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Infection and Immunity, April 2002, p. 2057-2064, Vol. 70, No. 4
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.4.2057-2064.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Lipoteichoic Acids from Lactobacillus johnsonii Strain La1 and Lactobacillus acidophilus Strain La10 Antagonize the Responsiveness of Human Intestinal Epithelial HT29 Cells to Lipopolysaccharide and Gram-Negative Bacteria

Karine Vidal,^1* Anne Donnet-Hughes,¹ and Dominique Granato²

Food Immunology Group,¹ Digestive Health Group, Nestlé Research Center, Nestec Limited, Vers-chez-les-Blanc, Lausanne, Switzerland²

Received 2 October 2001/ Returned for modification 1 November 2001/ Accepted 21 December 2001

Top Abstract Introduction Materials and Methods Results Discussion References

 
Intestinal epithelial cells (IECs) respond to lipopolysaccharide (LPS) from gram-negative bacteria in the presence of the soluble form of CD14 (sCD14), a major endotoxin receptor. Since sCD14 is also known to interact with gram-positive bacteria and their components, we looked at whether sCD14 could mediate their effects on human IECs. To this end, we examined the production of proinflammatory cytokines following exposure of the IECs to specific gram-positive bacteria or their lipoteichoic acids (LTAs) in the absence and presence of human milk as a source of sCD14. In contrast to LPS from Escherichia coli or Salmonella enteritidis, neither the gram-positive bacteria Lactobacillus johnsonii strain La1 and Lactobacillus acidophilus strain La10 nor their LTAs stimulated IECs, even in the presence of sCD14. However, both LTAs inhibited the sCD14-mediated LPS responsiveness of IECs. We have previously hypothesized that sCD14 in human milk is a means by which the neonate gauges the bacterial load in the intestinal lumen and liberates protective proinflammatory cytokines from IECs. The present observations suggest that gram-positive organisms, via their LTAs, temper this response and prevent an exaggerated inflammatory response.

Top Abstract Introduction Materials and Methods Results Discussion References

 
At parturition, there is heavy colonization of the previously sterile fetal intestine by a vast microbial inoculum. The establishment of the intestinal microflora is a very dynamic process during the first days of life before stable populations are established in defined regions of the gastrointestinal tract (34). There is sequential colonization first by Escherichia coli and Streptococcus species (35) and then by a bifidogenic microflora, which is highly dominant in breast-fed babies and offers some protection against potential pathogens (23). However, the predominance of endotoxin-producing gram-negative bacteria compromises the neonate and may contribute to the pathogenesis of a variety of immune and inflammatory neonatal conditions, such as necrotizing enterocolitis and gut-derived sepsis.

Lactic acid bacteria (LAB), such as lactobacilli and bifidobacteria, are normal inhabitants of the human adult gastrointestinal tract. Selected strains from these genera, termed probiotic organisms, have health benefits when administered orally to the host (8). These LAB survive gastrointestinal passage, and like LAB present in the microbial flora, they are thought to offer protection against potential pathogens by vying for nutrients, by producing antimicrobial substances such as bacteriocins, and/or by stimulating immune responses. Although little is known about the precise mechanisms which underlie these biological effects, it is widely accepted that strains which most likely exert health benefits are those which can transiently reside in the juxtamucosal niche, i.e., in the mucus or on the intestinal epithelium. It has been recently shown that the probiotic strain Lactobacillus johnsonii La1 can adhere to intestinal epithelial cells (IECs) via its lipoteichoic acid (LTA) (24).

LTA is a complex glycerophosphate polymer linked to a hydrophobic lipid moiety anchored in the cytoplasmic membrane of gram-positive bacteria, which extends through the cell wall peptidoglycan onto the surface of the cells (18). It is a major component of the cell wall of most gram-positive bacteria and although there is great diversity in the LTAs from different bacteria, it has structural similarities to lipopolysaccharide (LPS) found in the cell walls of gram-negative organisms. While LPS is renowned as a potent activator of innate immune responses that result in the production of both pro- and anti-inflammatory mediators from various cell types (43), less work has been done using LTA. Nevertheless, it appears that only LTA from particular species of bacteria mediate such effects (6, 28, 30, 41).

