Open Access

Cell surface proteins play an important role in probiotic activities of Lactobacillus reuteri

  • Tejinder Pal Singh1Email author,
  • Ravinder Kumar Malik1 and
  • Gurpreet Kaur1
Nutrire201641:5

DOI: 10.1186/s41110-016-0007-9

Received: 25 September 2015

Accepted: 16 May 2016

Published: 28 June 2016

Abstract

Background

Eight Lactobacillus reuteri strains, previously isolated from breast-fed human infant feces, were selected to assess the potential contribution of their surface proteins in probiotic activity. These strains were treated with 5 M LiCl to remove their surface proteins, and their tolerance to simulated stomach-duodenum passage, cell surface characteristics, autoaggregation, adhesion, and inhibition of pathogen adhesion to Caco-2 cells were compared with untreated strains.

Results

The survival rates, autoaggregation, and adhesion abilities of the LiCl-treated L. reuteri strains decreased significantly (p < 0.05) compared to that of the untreated cells. The inhibition ability of selected L. reuteri strains, untreated or LiCl treated, against adherence of Escherichia coli 25922 and Salmonella typhi NCDC113 to Caco-2 was evaluated in vitro with L. reuteri ATCC55730 strain as a positive control. Among the selected eight strains of L. reuteri, LR6 showed maximum inhibition against the E. coli ATCC25922 and S. typhi NCDC113. After treatment with 5 M LiCl to remove surface protein, the inhibition activities of the lactobacilli against pathogens decreased significantly (p < 0.05). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that LR6 strains had several bands with molecular weight ranging from 10 to 100 KDa, and their characterization and functions need to be confirmed.

Conclusions

The results revealed that the cell surface proteins of L. reuteri play an important role in their survivability, adhesion, and competitive exclusion of pathogen to epithelial cells.

Keywords

Lactobacillus reuteri Cell surface proteins Adhesion Pathogen inhibition

Background

Lactobacillus are natural inhabitant of human gut of healthy individuals, and some of these were clearly assessed for their probiotic characteristics [13]. The main properties for probiotic microorganisms consist in equilibrating the endogenous microflora, in protecting the gut from pathogens invasion by competitive exclusion and production of antimicrobial molecules, and in stimulating mucosal immunity. The ability to adhere to the intestinal epithelial cells is considered important in the selection of lactobacilli for probiotic use. Mechanisms of competitive exclusion of pathogens include the ability to adhere to host cells, often exerted through the same type of adhesins employed by pathogens as a strategy for gut colonization. In addition to other factors, probiotic bacterial adherence is often associated with the immunological effects of probiotic bacteria and with the interference of the adhesion of pathogenic bacteria [4].

The Gram-positive cell envelope consists of two main layers, the cytoplasmic membrane and peptidoglycan (or cell wall). Both of these layers are spanned by various proteins, such as transporters, and also, there are proteins attached on the cell surface. Several reports have appeared describing or assuming functions of cell surface proteins which includes its function as a protective sheath against hostile environmental agents, a cell shape determinant, and a sheath to mask properties of the underlying cell wall such as charge and phage receptors [5, 6]. There is also increasing evidence that bacteria may employ variation in surface proteins, by expressing alternative cell surface protein genes, for adaptation to different stress factors, such as the immune response of the host for pathogens and drastic changes in the environmental conditions for nonpathogens [6, 7]. It has been proposed that cell surface proteins are involved in cell protection and surface recognition and that they could be potential mediators in the initial steps involved in autoaggregation and adhesion [810]. In cell envelope proteome studies of potentially probiotic bacteria, the cell wall protein fraction has typically been extracted by lysozyme-containing buffer [1116], lithium chloride [16, 17], or some other cell surface molecule or protein-solubilizing agent [1518].

In our efforts to study the importance of cell surface proteins in probiotic activity of Lactobacillus reuteri strains, recovered from breast-fed human infant feces [19], our data demonstrates that cell surface proteins of L. reuteri play an important role in survivability, adhesion, and competitive exclusion of pathogen to epithelial cells.

Methods

Bacterial strains and growth conditions

L. reuteri LR5, LR6, LR9, LR11, LR19, LR20, LR26, and LR34 were the laboratory strains, recovered from the breast-fed human infant feces, selected for this study. The reference strain of L. reuteri ATCC55730 was obtained from Biogaia, Sweden. All the L. reuteri strains were grown in MRS broth (deMan, Ragosa, and Sharp broth; Himedia, Mumbai, India) at 37 °C for 18–24 h and maintained as glycerol stocks until further use. From these stocks cultures, working cultures were prepared and propagated twice prior to use by sub-culturing in MRS broth.

Removal of cell surface proteins

To remove the cell surface proteins, the bacterial cells were collected by centrifugation at 5000g for 15 min followed by washing with sterile distilled water and then incubating the cells in 5 M LiCl for 30 min.

