PC, Gopinath S, Naidu N, Choudhury B, Weimer BC, Monack DM, et al. Microbiota-liberated host sugars facilitate post-antibiotic expansion of enteric pathogens. Nature. 2013;502(7469):96–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Conway T, Cohen PS. Commensal and pathogenic Escherichia coli metabolism in the gut. Microbiol Spectr. 2015. https://doi.org/10.1128/microbiolspec.MBP-0006-2014

    Article  PubMed  PubMed Central  Google Scholar 

  10. Fabich AJ, Jones SA, Chowdhury FZ, Cernosek A, Anderson A, Smalley D, McHargue JW, Hightower GA, Smith JT, Autieri SM, et al. Comparison of carbon nutrition for pathogenic and commensal Escherichia coli strains in the mouse intestine. Infect Immun. 2008;76(3):1143–52.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Barnhart MM, Lynem J, Chapman MR. GlcNAc-6P levels modulate the expression of Curli fibers by Escherichia coli. J Bacteriol. 2006;188(14):5212–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Pacheco AR, Curtis MM, Ritchie JM, Munera D, Waldor MK, Moreira CG, Sperandio V. Fucose sensing regulates bacterial intestinal colonization. Nature. 2012;492(7427):113–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Sohanpal BK, El-Labany S, Lahooti M, Plumbridge JA, Blomfield IC. Integrated regulatory responses of fimB to N-acetylneuraminic (sialic) acid and GlcNAc in Escherichia coli K-12. Proc Natl Acad Sci USA. 2004;101(46):16322–7.

    Article  CAS  PubMed  Google Scholar 

  14. Vogt SL, Pena-Diaz J, Finlay BB. Chemical communication in the gut: effects of microbiota-generated metabolites on gastrointestinal bacterial pathogens. Anaerobe. 2015;34:106–15.

    Article  CAS  PubMed  Google Scholar 

  15. Le Bihan G, Jubelin G, Garneau P, Bernalier-Donadille A, Martin C, Beaudry F, Harel J. Transcriptome analysis of Escherichia coli O157:H7 grown in vitro in the sterile-filtrated cecal content of human gut microbiota associated rats reveals an adaptive expression of metabolic and virulence genes. Microbes Infect. 2015;17(1):23–33.

    Article  CAS  PubMed  Google Scholar 

  16. Le Bihan G, Sicard JF, Garneau P, Bernalier-Donadille A, Gobert AP, Garrivier A, Martin C, Hay AG, Beaudry F, Harel J, et al. The NAG sensor NagC regulates LEE gene expression and contributes to gut colonization by Escherichia coli O157:H7. Front Cell Infect Microbiol. 2017;7:134.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Bollinger RR, Barbas AS, Bush EL, Lin SS, Parker W. Biofilms in the normal human large bowel: fact rather than fiction. Gut. 2007;56(10):1481–2.

    PubMed  PubMed Central  Google Scholar 

  18. Bollinger RR, Everett ML, Wahl SD, Lee YH, Orndorff PE, Parker W. Secretory IgA and mucin-mediated biofilm formation by environmental strains of Escherichia coli: role of type 1 pili. Mol Immunol. 2006;43(4):378–87.

    Article  CAS  PubMed  Google Scholar 

  19. Reisner A, Krogfelt KA, Klein BM, Zechner EL, Molin S. In vitro biofilm formation of commensal and pathogenic Escherichia coli strains: impact of environmental and genetic factors. J Bacteriol. 2006;188(10):3572–81.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Rossi E, Cimdins A, Luthje P, Brauner A, Sjoling A, Landini P, Romling U. “It’s a gut feeling”—Escherichia coli biofilm formation in the gastrointestinal tract environment. Crit Rev Microbiol. 2017;44:1–30.