The CD14 molecule, which is expressed primarily on monocytes and macrophages, serves as a high-affinity receptor for LPS, mediating LPS-induced cell activation (43, 46). It exists both as a glycosylphosphatidylinositol-anchored membrane protein (mCD14) and as a soluble molecule (sCD14) found in serum (4) and in milk (17, 31). There is increasing evidence that sCD14 participates in LPS-induced activation of cell types that normally do not express mCD14 (3, 14, 22, 25, 26, 33, 38). The results of recent in vitro experiments have shown that CD14 is a pattern recognition receptor (37), which can bind not only LPS but also other bacterial components, such as LTA, peptidoglycan, lipoarabinomannan, and mannuronic acid polymers (16). Recently, the Toll-like receptors (TLRs), a family of pattern recognition receptors, that recognize LPS and other bacterial products have been postulated to act as transmembrane coreceptors to CD14 (for reviews, see references 1, 5, and 29).

We have recently reported that sCD14 present in human breast milk (HM) mediates E. coli- and LPS-induced cytokine production by mCD14-negative human IECs (31, 44). In the present study, we examined the capacity of gram-positive bacteria or their purified LTAs to stimulate cytokine production in IECs in the presence of sCD14. No effect was observed with different LAB strains, including Lactobacillus johnsonii strain La1 and Lactobacillus acidophilus strain La10, as well as with their purified LTAs. However, an antagonistic effect of the LTA on the responsiveness of IECs to either whole gram-negative bacteria or their purified LPSs was obtained. These data suggest that gram-positive bacteria, via their LTA, may prevent exaggerated inflammatory responses caused by gram-negative bacteria in the gut.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Reagents. Phenol-extracted LPS from E. coli O55:B5 and Salmonella enteritidis strains were purchased from Sigma Chemical Co. (St. Louis, Mo.) (catalog no. L-2880 and L-6011, respectively). LTA from Staphylococcus aureus was purchased from Sigma. Dulbecco modified Eagle medium (DMEM) and RPMI 1640 medium were purchased from Life Technologies AG (Basel, Switzerland). Fetal calf serum (FCS) (Amimed BioConcept, Allschwill, Switzerland) was inactivated by heating at 56°C for 45 min before use. Murine anti-CD14 monoclonal antibody MY4 (immunoglobulin G2b) was purchased from Coulter (Instrumentation Laboratory, Schlieren, Switzerland). The isotype-matched control monoclonal antibody was mouse immunoglobulin G2b (kappa chains) derived from MOPC 141 (Sigma). HM samples were obtained from healthy mothers. Samples were obtained up to 70 days postpartum by breast pump expression into sterile centrifugation tubes and processed within 2 h of collection. After centrifugation at 200 x g for 30 min, the acellular lipid-free fraction was frozen at -20°C until used.

All reagents to which cells were exposed were tested for LPS contamination by a turbidimetric kinetic Limulus amebocyte lysate clot assay (Charles River Endosafe, Charleston, S.C.). The test had a sensitivity of 0.05 to 0.1 endotoxin unit (E. coli O55:B5 LPS) per ml. The different media used in this study were found to be inactive or to contain <50 pg of endotoxin per ml.

Bacterial strains and growth conditions. Gram-positive bacteria Staphylococcus aureus, Staphylococcus epidermidis, Lactobacillus sakei, Lactobacillus casei, L. johnsonii strain La1 (NCC 533; Nestle Culture Collection), and L. acidophilus strain La10 (NCC 90) were grown separately under anaerobic conditions in Man-Rogosa-Sharpe (MRS) broth (Difco, Detroit, Mich.) overnight at 37°C. Bacterial cells were harvested by centrifugation for 10 min at 4,000 x g at 4°C and washed in phosphate-buffered saline (PBS). Bacterial cells were then resuspended in either DMEM or RPMI 1640 medium.