Survival in simulated stomach and duodenum passage

This assay represents a simplified and standardized test system giving predictive values for the assumed survival of lactobacilli in the human stomach and duodenum under “normal” conditions [19]. The principle involves first a simulation of the stomach containing ingested lactobacilli after a meal. After 1 h, bile and artificial duodenal secretions are added in order to simulate the further passage. The MRS broth was prepared following the manufacturer’s instructions, the pH adjusted to 3.0 with 5 M HCl, and then it was dispersed in the flasks containing the required volume for the test setup, followed by sterilization at 121 °C for 15 min. Synthetic duodenal juice was prepared by completely dissolving NaHCO3 (6.4 g/L), KCl (0.239 g/L), and NaCl (1.28 g/L) in distilled water. The pH was adjusted to 7.4 with 5 M HCl before sterilizing at 121 °C for 15 min. The oxgal solution was prepared by reconstituting 10 g of oxgal in 100 mL water and sterilizing at 121 °C for 15 min. The required volumes of the overnight cultures and MRS broth adjusted to pH 3.0 were aseptically mixed in sterile flasks to give a final concentration of 108 cfu/mL in MRS, and the counts were determined by spread plating. Samples were withdrawn after 1 h of incubation at 37 °C and viable counts were determined. Four milliliters of oxgal solution was added to the culture in the flasks, followed by 17 mL of duodenal juice. After mixing, the flasks were further incubated at 37 °C. Samples were withdrawn after 2 and 3 h and counts established as described above. Three independent experiments were carried out in duplicate for all the L. reuteri strains before and after LiCl treatment.

Determination of bacterial hydrophobicity

Microbial adhesion to solvents (MATS) was measured according to the method of Kos et al. [20]. Three different solvents were tested in this study: n-hexadecane (Himedia, Mumbai, India), which is an apolar solvent; chloroform (Himedia, Mumbai, India), a monopolar and acidic solvent; and ethyl acetate (Himedia, Mumbai, India), a monopolar and basic solvent. Only bacterial adhesion to n-hexadecane reflects cell surface hydrophobicity or hydrophilicity. The values of MATS obtained with the two other solvents, chloroform and ethyl acetate, were regarded as a measure of electron donor (basic) and electron acceptor (acidic) characteristics of bacteria, respectively [21].

Bacteria were harvested in the stationary phase by centrifugation at 5000 g for 15 min, washed twice, and resuspended in 0.1 mol/L KNO3 (pH 6.2) to approximately 108 CFU/ml. The absorbance of the cell suspension was measured at 600 nm (A 0). One milliliter of solvent was added to 3 ml of cell suspension. After a 10-min preincubation at room temperature, the two-phase system was mixed by vortexing for 2 min. The aqueous phase was removed after 20 min of incubation at room temperature, and its absorbance at 600 nm (A 1) was measured. The percentage of bacterial adhesion to solvent was calculated as (1 − A 1/A 0)*100. The experiment was also carried out for L. reuteri strains after LiCl treatment.

Autoaggregation assay

Autoaggregation assay was performed according to Kos et al. [20] with some modifications. Bacterial cells were grown for 18 h at 37 °C in MRS broth. The cells (with and without LiCl treatment) were harvested by centrifugation at 5000 g for 15 min, washed twice, and resuspended in phosphate-buffered saline (PBS; pH 7.4) to give viable counts of approximately 108 CFU/ml. Cell suspensions (4 ml) were mixed by vortexing for 10 s and autoaggregation was determined during 5 h of incubation at room temperature. Every hour, 0.1 ml of the upper suspension was transferred to another tube with 3.9 ml of PBS and the absorbance (A) was measured at 600 nm. The autoaggregation percentage is expressed as: 1 − (A t /A 0)*100, where A t represents the absorbance at time t = 1, 2, 3, 4, or 5 h and A 0 the absorbance at t = 0.

Caco-2 cell culture and adherence assay of L. reuteri

Caco-2 cell culture

The Caco-2 cell line was procured from the National Center of Cell Science, Pune, India. Cells were routinely grown in Dulbecco’s modified Eagle’s minimal essential medium (DMEM; Sigma, USA), supplemented with 10 % fetal bovine serum (FBS; Sigma, USA), and 100 μg/ml streptomycin (Sigma, USA) and 100 U/ml penicillin (Sigma, USA) at 37 °C in a 10 % CO2 atmosphere. For adhesion assays, Caco-2 monolayers were prepared in 6-well tissue plates. Cells were inoculated at a concentration of 7 × 104 cells per well to obtain confluence and cultured for 21 days prior to the adhesion assay. The cell culture medium was changed on alternate days, and the last two media changes were without penicillin/streptomycin.

In vitro adherence assay

Overnight cultures of lactobacilli grown in DMEM (without FBS and antibiotics) were centrifuged, washed, and re-suspended in DMEM. Viable counts were determined by plating on MRS agar. A 1.0-ml aliquot of the bacterial suspension (adjusted to 1 × 108 cfu/ml) was added to each well of the tissue culture plate and incubated in a 5 % CO2 atmosphere. After 2 h of incubation, the Caco-2 monolayers were washed three times with sterile PBS (pH 7.4). The cells from monolayers were detached by tripsinization. One millimeter 0.25 % trupsin-EDTA solution (Sigma, USA) was added to each well, and plate was incubated for 5 min at 37 °C. The detached cells were repeatedly but gently aspirated to make homogeneous suspension. The cell suspension was then serially diluted with saline solution and plated on MRS agar. The plates were then incubated for 24–48 h at 37 °C, and colonies were counted (B1 cfu/ml). Bacterial cells initially added to each well were also counted (B0 cfu/ml). The adhesion percentage was then calculated as % adhesion = (B 1/B 0)*100. The adhesion experiment was also performed for all the L. reuteri strains after LiCl treatment.

