Antagonistic activity of Lactobacillus acidophilus ATCC 4356 S-layer protein on Salmonella enterica subsp. enterica serovar Typhimurium in Caco-2 cells

Abstract

To gain insight into the mechanism of Lactobacillus S-layer protein in antimicrobial activity, we examined how the Lactobacillus acidophilus ATCC 4356 S-layer protein inhibited the adhesion and invasion of Salmonella enterica subsp. enterica serovar Typhimurium SL1344 (Salmonella Typhimurium SL1344) in vitro in cultured Caco-2 cells. The results showed that S-layer protein reduced Salmonella Typhimurium SL1344 association in Caco-2 cells by 69–88% in the adhesive experiments (competition, exclusion, and displacement assays). Although the antagonistic activity was demonstrated in the exclusion assay (preincubated with the S-layer protein), a greater effect for the S-layer protein on Salmonella Typhimurium was observed when Salmonella Typhimurium was coincubated with the S-layer protein. The S-layer protein could be bound directly to the Caco-2 cell line or be associated with Salmonella Typhimurium surface, thereby blocking Salmonella attachment. The data support the antimicrobial mechanisms of Lactobacillus S-layer protein, which are involved not only in competition for binding sites on the surface of host epithelial cells, but also in a direct interaction between this protein and Salmonella Typhimurium surface.

Introduction

Adhesion of pathogenic bacteria to host epithelial cells is the first step—and a prerequisite—in the initiation of the infectious process (Bhavsar et al. 2007; Galan and Bliska 1996). Salmonella enterica subsp. enterica serovar Typhimurium (Salmonella Typhimurium) is an important intracellular pathogen that causes a variety of illnesses, ranging from localized gastroenteritis to severe, life-threatening typhoid fever (Rugbjerg et al. 2004). Salmonella Typhimurium intimately adheres to, invades, and interacts with host epithelial cells to cause cytoskeletal rearrangements, increase epithelial monolayer permeability, and elicit proinflammatory responses (Galan and Bliska 1996).

Current therapy for Salmonella Typhimurium infection is limited to antibiotic treatment because antibiotics may increase the more diverse multidrug-resistant Salmonella Typhimurium strains and increase the risk of therapeutic failure in cases of life-threatening salmonellosis (Cloeckaert and Schwarz 2001; Quinn et al. 2006; Yu et al. 2008). The lactobacilli are a group of nonpathogenic microorganisms that have beneficial effects on health. In recent years, lactobacilli have been reported to prevent the adhesion, establishment, and invasion of numerous enteropathogens, such as Escherichia coli, Salmonella, Streptococcus, and Shigella (Moorthy et al. 2007; Otero and Nader-Macias 2006; Voravuthikunchai et al. 2006). Surface layer (S-layer) protein consists of crystalline arrays of proteinaceous subunits present as the outermost component of the cell wall in several Lactobacillus species. The S-layer protein has been shown to be a key adhesion factor of the cell and could play a role in the probiotic activity of certain lactobacilli (Jakava-Viljanen and Palva 2007; Wang et al. 2008). The S-layer protein of such species as Lactobacillus helveticus, L. crispatus, and L. kefir are involved in tissue adhesion and are able to inhibit the adhesion of pathogens, such as enterohemorrhagic E. coli (Johnson-Henry et al. 2007) and Salmonella enterica serovar Enteritidis (Salmonella enteritidis) (Golowczyc et al. 2007), to host epithelial cells.

However, the ability of S-layer protein to ameliorate the pathogenesis of Salmonella Typhimurium infection has not been previously investigated. Therefore, the aim of this study was to determine the potential for S-layer protein extracts from lactobacilli to reduce epithelial cell adhesion and invasion by this pathogen and also to investigate the possible antimicrobial mechanisms of S-layer protein. The results support the usefulness of Lactobacillus S-layer protein in inhibiting the ability of Salmonella Typhimurium to invade human intestinal epithelial cells, but they also highlight the S-layer protein in this antagonistic activity, which limits the capacity to generalize findings in probiotics research from one setting to another.

Materials and methods

Cell culture

Caco-2 human colon cancer epithelial cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD). Caco-2 cells (passages 45–60) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 100 mg L^-1 penicillin, 100 mg L^-1 phytomycin, 10% (v/v) heat-inactivated foetal calf serum (FCS), 4.5 g L^-1 D-glucose, 25 mM HEPES, 1% nonessential amino acids, and 2 mM L-glutamine (Gibco, Carlsbad, CA). The confluent monolayers of Caco-2 cells were trypsinized, washed, and resuspended, then seeded into 24-well plates (2 × 10⁵ cells/well) and grown until confluent (differentiated cells; 37°C; 5% CO₂).