Isolation and purification of LTAs from LAB. LTAs from L. johnsonii strain La1 and L. acidophilus strain La10 were isolated by the method of Fischer et al. (19), a method by which LTA preparations devoid of protein, nucleic acid, polysaccharide, peptidoglycan, and teichoic acid contamination can be obtained. Briefly, bacteria were cultured overnight in MRS broth, harvested, and resuspended in 0.1 M sodium acetate (pH 4.5) at 800 mg (wet weight) per ml of buffer. To facilitate the extraction of LTA, the bacterial cells were delipidated by mixing with 2 volumes of methanol and 1 volume of chloroform overnight at room temperature. The delipidated bacterial cells were recovered by filtration, washed with 2 volumes of methanol, and resuspended in 0.1 M sodium acetate (pH 4.7) at a concentration of 500 mg of bacterial cells per ml of buffer. The suspension was mixed with an equal volume of hot 80% (wt/vol) aqueous phenol and stirred constantly for 45 min in a water bath at 65°C. On cooling, an emulsion was formed, which could be broken into two phases by centrifugation at 4°C at 5,000 x g for 30 min. The lower phenol layer contained all the lipids, and the insoluble residue contained cell wall peptidoglycan. The upper aqueous layer, which contained LTAs, nucleic acids, polysaccharides, teichoic acids, and proteins, was extensively dialyzed against 0.1 M sodium acetate (pH 4.7) and adjusted to 15% with 1-propanol. This solution was then applied to an octyl-Sepharose CL-4B column (Pharmacia) equilibrated in 0.1 M sodium acetate (pH 4.7) containing 15% 1-propanol, with a flow rate of 0.1 ml/min. The column was extensively washed with the same buffer until the absorption at 260 and 280 nm was close to the baseline level and the presence of carbohydrates was no longer detected by the phenol-sulfuric acid method of Dubois et al. (15). Elution of the LTA was done with a gradient of 15 to 80% 1-propanol in the same buffer, with a flow rate of 0.5 ml/min. Monitoring of 1-propanol concentration was done by measuring the refraction index. Each fraction was analyzed for its content of carbohydrates (15) and phosphorus (10). Nucleic acid contamination was assessed by the absorption at 260 and 280 nm. Protein content was measured by the absorption at 280 nm and the bicinchoninic acid test (Pierce Socochim SA, Lausanne, Switzerland). The peak fractions were concentrated with a rotavaporator to get rid of the propanol and then extensively dialyzed against water. After the fractions were concentrated, 0.1 M sodium acetate (pH 4.7) was added and aliquots were frozen at -20°C. The antigenic activity of La1 LTA was verified by enzyme-linked immunosorbent assay (ELISA) as described recently (24).

Deacylation of LTAs. Deacylation was performed by the method of Teti et al. (42). Briefly, 1 mg of La1 and La10 LTA was incubated for 16 h in 15% ammonium hydroxide. After evaporation, the samples were subjected to Folch partition (21). Briefly, the samples were resuspended in 1 volume of distilled water and 19 volumes of chloroform-methanol (2:1). After a vigorous vortexing, 0.2 volume of distilled water was added, leading to the appearance of two phases. To confirm deacylation, the presence of free fatty acids in the organic phase was assessed by gas chromatography (GC)-mass spectrometry (MS), as described previously (24). The deacylated LTA present in the aqueous phase was dialyzed against water, concentrated by using a rotavaporator, and adjusted to the same concentration of carbohydrate as the nondeacylated LTA.

Culture of human IECs. The human colonic adenocarcinoma cell line HT29 (ATCC HTB-38) was obtained from the American Type Culture Collection (Manassas, Va.). Undifferentiated cells were maintained in glucose-containing DMEM supplemented with 10% FCS at 37°C in an incubator containing 5% CO₂, while differentiated cells were grown in glucose-free DMEM. The culture medium was changed every 2 days until the cell monolayers reached 90% confluency. For stimulation assays, HT29 cells were plated at 10⁴ cells/well in 96-well flat-bottom plates. After incubation for 5 days, HT29 cells were washed twice with serum-free medium before the addition of HM, LPS, and/or LTA in 200 µl of DMEM. In some wells, anti-CD14 monoclonal antibodies at a final concentration of 20 µg/ml were also added. The cell viability was examined using a cytotoxicity detection kit (Roche Diagnostics, Rotkreuz, Switzerland), which measured the lactate dehydrogenase activity released from the cytosol of damaged cells into the supernatant.