Inhibition of Escherichia coli ATCC25922 and Salmonella typhi NCDC113 adherence to Caco-2 cells by L. reuteri

The inhibition ability of Lactobacillus to pathogens adherence was performed according to the previous method with some modification [22]. Eight lactobacilli as mentioned above were used. The optical density was adjusted to 1 × 108 cfu/ml with PBS (pH 7.4). Three different procedures, competition, exclusion, and displacement, were used to evaluate the inhibition ability of lactobacillus with or without surface proteins to pathogen adherence to Caco-2. E. coli ATCC25922 was from American Type Culture Collection (ATCC, USA) and S. typhi NCDC113 was from National Collection of Dairy Cultures (NCDC, India), respectively. The selected pathogens were propagated in Brain Heart Infusion broth (BHI; Himedia) and maintained as glycerol stocks.

For competition assays, 200 μl (approximately 1 × 106 cfu) of lactobacillus and 200 μl (approximately 1 × 106 cfu) of pathogens were co-cultured with Caco-2 cells in DMEM for 2 h. For exclusion assays, lactobacilli were cultured with Caco-2 cells in DMEM for 1 h. After 1 h, Caco-2 cells were washed three times with PBS (pH 7.4) and pathogens were added for further incubation for 1 h. For displacement assays, pathogens were added and cultured for 1 h, and then, the lactobacillus were added and cultured for 1 h. After culture, the cells were lysed by addition of 0.25 % (v/v) trypsin-EDTA solution at 37 °C for 5 min and the number of viable adhering E. coli and S. typhi were determined by plating on eosin methylene blue (EMB) and Salmonella Shigella (SS) agar plates after serial dilutions, respectively. The inhibition of pathogens by lactobacillus without surface proteins was also conducted as above.

Observation by scanning and transmission electron microscopy

Aliquots of bacterial aggregates were fixed with 2.5 % (v/v) glutaraldehyde in PBS buffer. After 1 h of fixation, the cells were washed with PBS and refixed for 1 h in the dark at room temperature with PBS buffer containing 1 % osmium tetroxide. Cells were then washed three times with the same mixture and dehydrated in a concentration series (30, 50, 70, and 80 %) of ethanol solutions for 10 min each. The cells were then washed in 100 % ethanol for 10 min before being dried in a critical-point dryer (Balzers CPD 020) and coated with gold. All preparations were observed under a ZEISS EVO 18 scanning electron microscope.

To observe the surface structure of the strain, bacteria for thin section were prefixed in glutaraldehyde (3 % in phosphate buffer, pH 7.2) for 2 h at room temperature. The micrographs were taken by JEM-100CX transmission electron microscopes at an operating voltage of 100 kV.

Isolation and SDS-PAGE analysis of cell surface proteins from L. reuteri

Cell surface proteins of lactobacillus were extracted by 5 M LiCl according to the method reported by Zhang et al. [22]. L. reuteri LR6 showing maximum tolerance to simulated stomach and duodenum conditions and higher adherence to Caco-2 cell lines were incubated in 30 ml MRS broth. After culturing for 18 h, cells were collected and washed twice with ice-cold sterile water. Six millimeters of 5 M LiCl was used to mix with lactobacilli. Supernatant was collected and dialyzed with PBS and then freeze dried. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed with a 5 % (w/v) stacking gel and a 12 % (w/v) separating gel. Samples of the surface proteins were dissolved in denaturing buffer and subjected to SDS-PAGE gel. Gel was stained by Coomassie brilliant blue R-250 (Sigma, USA).

Statistical analysis

The results are expressed as the mean ± SD of three independent experiments. Statistical analysis was done by StatGraphicPlus software. Data were subjected to a one-way analysis of variance (ANOVA). Differences were considered statistically significant when p < 0.05.