    Article  PubMed  Google Scholar 

  21. Martinez-Medina M, Naves P, Blanco J, Aldeguer X, Blanco JE, Blanco M, Ponte C, Soriano F, Darfeuille-Michaud A, Garcia-Gil LJ. Biofilm formation as a novel phenotypic feature of adherent-invasive Escherichia coli (AIEC). BMC Microbiol. 2009;9:202.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Swidsinski A, Weber J, Loening-Baucke V, Hale LP, Lochs H. Spatial organization and composition of the mucosal flora in patients with inflammatory bowel disease. J Clin Microbiol. 2005;43(7):3380–9.

    Article  PubMed  PubMed Central  Google Scholar 

  23. Swidsinski A, Loening-Baucke V, Herber A. Mucosal flora in Crohn’s disease and ulcerative colitis—an overview. J Physiol Pharmacol. 2009;60(Suppl 6):61–71.

    PubMed  Google Scholar 

  24. Kaniga K, Delor I, Cornelis GR. A wide-host-range suicide vector for improving reverse genetics in Gram-negative bacteria: inactivation of the blaA gene of Yersinia enterocolitica. Gene. 1991;109(1):137–41.

    Article  CAS  PubMed  Google Scholar 

  25. Tremblay YD, Vogeleer P, Jacques M, Harel J. High-throughput microfluidic method to study biofilm formation and host-pathogen interactions in pathogenic Escherichia coli. Appl Environ Microbiol. 2015;81(8):2827–40.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Naves P, del Prado G, Huelves L, Gracia M, Ruiz V, Blanco J, Rodriguez-Cerrato V, Ponte MC, Soriano F. Measurement of biofilm formation by clinical isolates of Escherichia coli is method-dependent. J Appl Microbiol. 2008;105(2):585–90.

    Article  CAS  PubMed  Google Scholar 

  27. Vogeleer P, Tremblay YD, Jubelin G, Jacques M, Harel J. Biofilm-forming abilities of shiga toxin-producing Escherichia coli isolates associated with human infections. Appl Environ Microbiol. 2015;82(5):1448–58.

    Article  CAS  PubMed  Google Scholar 

  28. Baumler AJ, Sperandio V. Interactions between the microbiota and pathogenic bacteria in the gut. Nature. 2016;535(7610):85–93.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Dwivedi R, Nothaft H, Garber J, Xin Kin L, Stahl M, Flint A, van Vliet AH, Stintzi A, Szymanski CM. l-Fucose influences chemotaxis and biofilm formation in Campylobacter jejuni. Mol Microbiol. 2016;101(4):575–89.

    Article  CAS  PubMed  Google Scholar 

  30. Baumler DJ, Peplinski RG, Reed JL, Glasner JD, Perna NT. The evolution of metabolic networks of E. coli. BMC Syst Biol. 2011;5:182.

    Article  PubMed  PubMed Central  Google Scholar 

  31. Meador JP, Caldwell ME, Cohen PS, Conway T. Escherichia coli pathotypes occupy distinct niches in the mouse intestine. Infect Immun. 2014;82(5):1931–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Chang DE, Smalley DJ, Tucker DL, Leatham MP, Norris WE, Stevenson SJ, Anderson AB, Grissom JE, Laux DC, Cohen PS, et al. Carbon nutrition of Escherichia coli in the mouse intestine. Proc Natl Acad Sci USA. 2004;101(19):7427–32.

    Article  CAS  PubMed  Google Scholar 

  33. Maltby R, Leatham-Jensen MP, Gibson T, Cohen PS, Conway T. Nutritional basis for colonization resistance by human commensal Escherichia coli strains HS and Nissle 1917 against E. coli O157:H7 in the mouse intestine. PLoS ONE. 2013;8(1):e53957.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Plumbridge J, Kolb A. CAP and Nag repressor binding to the regulatory regions of the nagE-B and manX genes of Escherichia coli. J Mol Biol. 1991;217(4):661–79.

    Article  CAS  PubMed  Google Scholar 

  35. Plumbridge J, Kolb A. DNA loop formation between Nag repressor molecules bound to its two operator sites is necessary for repression of the nag regulon of Escherichia coli in vivo. Mol Microbiol. 1993;10(5):973–81.