ELISA. The amounts of interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-) generated in cell culture supernatants were measured by ELISA. Briefly, the wells of 96-well Maxisorp plates (Nunc) were coated with monoclonal antibodies against IL-8 (2 µg/ml) (ImmunoKontact; AMS Biotechnology, Lugano, Switzerland) by overnight incubation at 4°C. The wells were then washed twice with PBS containing 0.05% Tween 20. Nonspecific binding was blocked by incubating the plates with PBS containing 10% FCS for a further 2 h at room temperature. Samples or standard concentrations of recombinant cytokine (15,625 to 2,000 pg/ml; ImmunoKontact) in PBS with 10% FCS were then added for 3 h at room temperature. The plates were washed four times with PBS containing 0.05% Tween 20 before the addition of biotin-labeled mouse anti-human IL-8 monoclonal antibody (1 µg/ml; ImmunoKontact) for another hour at room temperature. After the plates were washed four times, streptavidin-peroxidase (0.5 µg/ml) (KPL Bioreba, Reinach, Switzerland) was added for 1 h at room temperature. Plates were then washed, and the substrate (TMB; KPL) was added for 10 to 30 min. The enzymatic reaction was stopped by the addition of 1 N HCl. Absorbance was read at 450 nm in an ELISA reader (Dynex Technologies, Denkendorf, Germany). The detection limit was approximately 30 pg/ml. The amounts of TNF- released into cell culture supernatants were measured by a commercially available ELISA kit which has a detection limit of approximately 0.2 pg/ml (Quantikine High Sensitivity Human TNF- kit; R&D Systems, Oxon, England).

RNA isolation, RT, and semiquantitative PCR. Total cellular RNA was extracted from HT29 cells in tissue culture dishes by using the Trizol method (Gibco-BRL Life Technologies, Basel, Switzerland). RNA isolated from IECs was reverse transcribed with Moloney murine leukemia virus reverse transcriptase (Perkin-Elmer, Huenenberg, Switzerland). Briefly, RNA samples (0.5 µg of total RNA), 0.5 U of RNase inhibitor, 1 mM (each) deoxynucleoside triphosphate, 0.5 nmol of 3' primer per ml, 5 mM MgCl_2, and 1.25 U of reverse transcriptase were incubated in 10 µl (total volume) of reaction mixture containing the enzyme buffer supplied by the manufacturer. The reaction mixture was incubated for 30 min at 42°C and then heated for 5 min at 95°C. The reverse-transcribed products were then amplified with Gold DNA polymerase (Perkin-Elmer) on a thermocycler (Biolabo Scientific Instruments, Chatel-St-Denis, Switzerland). The PCR was performed in a total volume of 50 µl using 10 µl of the reverse-transcribed products in PCR buffer, 2 mM MgCl₂, 5 µM (each) deoxynucleoside triphosphate, 0.2 nmol of both epithelial cell-derived neutrophil-activating protein 78 (ENA-78)-specific antisense and sense primers per ml (5'-CGTTCTCAGGGAGGCTC-3' and 5'-TCCTTCGAGCTCCTTGTG-3', respectively [27]), and 1.25 U of DNA polymerase. After an initial denaturation of 10 min at 95°C, samples were amplified by 35 cycles of PCR, with 1 cycle consisting of denaturation at 94°C for 45 s, annealing at 60°C for 1 min, and extension at 72°C of 1.5 min, followed by a final extension step at 72°C for 7 min. All samples were subjected to reverse transcription-PCR (RT-PCR) for ß-actin as a positive control. Samples of RT-PCR products were loaded onto 1.2% agarose gels (containing ethidium bromide) in Tris-acetate-EDTA buffer and separated by electrophoresis at 150 V for 1 h. RT-PCR products were visualized under UV light. The correct size of the bands was determined by comparison with DNA size markers (Boehringer Mannheim, Rotkreuz, Switzerland).

Top Abstract Introduction Materials and Methods Results Discussion References

 
LTAs from Lactobacillus species inhibit E. coli- and LPS-induced IL-8 release by HT29 cells. We recently demonstrated that the human IEC line HT29 secretes IL-8 in response to gram-negative bacteria or LPS and that this stimulation is sCD14 dependent (31). Since sCD14 is known to interact with bacterial products from both gram-negative and gram-positive bacteria, we examined whether gram-positive bacteria or their purified components could also stimulate HT29 cells. In the first instance, different whole gram-positive organisms were incubated with HT29 cells in the presence or absence of human milk as a source of sCD14. In contrast to E. coli, the gram-positive bacteria L. sakei, L. casei, L. acidophilus strain La10, and L. johnsonii strain La1, as well as Staphylococcus aureus and Staphylococcus epidermidis were unable to stimulate the release of IL-8 by HT29 cells, even in the presence of sCD14 (data not shown).