Results

Survival in simulated stomach and duodenum passage

Among eight test strains, LR6 showed the maximum survival when exposed to simulated gastric and duodenum conditions for longer period. To evaluate the survival of the L. reuteri strains after removal of the cell surface proteins, all the test strains and reference culture were treated with 5 M LiCl. The survival rates of the LiCl treated L. reuteri strains decreased significantly (p < 0.05) compared to that of the untreated cells as the removal of cell surface proteins decreased the survival by two to four logs after 3 h of exposure period (Table 1).
Table 1

Survival (log10 cfu/mL) of L. reuteri strains (with and without LiCl treatment) under simulated stomach–duodenum passage (SSDP) conditions after 1, 2, and 3 h of incubation

Strains

0 h

1 h

2 h

3 h

Untreated

LiCl treated

Untreated

LiCl treated

Untreated

LiCl treated

Untreated

LiCl treated

LR5

8.16 ± 0.25W

8.16 ± 0.32w

7.05 ± 0.33X**

5.99 ± 0.10x*

4.70 ± 0.17Y**

3.86 ± 0.50y*

4.49 ± 0.29DZ**

2.26 ± 0.13Ez*

LR6

8.44 ± 0.14W

8.44 ± 0.30w

7.26 ± 0.23X**

6.97 ± 0.27x*

6.53 ± 0.44Z**

3.81 ± 0.38y*

6.91 ± 0.15AY**

2.48 ± 0.36BCxz*

LR9

8.37 ± 0.42W

8.37 ± 0.45w

7.65 ± 0.3X**

5.55 ± 0.19x*

5.09 ± 0.26Y**

3.32 ± 0.59y*

4.32 ± 0.54DZ**

2.47 ± 0.41BCDz*

LR11

8.44 ± 0.18W

8.43 ± 0.38w

7.69 ± 0.13X**

5.89 ± 0.18x*

6.63 ± 0.15Y**

3.13 ± 0.18y*

5.09 ± 0.42BCZ**

2.58 ± 0.63Bz*

LR19

8.37 ± 0.19W

8.37 ± 0.20w

7.19 ± 0.12X**

6.83 ± 0.16x*

4.05 ± 0.29Z**

3.62 ± 0.15y*

4.60 ± 0.33DY**

2.31 ± 0.14DEz*

LR20

8.83 ± 0.13W

8.83 ± 0.32w

7.32 ± 0.2X**

6.71 ± 0.27x*

5.09 ± 0.26Y**

3.15 ± 0.17y*

4.32 ± 0.41DZ**

2.60 ± 0.46Bz*

LR26

8.54 ± 0.18W

8.54 ± 0.31w

7.67 ± 0.53X**

6.24 ± 0.18x*

4.74 ± 0.27Y**

3.81 ± 0.57y*

4.65 ± 0.53CDY**

2.39 ± 0.12CDEz*

LR34

8.59 ± 0.16W

8.59 ± 0.46w

7.63 ± 0.37X**

5.96 ± 0.27x*

6.33 ± 0.51Y**

3.79 ± 0.44y*

5.22 ± 0.29BZ**

2.49 ± 0.17BCz*

L. reuteri ATCC55730

8.60 ± 0.16W

8.60 ± 0.33w

7.03 ± 0.31X**

7.15 ± 0.17x*

6.69 ± 0.23XY**

3.84 ± 0.13y*

6.93 ± 0.1AY**

2.78 ± 0.15Az*

Data are mean ± standard deviation of three independent experiments

A, B, C, D, EDifferent symbol means statistically significant difference (p < 0.05) within the same column for 3 h

*,**Different symbol means statistically significant difference (p < 0.05) within the same row between the treatments at particular time

WXYZDifferent symbol means statistically significant difference (p < 0.05) within the same row for untreated strains at different time.

wxyzDifferent symbol means statistically significant difference (p < 0.05) within the same row for LiCl treated strains at different time.

Influence of surface properties on cell surface hydrophobicity and autoaggregation of L. reuteri strains

To evaluate the hydrophobic/hydrophilic and Lewis acid–base properties in the cell surfaces of L. reuteri strains, three solvents such as n-hexadecane, chloroform, and ethyl acetate were employed by using the MATS method. As listed in Table 2, a strong affinity to n-hexadecane and chloroform as well as a low adherence to ethyl acetate indicated the hydrophobic and basic phenotype of these strains.
Table 2