Bacterial strains

The Salmonella enterica subsp. enterica serovar Typhimurium SL1344 (Salmonella Typhimurium SL1344) was a generous gift from Prof. Liu (Peking University, Beijing, China). Lactobacillus acidophilus ATCC 4356 was purchased from the China Committee for Culture Collection of Microorganisms. For infection of the Caco-2 cells, Salmonella Typhimurium was routinely grown in Luria–Bertani medium overnight. The bacteria were collected by centrifugation, washed with sterile phosphate-buffered saline (PBS, pH 7.4), then resuspended in tissue culture medium without antibiotics. ATCC 4356 was grown in De Man–Rogosa–Sharpe static cultures with minimal aeration at 37°C for 24 h and harvested by centrifugation. After two washes with PBS, cell pellets were resuspended directly in tissue culture medium without antibiotics for the adhesion assay

S-layer protein

S-layer protein was obtained from L. acidophilus ATCC 4356 as previously reported (Li et al. 2010). Briefly, S-layer protein was extracted from L. acidophilus ATCC 4356 by 4.0 M guanidine hydrochloride (Boot et al. 1993) and subsequently purified on an anion–exchange column (DE52; Whatman, Kent, UK).

Salmonella Typhimurium association with S-layer protein assay

The association assays were investigated on Caco-2 cell monolayers in 24-well plates (Ni et al. 2008). Tissue culture medium was replaced with antibiotic-free medium 1 day before bacterial infection. Different types of experiments were performed:

• exclusion assay: Caco-2 cells were preincubated with the S-layer protein (100 μg mL^-1) for 1 h at 37°C, and then Salmonella Typhimurium SL1344 [multiplicity of infection (MOI) 20:1] was added to each well and incubated for 1 h at 37°C;

• competition assay: the S-layer protein (100 μg mL^-1) and SL1344 (MOI 20:1) were mixed and coincubated simultaneously with Caco-2 monolayers for 2 h at 37°C;

• displacement assay: SL1344 (MOI 20:1) was added to Caco-2 monolayers and incubated for 1 h at 37°C before challenge with the S-layer protein (100 μg mL^-1); the plates were then incubated 1 h at 37°C; finally, the cells were washed three times with sterile PBS and lysed with sterile distilled water.

Each treatment was carried out in triplicate wells. Salmonella Typhimurium colony counts were performed on the number of associated (adhering plus invading) viable cells. All assays were performed in triplicate on three consecutive cell passages.

In vitro bactericidal effect

To determine whether the S-layer protein had a bactericidal effect, Salmonella Typhimurium (10⁸ CFU mL^-1) was coincubated with various concentrations of the S-layer protein (0, 0.01, 0.02, 0.05, and 0.1 mg mL^-1) for 2 h at 37°C in Caco-2 tissue culture medium. Appropriate dilutions of the bacteria were made and plated onto Salmonella–Shigella agar plates for 24 h, following which the bacterial colonies were measured.

Scanning electron microscopy

Caco-2 cells were differentiated on glass coverslips, which were placed in 24-well tissue culture plates for 2 weeks with changes of medium every 3 days. The differentiated Caco-2 cells were then incubated in Dulbecco’s modified Eagle’s medium–10% FCS, infected with Salmonella Typhimurium strains (MOI 20:1) for 30 min (37°C; 5% CO₂), and washed three times with PBS. The cells were then fixed in glutaraldehyde, postfixed in OsO₄ for 1 h, dehydrated with a graded series of ethanol, and then freeze-dried by t-butyl alcohol substitution. The dried samples were examined with a Hitachi S-3000 N (Tokyo, Japan) scanning electron microscope.

Data analysis

Results were expressed as means ± standard error of mean (SEM). Analysis of variance (ANOVA) and the unpaired Student’s t test were employed to determine statistical differences among multiple groups.

Results

Effect of S-layer protein on inhibition of Salmonella Typhimurium adhesion and invasion in Caco-2 cells

Pretreatment, coincubation, and post-treatment of Salmonella Typhimurium-infected Caco-2 cells with the S-layer protein produced a significant decrease in association (Fig. 1). These results indicated that the association ability of Salmonella Typhimurium in Caco-2 cells was impaired by the S-layer protein (88.83 ± 5.97, 71.29 ± 1.36, and 69.44 ± 0.92% reduction, respectively in the competition, exclusion, and displacement assays, relative to Salmonella Typhimurium association without the S-layer protein; ANOVA P < 0.01; Fig. 1). The level of inhibition of association was stronger in the competition assay than in the exclusion and displacement assays.

Fig. 1

Treatment of Caco-2 cells with S-layer protein reduces Salmonella Typhimurium association. The number of association bacteria was counted in the adhesion and invasion assays. **Analysis of variance (ANOVA) P < 0.01 compared with the control

Effect of microvillous structure of Salmonella Typhimurium-infected Caco-2 cells with the S-layer protein

To obtain more detailed information on the effect of S-layer protein on Salmonella Typhimurium adhesion and invasion, we examined infected Caco-2 monolayers by scanning electron microscopy. As evident in Fig. 2a, the intact monolayer of uninfected Caco-2 cells showed intact microvilli over the entire cell surface. In comparison, at 30-min postinfection, shortened microvilli and attached and agminated bacterial cells were observed in Salmonella Typhimurium-infected Caco-2 cells (Fig. 2b). Although complete protection was not achieved when Caco-2 infected with Salmonella Typhimurium was coincubated with S-layer protein, agminated bacterial cells were not observed. In addition, the number of adhered Salmonella Typhimurium coincubated with this protein even diminished (Fig. 2d). However, S-layer protein alone did not affect the microvillous structures (Fig. 2c).

