OUP user menu

Purification and characterization of a surface-binding protein from Lactobacillus fermentum RC-14 that inhibits adhesion of Enterococcus faecalis 1131

Christine Heinemann , Johan E.T. van Hylckama Vlieg , Dick B. Janssen , Henk J. Busscher , Henny C. van der Mei , Gregor Reid
DOI: http://dx.doi.org/10.1111/j.1574-6968.2000.tb09282.x 177-180 First published online: 1 September 2000

Abstract

Lactobacilli have been shown to be important in the maintenance of the healthy urogenital flora. One strain, Lactobacillus fermentum RC-14, releases surface-active components which can inhibit adhesion of uropathogenic bacteria. Using a quantitative method for determining inhibition of adhesion, a protein with high anti-adhesive properties against Enterococcus faecalis 1131 was purified. The N-terminal sequence of the 29-kDa protein was identical to that of a collagen-binding protein from Lactobacillus reuteri NCIB 11951, and exhibited close homology with a basic surface protein from L. fermentum BR11. The results suggest that this anti-adhesive cell surface protein of Lactobacillus could protect against uropathogens by preventing their adhesion.

Keywords
  • Lactobacillus
  • Enterococcus
  • Bacterial adhesion
  • Collagen

1 Introduction

Lactobacilli have been implicated for many years as contributing to the prevention of intestinal and urinary tract infections [1,2]. Several mechanisms have been proposed to be involved including competitive exclusion and displacement of uropathogens [3], production of hydrogen peroxide, lactic acid and growth inhibitors [4,5], and the release of biosurfactants [6]. The surface-active components of Lactobacillus fermentum RC-14 are believed to be powerful defense agents because they inhibit the adhesion of a wide variety of uropathogens [7]. The amount of material released by one adhering Lactobacillus suffices to coat a substratum surface area several times its geometrical area [8], rendering unfavorable conditions for uropathogens to adhere. Velraeds et al. [6] demonstrated that of 15 Lactobacillus strains tested, all released surface-active components when grown to their mid-exponential and stationary growth phases. The isolated components could reduce the surface tension of water and were found to be primarily proteinaceous in nature, based on infrared absorption and X-ray photoelectron spectroscopy [9]. The aim of the work described in this paper was to isolate and identify key components of the biosurfactant mixture released by L. fermentum RC-14 and determine their ability to interfere with Enterococcus faecalis 1131 adhesion.

2 Materials and methods

2.1 Strains and culture conditions

Lactobacillus fermentum RC-14, an isolate from the urogenital tract of a healthy woman [10], was stored in DeMan-Rogosa-Sharpe (MRS) broth (Merck, Germany) containing 7% (v/v) dimethyl sulfoxide at −20°C. For each experiment, the bacteria were thawed, streaked on MRS agar plates, and incubated at 37°C in an atmosphere containing 5% CO2 for further culturing.

The uropathogen Enterococcus faecalis 1131 was stored in brain heart infusion (BHI) broth (Oxoid, UK) containing 7% (v/v) dimethyl sulfoxide at −20°C. From frozen stocks, blood agar plates were inoculated and incubated aerobically at 37°C to obtain cultures. Precultures were prepared by inoculating 5 ml of BHI broth and subsequent aerobic overnight incubation at 37°C.

2.2 Biosurfactant isolation

The production of the biosurfactant was based on the method by Velraeds et al. [6]. A starter culture of L. fermentum RC-14 was grown at 37°C overnight in 30 ml MRS medium in the presence of 5% CO2. An amount of 1200 ml of MRS broth was inoculated with the overnight culture and incubated for 18 h. The bacteria were harvested by centrifugation (10 000×g, 5 min, 10°C), washed twice in demineralized water and resuspended in 200 ml of PBS (10 mM KH2PO4/K2HPO4, 150 mM NaCl, pH 7.0). To obtain biosurfactant release, the bacteria were kept at room temperature for 2 h with gentle stirring. After centrifugation (10 000×g, 10 min, 10°C) the supernatant containing the biosurfactant was filtered (0.22-μm, Millipore) and dialyzed against demineralized water at 4°C (Spectrapor membrane tube, MWCO, 60 00–8 000, Spectrum Medical Industries, California). Finally, the dialysate was freeze-dried and stored at −20°C.

2.3 Anti-adhesion assay

The anti-adhesive activity against E. faecalis 1131 was quantified using a previously reported adhesion assay [11]. Briefly, the wells of tissue culture treated, polystyrene microtiter plates (Corning Glassworks, Corning, NY) were treated with 200 μl of the solution to be tested for anti-adhesive activity. The plate was incubated for 18 h at 4°C and subsequently washed twice with PBS. Control wells contained buffer only. E. faecalis 1131 was grown at 37°C overnight under aerobic conditions. An amount of 200 μl of a washed suspension of E. faecalis 1131 (3×108 bacteria ml−1 in PBS) was added and incubated in the wells for 4 h at 4°C. Unattached organisms were removed by washing the wells three times with PBS. The adherent bacteria were stained with crystal violet and optical density readings taken at 595 nm. The change in adherence was used to calculate the percentage inhibition of adhesion. From various dilutions of the crude biosurfactant mixture and a calibration curve, the amount of material that caused a 50% reduction of adhesion was calculated. This was defined as one inhibitory unit.