Next, we tested whether the LTA cell wall component of gram-positive organisms could stimulate HT29 cells. We purified LTAs from two strains of lactobacilli, L. acidophilus strain La10 and L. johnsonii strain La1, which have recently been shown to differ in their adhesive properties to IECs (24). To obtain purified LTAs without protein, polysaccharide, peptidoglycan, and nucleic acid contamination, the crude extract preparations were subjected to hydrophobic interaction chromatography by the method of Fischer et al. (19). The conditions were chosen to allow (i) all hydrophilic compounds to be eluted from the column in a buffer containing 15% 1-propanol and (ii) the amphiphilic molecules to be retained and then eluted with a gradient of 15 to 80% 1-propanol. Both LTAs were eluted from the column at a 25% concentration of 1-propanol, which was in accordance with the concentration reported for other LTAs (19). Contamination by nucleic acids as well as proteins was minimal, as previously discussed by Fischer et al. (19). The LTA preparation exhibited no activity in the Limulus assay, indicating low or no LPS contamination. For comparison, we also tested a commercial LTA purified from Staphylococcus aureus. None of the LTAs tested, at concentrations of up to 100 µg/ml, stimulated IL-8 release by HT29 cells, even in the presence of sCD14 (data not shown). However, both LTAs (5 µg/ml) were able to induce the release of IL-8 from peripheral blood mononuclear cells (data not shown), indicating that these LTAs can exhibit stimulatory activity.

It has recently been shown that LTA can act as an agonist or antagonist of LPS (7, 30, 41). We therefore tested whether LTAs purified from L. johnsonii strain La1 and L. acidophilus strain La10 could antagonize the response of IECs to gram-negative bacteria. To this end, HT29 cells were challenged with E. coli LPS (10 and 100 ng/ml) in the presence or absence of HM as the source of sCD14 and various amounts of LTA purified from either La1 (Fig. 1A) or La10 (Fig. 1B). As expected, HT29 cells exposed to 10 or 100 ng of E. coli LPS per ml in the presence of sCD14 released significant amounts of IL-8 (Fig. 1). The addition of LTA from either La1 (Fig. 1A) or La10 (Fig. 1B) caused a marked decrease in the LPS-induced IL-8 secretion. This inhibitory activity by LTA was observed in a dose-dependent manner irrespective of the dose of the LPS agonist (10 and 100 ng/ml) and with complete inhibition being observed with 100- to 1,000-fold excess LTA compared to LPS. Similar inhibitory activity was observed with LTA purified from Staphylococcus aureus (data not shown). In addition, similar inhibitory activity of LTA from La1 was observed when the HT29 cells were challenged with LPS from Salmonella enteritidis (data not shown), indicating that it was a general phenomenon. More interestingly, LTA from L. johnsonii strain La1 antagonized the sCD14-dependent IL-8 release induced by whole E. coli bacteria on HT29 cells in a dose-dependent manner (Fig. 2). Furthermore, as seen with the undifferentiated HT29 cells, purified LTAs from both Lactobacillus strains failed to stimulate IL-8 production in differentiated HT29 cells but rather they caused the decrease of LPS- or CD14-induced IL-8 secretion (data not shown). Of note, the observed inhibitory activities of LTAs were not due to a cytotoxic effect of the LTA preparations on the HT29 cells, as no significant release of lactate dehydrogenase could be detected in culture supernatants (data not shown). Taken together, these data demonstrate that LTAs from the two LAB strains tested do not stimulate HT29 cells but suppress the capacity of gram-negative bacteria to induce IEC responsiveness.

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FIG. 1. Effects of LTA from L. johnsonii strain La1 and L. acidophilus strain La10 on the release of IL-8 by HT29 cells challenged with LPS purified from E. coli. IL-8 production was measured by ELISA in supernatants of HT29 cells incubated for 24 h in medium supplemented with 2% HM in the presence of E. coli LPS at 10 ng/ml (•) or 100 ng/ml () and various amounts of LTA from L. johnsonii strain La1 (A) or L. acidophilus strain La10 (B). Activation of HT29 cells by LPS plus HM alone is depicted (dashed lines). The standard deviations are indicated by the error bars. The results are representative of seven independent experiments for La1 LTA and three independent experiments for La10 LTA.