Effect of LiCl treatment on cell surface hydrophobicity of L. reuteri strains

Strains

n-Hexadecane

Chloroform

Ethyl acetate

Untreated

LiCl treated

Untreated

LiCl treated

Untreated

LiCl treated

LR5

30.05 ± 0.03bx

17.42 ± 0.02cy

25.30 ± 0.01hy

37.61 ± 0.02hx

37.54 ± 0.04bx

7.38 ± 0.01ey

LR6

19.13 ± 0. 03ix

4.66 ± 0.01gy

60.18 ± 0.03ey

65.36 ± 0.04ex

14.38 ± 0.09fx

13.79 ± 0.01cx

LR9

23.54 ± 0.02dx

5.21 ± 0.04gy

64.14 ± 0.02dx

64.42 ± 0.01fx

11.93 ± 0.03gx

1.72 ± 0.01gy

LR11

35.97 ± 0.07ax

22.74 ± 0.01ay

49.35 ± 0.03fy

75.08 ± 0.01cx

20.69 ± 0.06dx

6.23 ± 0.01fy

LR19

12.02 ± 0.02hx

9.17 ± 0.01fy

69.02 ± 0.01cy

77.84 ± 0.02bx

3.36 ± 0.04iy

12.09 ± 0.01dx

LR20

22.07 ± 0.05ex

21.53 ± 0.01bx

32.86 ± 0.04gy

70.18 ± 0.03dx

30.02 ± 0.01cx

24.36 ± 0.06by

LR26

13.74 ± 0.03gx

10.58 ± 0.01ey

74.87 ± 0.01bx

75.66 ± 0.01cx

17.65 ± 0.00ey

58.86 ± 0.01ax

LR34

15.07 ± 0.04fx

12.43 ± 0.02dy

49.37 ± 0.05fy

51.34 ± 0.01gx

8.92 ± 0.03hy

12.50 ± 0.01dx

L. reuteri ATCC55730

25.04 ± 0.01cx

18.12 ± 0.01cy

80.45 ± 0.02ax

80.29 ± 0.03ax

38.52 ± 0.01ay

59.08 ± 0.02ax

Data are mean ± standard deviation of results from three separate experiments

abcdefghiDifferent symbol means statistically significant difference (p < 0.05) within the same column

xyDifferent symbol means statistically significant difference (p < 0.05) within the same row between the treatments

The maximum autoaggregation was showed by LR6 (38.89 %) followed by LR11 (28.57 %) (Table 3). The autoaggregation of the strains decreased significantly after LiCl treatment compared to untreated strains (p < 0.05), indicating that cell surface proteins could be associated with the autoaggregation (Table 3). The differences in the aggregative properties of untreated and LiCl L. reuteri strains were also illustrated by qualitative scanning and transmission electron microscopy observations (Fig. 1). Micrographs showed the spatial arrangement of microbial aggregates and also highlighted the presence of exopolymeric substances which probably act as cement between cells (L. reuteri without LiCl treatment). The ultrastructure of the L. reuteri strains were observed by transmission electron microscopy. The changes in the cell surface after treatment with 5 M LiCl could be distinctly visible in a thin-sectioned cell.
Table 3

Comparison of autoaggregation and adhesion to Caco-2 cells of L. reuteri strains (with and without LiCl treatment)

Strains

Autoaggregation

Adhesion to Caco-2 cells

Untreated

LiCl treated

Untreated

LiCl treated

LR5

25.86 ± 0.58d**

5.08 ± 0.16c*

21.92 ± 0.54f**

8.34 ± 0.05cd*

LR6

38.89 ± 0.64b**

3.33 ± 0.24e*

50.62 ± 0.88a**

7.75 ± 0.18de*

LR9

21.05 ± 0.99e**

7.69 ± 0.21a*

25.64 ± 0.10d**

11.92 ± 0.91a*

LR11

28.57 ± 0.48c**

6.52 ± 0.56b*

23.50 ± 0.24e**

11.33 ± 0.94a*

LR19

20.00 ± 1.95ef**

4.44 ± 0.59cd*

23.95 ± 0.29e**

8.50 ± 0.35c*

LR20

19.57 ± 1.15f**

4.25 ± 0.47d*

45.58 ± 0.35b**

8.70 ± 0.06c*

LR26

21.95 ± 0.83e**

7.14 ± 0.35ab*

24.09 ± 3.21e**

8.54 ± 0.64c*

LR34

21.05 ± 1.22e**

3.57 ± 0.48e*

39.50 ± 0.71c**

9.45 ± 0.64b*

L. reuteri ATCC55730

42.67 ± 0.57a**

2.50 ± 0.59f*

52.00 ± 0.94a**

7.36 ± 0.05d*

Data are mean ± standard deviation of results from three separate experiments

*,** Indicates the differences at p < 0.05 level existed between the LiCl treated cells and untreated ones of the same strain

abcdefDifferent symbol means statistically significant difference (p < 0.05) within the same column

Fig. 1

i Examination of L. reuteri LR6 by scanning electron microscopy. a L. reuteri LR6 (without LiCl treatment) showing aggregation. b LiCl-treated L. reuteri LR6 showing separated cells. ii Examination of L. reuteri LR6 by transmission electron microscopy. a L. reuteri LR6 (without LiCl treatment). b LiCl-treated L. reuteri LR6. The arrows indicate the surface proteins of L. reuteri LR6

In vitro adhesion assay to Caco-2 cells

The adhesion of L. reuteri strains showed a great variability depending on the strain (Table 3) and varied from 21.92 % to 52 %. Among the tested strains, the most adhesive strains were L. reuteri LR6 (50.62 %) and LR20 (45.58 %), while the least adhesive strain was LR5 (21.92 %). Also, a significant (p < 0.05) reduction in adhesion values was observed after LiCl treatment of the strains.