    Article  CAS  PubMed  Google Scholar 

  36. El Qaidi S, Plumbridge J. Switching control of expression of ptsG from the Mlc regulon to the NagC regulon. J Bacteriol. 2008;190(13):4677–86.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Schembri MA, Christiansen G, Klemm P. FimH-mediated autoaggregation of Escherichia coli. Mol Microbiol. 2001;41(6):1419–30.

    Article  CAS  PubMed  Google Scholar 

  38. Pratt LA, Kolter R. Genetic analysis of Escherichia coli biofilm formation: roles of flagella, motility, chemotaxis and type I pili. Mol Microbiol. 1998;30(2):285–93.

    Article  CAS  PubMed  Google Scholar 

  39. Barnich N, Boudeau J, Claret L, Darfeuille-Michaud A. Regulatory and functional co-operation of flagella and type 1 pili in adhesive and invasive abilities of AIEC strain LF82 isolated from a patient with Crohn’s disease. Mol Microbiol. 2003;48(3):781–94.

    Article  CAS  PubMed  Google Scholar 

  40. Nickerson KP, McDonald C. Crohn’s disease-associated adherent-invasive Escherichia coli adhesion is enhanced by exposure to the ubiquitous dietary polysaccharide maltodextrin. PLoS ONE. 2012;7(12):e52132.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. El Qaidi S, Allemand F, Oberto J, Plumbridge J. Repression of galP, the galactose transporter in Escherichia coli, requires the specific regulator of N-acetylglucosamine metabolism. Mol Microbiol. 2009;71(1):146–57.

    Article  CAS  PubMed  Google Scholar 

  42. Plumbridge J. Co-ordinated regulation of amino sugar biosynthesis and degradation: the NagC repressor acts as both an activator and a repressor for the transcription of the glmUS operon and requires two separated NagC binding sites. EMBO J. 1995;14(16):3958–65.

    Article  CAS  Google Scholar 

  43. Agus A, Massier S, Darfeuille-Michaud A, Billard E, Barnich N. Understanding host-adherent-invasive Escherichia coli interaction in Crohn’s disease: opening up new therapeutic strategies. Biomed Res Int. 2014;2014:567929.

    Article  PubMed  PubMed Central  Google Scholar 

  44. Strugala V, Dettmar PW, Pearson JP. Thickness and continuity of the adherent colonic mucus barrier in active and quiescent ulcerative colitis and Crohn’s disease. Int J Clin Pract. 2008;62(5):762–9.

    Article  CAS  PubMed  Google Scholar 

  45. Azuma K, Osaki T, Kurozumi S, Kiyose M, Tsuka T, Murahata Y, Imagawa T, Itoh N, Minami S, Sato K, et al. Anti-inflammatory effects of orally administered glucosamine oligomer in an experimental model of inflammatory bowel disease. Carbohydr Polym. 2015;115:448–56.

    Article  CAS  PubMed  Google Scholar 

  46. Bak YK, Lampe JW, Sung MK. Effects of dietary supplementation of glucosamine sulfate on intestinal inflammation in a mouse model of experimental colitis. J Gastroenterol Hepatol. 2014;29(5):957–63.

    Article  CAS  PubMed  Google Scholar 

  47. Blattner FR, Plunkett G 3rd, Bloch CA, Perna NT, Burland V, Riley M, Collado-Vides J, Glasner JD, Rode CK, Mayhew GF, et al. The complete genome sequence of Escherichia coli K-12. Science. 1997;277(5331):1453–62.

    Article  CAS  PubMed  Google Scholar 

  48. Perna NT, Plunkett G 3rd, Burland V, Mau B, Glasner JD, Rose DJ, Mayhew GF, Evans PS, Gregor J, Kirkpatrick HA, et al. Genome sequence of enterohaemorrhagic Escherichia coli O157:H7. Nature. 2001;409(6819):529–33.