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FIG. 2. Effect of LTA from L. johnsonii strain La1 on the release of IL-8 by HT29 cells challenged with whole E. coli bacteria. IL-8 production was measured by ELISA in supernatants of HT29 cells incubated for 24 h in medium supplemented with 2% HM and whole E. coli bacteria (2.5 x 105/ml) () or E. coli LPS (100 ng/ml) (•) in the presence of various amounts of LTA from L. johnsonii strain La1. Activation of HT29 cells in the absence of LTA is depicted (dashed lines).The standard deviations are indicated by the error bars. The results are representative of two independent experiments.

Inhibition of the ENA-78 and TNF- induction activity of E. coli LPS by LTAs from Lactobacillus species. To further examine the antagonistic activity of LTAs from LAB on the response of IECs to gram-negative bacteria, the effects of LTAs on the production of various immune molecules was assessed. For example, we reported that gram-negative bacteria cause a CD14-dependent production of the proinflammatory cytokine TNF- and the CXC chemokine ENA-78 in HT29 cells (31). As shown in Fig. 3A, the LPS-induced expression of mRNA coding for ENA-78 was markedly inhibited by purified LTA from Lactobacillus strain La1. Similarly, both LTAs purified from Lactobacillus strain La1 and La10 inhibited the TNF- production by HT29 cells stimulated by E. coli LPS, as shown in Fig. 3B. Interestingly, LTA from La10 appeared to be more inhibitory than that from La1 for TNF- production by HT29 cells but not for IL-8 release (Fig. 1). Significant inhibition of TNF- production was observed at a 100-fold excess of LTA from La1 compared to LPS. However, LTA from La10 completely blocked TNF- production at all doses tested. Overall, these data reveal that LTAs from the two LAB tested suppress the capacity of gram-negative bacteria to induce the proinflammatory response of IECs.

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FIG. 3. Effects of LTA from L. johnsonii strain La1 and L. acidophilus strain La10 on LPS-induced ENA-78 mRNA expression and TNF- cytokine release by HT29 cells. (A) ENA-78 expression was assessed by RT-PCR on total RNA of HT29 cells challenged with 100 ng of E. coli LPS per ml in the absence (lane 1) or presence of 2% HM (lanes 2 to 6) with the addition of MY4 anti-CD14 monoclonal antibody (lane 3), isotype-matched antibody control (lane 4), or LTA from L. johnsonii strain La1 at 1 µg/ml (lane 5) or 50 µg/ml (lane 6). The expected PCR product size for ENA-78 transcripts was 220 bp. Amplified bands for ß-actin (460 bp) were used as the housekeeping gene. SM, size marker. (B) TNF- production was measured by ELISA in supernatants of HT29 cells incubated for 24 h in medium supplemented with 2% HM, 100 ng of E. coli LPS per ml, and various amounts of LTA from L. johnsonii strain La1 () or L. acidophilus strain La10 (•). Production of TNF- by HT29 cells in the absence of LTA is shown (dashed line). The standard deviations are indicated by the error bars. The results are representative of three independent experiments.

Influence of LTA deacylation on its biological activity. It has been shown that deacylation of LTA results in a complete loss of activity (6, 20, 28). This implies that the presence of the lipid moiety of LTA is critical for immunomodulatory activity. In order to obtain more information about the suppressive activity of the LTAs from the two lactobacilli, we examined the effects of their deacylation on the antagonism of LPS in our cellular model. LTAs from L. johnsonii strain La1 and L. acidophilus strain La10 were deacylated as previously described (42). GC-MS analysis of the material liberated in the organic phase revealed the presence of 10:0, 12:0, 14:0, 16:0, and 18:0 free fatty acids, indicating that deacylation had been effective. As shown in Fig. 4, the antagonistic activity of LTAs from both L. johnsonii strain La1 (Fig. 4A) and L. acidophilus strain La10 (Fig. 4B) toward the LPS or sCD14 induction of IL-8 by HT29 cells was significantly weaker after deacylation. Therefore, it was the lipid moiety of Lactobacillus LTAs that mediated the inhibitory effects we observed in our cellular system.