Inhibition of E. coli ATCC25922 and S. typhi NCDC113 adherence to Caco-2 cells by L. reuteri

Inhibition of E. coli ATCC25922 and S. typhi NCDC113 adherence to Caco-2 cells by L. reuteri strains, with or without surface proteins, is shown in Tables 4 and 5, respectively. All the lactobacillus strains significantly inhibited the adhesion of E. coli ATCC25922 and S. typhi NCDC113 to Caco-2 cells (p < 0.05).
Table 4

Competition, exclusion and displacement of E. coli ATCC25922 adhering to Caco-2 cells by the lactobacillus strains (with or without LiCl treatment)

Strains

Competition

Exclusion

Displacement

Untreated

LiCl treated

Untreated

LiCl treated

Untreated

LiCl treated

LR5

85.0 ± 1.0bcy

91.2 ± 1.2ax

67.5 ± 1.0dy

89.5 ± 1.0cdx

82.5 ± 1.3cy

88.2 ± 0.6bx

LR6

59.5 ± 2.3fy

89.4 ± 1.2ax

56.0 ± 1.1fy

93.1 ± 1.2abx

59.5 ± 1.7ey

93.2 ± 1.2ax

LR9

67.5 ± 1.2ey

83.7 ± 1.8bx

67.5 ± 1.5dy

87.4 ± 0.8dx

71.5 ± 1.5dy

87.3 ± 1.5bx

LR11

72.0 ± 1.5dy

83.9 ± 1.3bax

80.5 ± 0.8ay

88.4 ± 1.1cdx

87.5 ± 1.7by

86.0 ± 2.7bx

LR19

82.5 ± 2.1cy

91.3 ± 1.8ax

74.0 ± 0. 4by

93.1 ± 1.3abx

74.5 ± 2.1dy

78.5 ± 0.7cx

LR20

87.5 ± 1.7aby

93.2 ± 1.5ax

69.5 ± 0.9cdy

90.7 ± 0.9bcx

93.5 ± 1.2ay

94.1 ± 0.8ax

LR26

89.0 ± 1.0ay

90.8 ± 2.6ax

59.5 ± 1.1ey

93.7 ± 1.1ax

83.5 ± 2.1cy

91.8 ± 0.7ax

LR34

86.0 ± 1.2by

92.1 ± 0.9ax

81.0 ± 0.8ay

92.2 ± 1.2abx

73.0 ± 1.2dy

92.3 ± 0.5ax

L. reuteri ATCC55730

51.0 ± 0.8gy

89.6 ± 2.5ax

71.0 ± 0.7cy

87.2 ± 1.1dx

57.5 ± 1.8ey

88.2 ± 0.9bx

Data are adherence ratio of E. coli ATCC25922 to Cao-2 cells = (test/control) × 100 %, shown as mean ± standard deviation of three independent experiments

abcdefgDifferent symbol means statistically significant difference (p < 0.05) within the same column

xyDifferent symbol means statistically significant difference (p < 0.05) within the same row between the treatments

Table 5

Competition, exclusion and displacement of S. typhi NCDC113 adhering to Caco-2 cells by the lactobacillus (with or without LiCl treatment)

Strains

Competition

Exclusion

Displacement

Untreated

LiCl treated

Untreated

LiCl treated

Untreated

LiCl treated

LR5

80.0 ± 1.5bxy

81.2 ± 1.2ax

72.5 ± 2.1ay

83.5 ± 3.6bcx

75.0 ± 1.7cy

80.2 ± 1.7cdx

LR6

47.5 ± 1.7fy

88.4 ± 1.6bx

49.0 ± 2.0dy

91.6 ± 1.9ax

49.5 ± 2.5fy

87.4 ± 1.5abx

LR9

55.0 ± 0.4ey

79.6 ± 2.8ax

55.0 ± 1.2cy

78.4 ± 2.4dx

61.5 ± 1.1ey

77.3 ± 2.0dx

LR11

61.0 ± 0.6dy

81.5 ± 1.3ax

75.0 ± 1.5ay

83.4 ± 1.7bcx

85.0 ± 1.7by

83.6 ± 2.0bcx

LR19

75.0 ± 0.2cy

88.7 ± 3.0bx

62.5 ± 2.5by

81.3 ± 1.6cdx

70.0 ± 2.1dy

87.8 ± 0.9ax

LR20

85.0 ± 2.0ay

92.3 ± 2.8bx

77.0 ± 2.2ay

90.7 ± 2.1ax

90.0 ± 1.2ay

90.3 ± 2.1ax

LR26

75.0 ± 2.4cy

89.1 ± 1.9bx

61.0 ± 3.1by

83.7 ± 2.0bcx

75.0 ± 2.0cy

89.1 ± 1.2ax

LR34

80.0 ± 2.3by

90.3 ± 1.3bx

76.5 ± 3.0ay

88.2 ± 2.1abx

67.5 ± 2.8dy

89.4 ± 1.9ax

L. reuteri ATCC55730

45.0 ± 2.5fy

82.6 ± 2.3ax

73.0 ± 2.2ay

81.7 ± 1.2cdx

48.5 ± 1.8fy

80.2 ± 2.1cdx

Data are adherence ratio of E. coli ATCC25922 to Cao-2 cells = (test/control) × 100 %, shown as mean ± standard deviation of three independent experiments

abcdefDifferent symbol means statistically significant difference (p < 0.05) within the same column

xyDifferent symbol means statistically significant difference (p < 0.05) within the same row between the treatments

In competition assay, the inhibition activity of strains LR6, LR9, LR11, and L. reuteri ATCC55730 against E. coli ATCC25922 and S. typhi NCDC113 was much higher than that of LR5, LR19, LR20, LR34, and LR26. The selected L. reuteri LR6, LR9, LR11, and L. reuteri ATCC55730 inhibited 40.5, 32.5, 28, and 49 % of the adherence of E. coli ATCC25922 to Caco-2 cells, respectively, while among test strains, L. reuteri LR6 showed the highest inhibition ability against S. typhi NCDC113 up to 52.5 %. After the surface proteins were removed by 5 M LiCl, the inhibition activity of L. reuteri strains against E. coli ATCC25922 and S. typhi NCDC113 were significantly reduced (p < 0.05).