    Article  CAS  PubMed  Google Scholar 

  49. Rich C, Favre-Bonte S, Sapena F, Joly B, Forestier C. Characterization of enteroaggregative Escherichia coli isolates. FEMS Microbiol Lett. 1999;173(1):55–61.

    Article  CAS  PubMed  Google Scholar 

  50. Kim SC, Tonkonogy SL, Albright CA, Tsang J, Balish EJ, Braun J, Huycke MM, Sartor RB. Variable phenotypes of enterocolitis in interleukin 10-deficient mice monoassociated with two different commensal bacteria. Gastroenterology. 2005;128(4):891–906.

    Article  CAS  PubMed  Google Scholar 

  51. Roland K, Curtiss R 3rd, Sizemore D. Construction and evaluation of a delta cya delta crp Salmonella typhimurium strain expressing avian pathogenic Escherichia coli O78 LPS as a vaccine to prevent airsacculitis in chickens. Avian Dis. 1999;43(3):429–41.

    Article  CAS  PubMed  Google Scholar 

  52. Amann E, Ochs B, Abel KJ. Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli. Gene. 1988;69(2):301–15.

    Article  CAS  PubMed  Google Scholar 

Download references

Authors’ contributions

The experiments were performed mainly by JFS with the help of PV. JFS and JH performed the data analysis. The study was designed by JH, JFS, GLB and PV. All authors read and approved the final manuscript.

Acknowledgements

We thank Joan Pena and Yaindrys Rodriguez Olivera for their help with qRT-PCR assays. We also thank Jean-Charles Côté for proofreading the manuscript.

Competing interests

The authors declare that they have no competing interests.

Funding

This research was supported by a Team Grant from the Fonds de Recherche du Québec, Nature et Technologies (FRQNT PT165375), to JH, MJ and FB and by the Discovery grant program of the Natural Sciences and Engineering Research Council of Canada (RGPIN-2015-05373 to JH and RGPIN-2016-04203 to MJ). JFS is a recipient of a scholarship from the NSERC Collaborative Research and Training Experience Program in Milk Quality; and PV is a recipient of a scholarship from the FRQNT Québec Wallonie program.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Author information

Authors and Affiliations

  1. Groupe de Recherche sur les Maladies Infectieuses en Production Animale, Centre de Recherche en Infectiologie Porcine et Avicole, Faculté de Médecine Vétérinaire, Université de Montréal, St-Hyacinthe, QC, J2S 2M2, Canada

    Jean-Félix Sicard, Philippe Vogeleer, Guillaume Le Bihan, Yaindrys Rodriguez Olivera, Francis Beaudry & Josée Harel

  2. Regroupement de Recherche Pour un Lait de Qualité Optimale (Op+Lait), Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, QC, J2S 2M2, Canada

    Mario Jacques

Authors
  1. Jean-Félix Sicard
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Philippe Vogeleer
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Guillaume Le Bihan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yaindrys Rodriguez Olivera
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Francis Beaudry
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Mario Jacques
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Josée Harel
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Josée Harel.

Additional files

Additional file 1: Table S1.

List of primers used in this study.

Additional file 2: Figure S1.

Monitoring of NAG consumption by LF82 WT, LF82ΔnagC and the complemented strain when grown under static condition. Concentration of NAG was measured by mass spectrometry.

Additional file 3: Figure S2.

Specific biofilm formation of LF82 was evaluated in the presence or absence of mannose in either in LB or LB supplemented with NAG.

Additional file 4: Figure S3.

The effect of the addition of mucus-derived sugars on growth of strains.

Additional file 5: Figure S4.

NagC consensus DNA binding sites and nucleotide BLAST of LF82 and MG1655.

Rights and permissions

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Sicard, JF., Vogeleer, P., Le Bihan, G. et al. N-Acetyl-glucosamine influences the biofilm formation of Escherichia coli. Gut Pathog 10, 26 (2018). https://doi.org/10.1186/s13099-018-0252-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s13099-018-0252-y

Keywords

Gut Pathogens

ISSN: 1757-4749