2.4 Size exclusion chromatography

For purification, the crude biosurfactant was dissolved in PBS (3 mg ml−1) and loaded onto a Sephacryl S-200 gel filtration column (700 ml, Pharmacia Biotech) previously equilibrated in PBS (pH 7.0). Elution was carried out with PBS (flow rate, 30 ml h−1; fractions, 10 ml). The protein content of the eluted fractions was determined with Coomassie brilliant blue using bovine serum albumin as the standard [12]. All fractions obtained from the various purification steps were also tested for anti-adhesive activity using the assay described above.

2.5 Ion exchange chromatography

For further purification, fractions 44–49 were pooled and dialyzed overnight against TMA buffer (10 mM Tris–HCl, l mM β-mercaptoethanol, 3 mM NaN3, pH 8.0) at 4°C. The dialysate was subsequently applied to a Resource Q anion-exchange column (6 ml, Pharmacia Biotech) connected to a LCC500 type FPLC system (Pharmacia Biotech). Bound protein was eluted with a 140-ml linear gradient from 0 to 0.45 M NaCl in TMA buffer (flow rate, 5 ml min−1; fractions, 2 ml).

2.6 SDS–PAGE and electroblotting

Sodium dodecyl sulfate polyacrylamide electrophoresis (SDS–PAGE) was performed as previously described [13]. The gels were either stained with Coomassie brilliant blue R-250 or the protein was transferred to a Polyvinylidene Difluoride (PVDF) membrane (Mini ProBlot™, Applied Biosystems) by electroblotting. Briefly, the unstained gel was soaked for 5 min in transfer buffer (25 mM Tris–HCl/192 mM glycine buffer, pH 8.3 containing 15% methanol) before blotting. A PVDF membrane was pre-wetted with 100% methanol and transferred to the blotting buffer. After electroblotting (200 mA, 2 h), the filter was washed in distilled water and stained with Coomassie brilliant blue R-250. The blot was partially destained with 50% methanol.

2.7 N-terminal sequencing

Following electroblotting, N-terminal sequencing was performed with an automated sequencer (Model 494 Procise/Model 477A, Applied Biosystems) by Eurosequence (Groningen, The Netherlands).

3 Results and discussion

An anti-adhesion component, which appeared to be a 29-kDa protein, was purified from the crude biosurfactant mixture in two steps. The first step involved size exclusion chromatography and most of the anti-adhesive activity co-eluted with the bulk of the protein, except for some later eluting fractions which had the highest specific activity (Fig. 1). Starting with 45 mg of crude material, containing 694 U mg protein−1, 4.1 mg protein was recovered with a specific activity of 1290 U mg protein−1. Further purification using ion exchange chromatography yielded a single peak of activity with a specific activity of 1716 U mg protein−1, although the yield was relatively low (1.8%), probably due to absorption losses (results not shown). SDS–PAGE correlated the activity to a protein with an apparent molecular mass of 29 kDa (Fig. 2). The N-terminal sequence of the purified protein was found to be ASSAVNSELVHKGELTIGLE. Database searches revealed 100% identity of the obtained sequence to a collagen-binding protein from L. reuteri NCIB 11951 [14], and 90% similarity to a basic surface protein from L. fermentum BR11 [15]. The collagen-binding protein of L. reuteri NCIB 11951 has been purified by affinity chromatography on collagen-Sepharose by Aleljung et al. [16]. Its biological function is still unknown, but it is thought to function as an adhesin [14]. The N-terminal sequence of the anti-adhesion protein was also similar to the sequence of a basic surface protein from L. fermentum BR11. This 32-kDa protein, which is also thought to function as an adhesin, was purified by removing it from the bacterial cell surface by extraction with 5 M LiCl [15]. The authors suggest that the protein is anchored to the cell surface by electrostatic interactions with acidic groups since it could easily be removed from the cell surface with acidic buffers. [15] In recent years, many microorganisms have been shown to mediate their adhesion to host tissues via binding to extracellular matrix proteins, such as fibronectin, laminin and collagen [17]. Several studies have shown that lactobacilli also have the ability to bind to collagen and fibronectin [18,19] which suggests that binding to extracellular matrix proteins might be an important feature in their ability to colonize the intestinal and urogenital tract. Interestingly, we isolated the same protein as Roos et al. [14], but based on a different activity, namely the ability to inhibit the adhesion of uropathogenic enterococci. These results suggest that binding of lactobacilli to extracellular matrix proteins is not only involved in the adhesion of these bacteria to host tissues, but that release of these proteins also plays an important role in the ability of lactobacilli to protect the host against invading pathogens.

1

Sephacryl S-200 elution profile of components from L. fermentum RC-14 with anti adhesion-activity. Both activity and specific activity of eluting fractions are showing. ○, activity; ●, specific activity against E. faecalis 1131 adhesion; dashed line, protein concentration.

2

SDS–PAGE of purified anti-adhesion protein present in a biosurfactant mixture of L. fermentum RC-14. Lane 1, size markers; lane 2, crude biosurfactant mixture; lane 3, fractions 44–49 from gel filtration; lane 4, purified anti-adhesion protein.

Acknowledgements

This work was supported, in part, by the Natural Sciences and Engineering Research Council of Canada.

Footnotes

  • 1 Also affiliated with University of Western Ontario.

  • 2 Also affiliated with Lawson Research Institute, 268 Grosvenor Street, London, Ont. N6A 4V2, Canada.

References

  1. [1].
  2. [2].
  3. [3].
  4. [4].
  5. [5].
  6. [6].
  7. [7].
  8. [8].
  9. [9].
  10. [10].
  11. [11].
  12. [12].
  13. [13].
  14. [14].
  15. [15].
  16. [16].
  17. [17].
  18. [18].
  19. [19].
View Abstract