In exclusion assay, the strains LR5, LR6, LR9, LR20, and LR26 have higher inhibition ability against E. coli ATCC25922 than LR11, LR19, LR34, and L. reuteri ATCC55730 (p < 0.05), whereas the strains LR6, LR9, LR19, and LR26 have higher inhibition against S. typhi NCDC113 than LR5, LR11, LR20, LR34, and L. reuteri ATCC55730. L. reuteri LR6 inhibited 44 % of E. coli ATCC25922 adhering to the cells, higher than LR5 (32.5 %), LR9 (32.5 %), LR20 (30.5 %), and LR26 (40.5 %). For S. typhi NCDC113, 51, 45, 37.5, and 39 % were inhibited by LR6, LR9, LR19, and LR26, respectively, with LR6 showing the highest inhibitive ability. Without surface proteins, the inhibition activity of LR6, LR9, LR19, and LR26 were significantly reduced (p < 0.05) against S. typhi NCDC113, and the inhibition activity of LR5, LR6, LR9, LR20, and LR26 were also significantly reduced against E. coli ATCC25922 (p < 0.05).

In displacement assay, LR6 and L. reuteri ATCC55730 inhibited 50.5 and 51.5 % of S. typhi NCDC113 to adhere to Caco-2, respectively. Also, LR6 and L. reuteri ATCC55730 showed the highest inhibition ability to decrease 40.5 % of the E. coli ATCC25922, respectively. The inhibition activity of strains LR6 and L. reuteri ATCC55730 against S. typhi NCDC113 and E. coli ATCC25922 was significantly reduced (p < 0.05) on treatment with 5 M LiCl.

SDS-PAGE analysis of surface proteins

Cell surface proteins from L. reuteri LR6, showing maximum survival in simulated gastrointestinal conditions and highest adhesion to Caco-2 cells, were extracted with 5 M LiCl and separated on SDS gel showed bands ranging 10 to 100 kDa, as shown in Fig. 2.
Fig. 2

SDS-PAGE analysis of surface proteins extracted with 5 M LiCl from L. reuteri LR6. M low molecular weight protein standards, S surface protein extract from LR6

Discussion

This study demonstrates the importance of cell surface proteins in probiotic activities of L. reuteri strains including survival in simulated gastrointestinal conditions, cell surface characteristics, aggregation properties, and adhesion abilities of selected probiotic strains and inhibition of selected pathogens to Caco-2 cells.

The survival of probiotic bacteria in the gastrointestinal ecosystem as well as adhesion to the intestinal mucosa is regarded as a prerequisite for transient colonization, stimulation of the immune system, and for antagonistic activity to enteropathogens [23, 24]. The hostile gastrointestinal conditions is the first hurdle that probiotic has to face on ingestion. The high acidic environment of stomach and high bile salts secretions in duodenum are not suitable for the survival of the bacteria. Therefore, the probiotic must be able to resist these harsh conditions. In the present study, LR6 showed maximum resistance to such unsuitable conditions. Several reports suggest that cell surface proteins act as a protective sheath against hostile environmental agents such as acid and bile salts [7]. Our study revealed that the survival of the L. reuteri strains significantly reduced on the removal of cell surface proteins with 5 M LiCl, confirming the protective role of their surface proteins against hostile gastrointestinal conditions.

In order to gain information on the structural properties of the cell surface of L. reuteri that are responsible for aggregation and adhesion, its hydrophobicity/hydrophilicity was determined. n-Hexadecane, chloroform, and ethyl acetate were used to assess the hydrophobic/hydrophilic, electron donor (basic), and electron acceptor (acidic) characteristics of bacterial surface, respectively (Table 2), which are attributed to carboxylic groups and Lewis acid–base interactions [20, 21]. L. reuteri LR11 showed higher hydrophobicity, while LR19 and LR26 showed lower hydrophobicity. The hydrophobic differences between probiotics may result in variability in their colonizing ability. Many previous studies on the physicochemistry of microbial cell surfaces have shown that the presence of (glyco-)proteinaceous material at the cell surface results in higher hydrophobicity, whereas hydrophilic surfaces are associated with the presence of polysaccharides [9, 25, 26]. The bacterial affinities to ethyl acetate were relatively low when compared to n-hexadecane and chloroform, indicating that probiotic strains have the nonacidic and poor electron acceptor property [25]. The autoaggregation of the probiotics varied between strains (Table 2), where L. reuteri LR6 showed strongest auto-aggregation ability suggesting specific binding capabilities of probiotics in the gastrointestinal tract. In most cases, autoaggregation ability was also related to cell adherence properties.