Fig. 2

Scanning electron microscopic analysis of Caco-2 cells infected with Salmonella Typhimurium strains. a Uninfected Caco-2 cells, b Salmonella-infected Caco-2 cells, c Caco-2 cells were treated with S-layer protein alone, d Caco-2 infected with Salmonella coincubated with S-layer protein

No bactericidal effect with S-layer protein to Salmonella Typhimurium

Pretreatment of Salmonella Typhimurium with L. acidophilus S-layer protein (0, 0.01, 0.02, 0.05, and 0.1 mg mL^-1) under these experimental conditions did not affect its viability because the incubation of Salmonella Typhimurium with S-layer protein did not have any effect on their concentration. This result indicates that the S-layer protein does not have bactericidal effects on Salmonella Typhimurium (data not shown).

Discussion

Lactobacilli are used as probiotic bacteria in both functional foods and antimicrobial agents (Vadillo-Rodriguez et al. 2005). An important factor of the antimicrobial activity of probiotic strains is considered to be their competition with enteric pathogens for binding sites (i.e., “competitive exclusion”) (Servin 2004). The location of S-layer protein at the outermost surface of some lactobacilli strains implies that it plays a role in adhesion. If the S-layer protein of lactobacilli is removed or damaged, the adhesion of lactobacilli to the host epithelial cells is reduced (Lee et al. 2003). Therefore, S-layer protein may have an important role in antimicrobial activity during competitive exclusion. The antagonistic ability of S-layer protein on some pathogens has recently been reported (Golowczyc et al. 2007; Jakava-Viljanen and Palva 2007; Johnson-Henry et al. 2007). However, the inhibition of L. acidophilus S-layer protein on Salmonella Typhimurium has not been studied, and the general mechanisms of the antagonistic activity remain unclear.

In this study, we investigated the effects of S-layer protein on its role in attenuating the adhesion and invasion of Salmonella Typhimurium in a Caco-2-cultured human intestinal cell line. The results further demonstrated that L. acidophilus S-layer protein is able to antagonize the adhesion and/or invasion of Salmonella Typhimurium in vitro in adhesive assays—and not because S-layer protein has a bactericidal effect on Salmonella Typhimurium. Changes in the distribution of cytoskeletal components and disorganization of microvilli have been reported during Salmonella invasion (Finlay and Falkow 1990; Finlay et al. 1989). To acquire information on the effect of Salmonella on microvillous structure, we examined infected Caco-2 monolayers by scanning electron microscopy and observed that microvilli of Caco-2 cells infected with Salmonella Typhimurium were clearly disorganized. In contrast, protection was clearly achieved when Salmonella Typhimurium was coincubated with L. acidophilus S-layer protein.

With respect to the antimicrobial mechanism, it has been reported that Lactobacillus S-layer protein may interact with receptors on host epithelial cells, thereby blocking receptor sites on the mucosal surface for the adhesion of pathogenic species (Borinski and Holt 1990). Other researchers have found that when Salmonella enteritidis is preincubated with L. kefir S-layer protein, there is a direct interaction between this protein and the surface of the S. enteritidis cells instead of a competition for binding sites on the surface of the enterocyte (Golowczyc et al. 2007). However, in our study, although pretreatment of Salmonella Typhimurium-infected Caco-2 cells with the S-layer protein produced a significant inhibitory effect, a higher inhibitory effect was observed when Salmonella Typhimurium was coincubated with the S-layer protein. These results indicate that competition for the same binding sites may also develop in the extracellular matrix. The results necessitate further revisions to the antimicrobial mechanisms of Lactobacillus S-layer protein, which involve not only a direct interaction between Lactobacillus S-layer protein and the pathogen surface, but also a competition for binding sites on the surface of the host epithelial cells. S-layer protein has the ability to be retained on bacterial surfaces (Antikainen et al. 2002; Golowczyc et al. 2007), which was also confirmed by our dot-blot assay (data not shown). In addition, we found that S-layer protein could bind directly to the Caco-2 cell line (Li et al. 2011). These results support the possibility that S-layer protein is able to interact with specific sites on Salmonella or the epithelial cell surface in the first step of mucosal infection; alternatively, the S-layer protein may either modify or mask Salmonella Typhimurium or the epithelial cell surface structures necessary for the invasion of cultured human enterocytes.

In recent years, lactobacilli have been used as probiotic bacteria in antimicrobial agents (Vadillo-Rodriguez et al. 2005). However, some problems exist in the industrial application of probiotic bacteria, such as limitations in viability which present difficulties for long-term preservation and transportation. Our study provides insight into the probiotic properties of Lactobacillus S-layer protein against enteropathogens. The characteristic feature of the S-layer protein is that it is a nonviable, abundant constituent (10–15% in lactobacilli) of whole, viable probiotic bacteria which has a good stability (Avall-Jaaskelainen and Palva 2005). Further studies are needed to characterize the adhesion inhibitory effects and in vivo activity of the S-layer protein against pathogenic bacterial infection.