Adhesion of lactobacillus strains to the enterocyte-like Caco-2 cell model is commonly used to investigate the adhesion, inhibition, displacement, and competitive inhibition because the adhesion ability to epithelial cells is primarily considered a functional criterion for the selection of potential probiotic strains [9]. The strains with high adhesion ability can efficiently occupy the adhesive sites on the intestinal cells and mucus to inhibit the adhesion of pathogens and protect the host cells from infections. L. reuteri LR6 and L. reuteri ATCC55730 strongly adhered to Caco-2 cells (Table 3) and effectively inhibited the adherence of pathogens to Caco-2 cells (Tables 4 and 5). The observation suggests that in vitro adhesion to Caco-2 cells is correlated with competitive inhibition, which is competitively excluding entropathogens. Bacterial adhesion to the gastrointestinal tract is a complex mechanism that involves extracellular and cell surface receptors [9, 20]. To assess the potential contribution of these proteins to autoaggregation and adherence, bacterial cells were extracted with 5 M LiCl to remove surface proteins. The results showed that these proteins are important for autoaggregation in L. reuteri strains.

The inhibition of adhesion of different pathogens was specifically depending on the strains and pathogens used as well as the methods of assessment [27, 28]. L. reuteri LR6 and the reference strain L. reuteri ATCC55730 showed higher inhibition efficiency against E. coli ATCC25922 and S. typhi NCDC113. Other strains with high adhesive ability did not show the same inhibition capacity against E. coli ATCC25922 and S. typhi NCDC113, but they efficiently inhibited the adhesion of both pathogenic bacteria to Caco-2 cell in all three assays. It is reported that L. casei rhamnosus 35 can interfere with the adhesion of enterotoxigenic and enteropathogenic E. coli [29]. L. reuteri LR6 and reference strain L. reuteri ATCC55730 with high adhesion ability generally showed much higher inhibition ability to the adherence of pathogens to Caco-2 cells, indicating that the inhibition capacity of lactobacillus against pathogenic bacteria may be related to the adhesion ability. In contrast, Collado et al. [30] found that some commercial strains with low adhesive ability had better inhibition ability compared with other high adhesive strains. Higher adhesion ability is not always associated with higher inhibition capacity against pathogens, suggesting that the inhibition capacity is complicated and many factors may be involved.

For some lactobacillus strains, surface proteins perform as adhesion medium binding lactobacillus to the intestinal epithelial cells and mucus, such as mucus-binding proteins MapA from L. reuteri [31] and surface protein from L. plantarum 423 [17]. Surface proteins of several lactobacilli, including L. crispatus and L. acidophilus whose ability to bind to host epithelial cells is decreased after removal or disruption of the S-layer proteins [3234], have been shown to confer tissue adherence. After the lactobacillus pretreated with LiCl to remove cell surface protein, the inhibition ability of lactobacillus against pathogens decreased [27, 3537]. In the present study, the inhibition capacity of the L. reuteri strains against E. coli ATCC25922 and S. typhi NCDC113 was significantly reduced when they were treated with 5 M LiCl (p < 0.05).

The SDS-PAGE of cell surface proteins of L. reuteri LR6 revealed the presence of several bands with molecular weight ranging from 10 to 100 KDa. It has been proposed that cell surface proteins are involved in cell protection and inhibition of pathogen adhesion, and they could be potential mediators in the initial steps involved in adhesion [810]. Recently, researchers have reported that the surface proteins from Lactobacillus kefir strains remained associated with S. enteritidis 50335 surface and could either modify or mask Salmonella structures necessary for the invasion of cultured human enterocytes instead of a competition for binding sites on the surface of the enterocyte [38]. On the other hand, surface proteins from L. kefir interact with the binding sites on host cell to inhibit the adhesion of E. coli K88 [39]. Therefore, the role of surface proteins may differ in the inhibition against pathogens. Further research is needed to explain the adhesion mechanism as the adhesins of lactobacillus strains and the receptors expressed on host involved in adhesion are still unclear.

Conclusions

L. reuteri LR6 can be exploited as a gastrointestinal probiotics because of its resistance to acidic condition and bile salt as well as its high adhesive ability. Our findings also indicate that the cell surface proteins contributed to its increased adhesion to the cultured cells and competitive exclusion of pathogens.

Declarations

Acknowledgements

We thank Dr. Sudhir Kumar Tomar, Dairy Microbiology Division, NDRI for providing the facility to carry out scanning electron microscopy.

Authors’ contributions

Tejinder and Gurpreet have made substantial contributions to the conception and design, acquisition of data, and analysis and interpretation of data. The article was written by Tejinder with assistance from RK Malik and Gurpreet, taking into account the comments and suggestions of the coauthors. All coauthors had the opportunity to comment on the analysis and interpretation of the findings and approved the final version for publication.

Competing interests

The authors declare that they have no competing interests.

Open AccessThis 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.

Authors’ Affiliations

(1)
Dairy Microbiology Division, National Dairy Research Institute

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