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FIG. 4. Effects of deacylation of the LTAs on their antagonistic activity. HT29 cells were challenged with E. coli LPS (100 ng/ml) in medium supplemented with 2% HM in the absence (dashed lines) or presence (solid lines) of various amounts of native LTA () or deacylated LTA (•) purified from either L. johnsonii strain La1 (A) or L. acidophilus strain La10 (B). After 24 h, the release of IL-8 in the culture supernatants was measured by ELISA. The standard deviations are indicated by the error bars. The results are representative of two independent experiments.

Effect of HT29 pretreatment with LTA or LPS on the inhibition of the effects of LPS-sCD14. To understand further the means by which LTA exerts its antagonistic effect on gram-negative bacteria, different sequential treatments were performed (Fig. 5). When HT29 cells were preincubated for 4 h with LTA from L. johnsonii strain La1 (100 µg/ml) with or without HM, washed twice with a serum-free medium, and then challenged for 20 h with LPS (100 ng/ml) in the presence of milk, the secretion of IL-8 was not abrogated (Fig. 5A). However, when the cells were preincubated for 4 h with LTA from strain La1 (50 µg/ml) in the presence of HM and then challenged for 24 h with LPS (100 ng/ml) without washing, the inhibition of IL-8 production was similar to that obtained when HT29 cells were incubated for 24 h with LTA, LPS, and HM together (Fig. 5B). The inhibitory effect obtained after the addition of LTA from strain La1 to HT29 cells preincubated for 4 h with LPS and HM was similar to the effect obtained when the cells were incubated for 24 h with LTA, LPS, and HM together (Fig. 5C).

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FIG. 5. Effects of different sequential treatments with LTA, LPS, and HM on IL-8 production by HT29 cells. (A) HT29 cells were preincubated for 4 h with LTA from L. johnsonii strain La1 (100 µg/ml) in the presence of 2% HM, washed twice with a serum-free medium, and then challenged for 20 h with E. coli LPS (100 ng/ml) in the presence or absence of HM. (B) HT29 cells were incubated with LTA from L. johnsonii strain La1 (50 µg/ml) in the presence of HM for 4 h before the addition of E. coli LPS (100 ng/ml). (C) HT29 cells were challenged with E. coli LPS (100 ng/ml) in the presence of HM for 4 h before the addition of LTA from L. johnsonii strain La1 (1, 10, and 50 µg/ml). IL-8 release in the supernatants after a total of 24 h of culture was measured by ELISA. The standard deviations are indicated by the error bars. The results are representative of two independent experiments.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The interaction between microorganisms and IECs is the first step in the sequence of events leading to a host immune response intended to eradicate potential pathogens. Our previous work showed that sCD14 in HM modulates the responses to gram-negative bacteria and their cell wall components (31, 44). The present study addressed whether gram-positive and gram-negative organisms could initiate differential responses from the intestinal epithelium and whether milk sCD14 also modulated the responses to components of gram-positive bacteria. We hypothesized that LTA, a major product of gram-positive bacteria, might be involved in the beneficial effects provided by probiotic bacteria. The L. johnsonii strain La1 used in this study is such a probiotic organism, which adheres to IECs through its LTA (24).

Although LTAs from gram-positive bacteria show great diversity from one bacterial strain to another, some physicochemical properties of LTAs are similar to those of LPS present in the cell walls of gram-negative organisms. Some researchers have reported that gram-positive bacterial cell wall components, such as peptidoglycan and LTA, induced the secretion of various proinflammatory cytokines, such as TNF-, IL-1ß, macrophage inflammatory protein 1, IL-6, and IL-12 from monocytes (2, 6, 11, 13, 28, 36, 40, 41, 45). However, only LTAs from specific species of bacteria were able to do so (6, 28, 30, 41). Thus, it appears that the biological activity of LTAs cannot be predicted.

IECs require sCD14 molecules to respond to LPS in vitro (38). As we described above, HM sCD14 mediated the action of gram-negative bacteria and LPS to induce proinflammatory cytokine production in IECs (31). However, here we showed that the IECs were unresponsive to various gram-positive bacteria as well as LTAs from different sources, even in the presence of sCD14. Since other studies suggest that binding of LTA to eukaryotic cell membranes requires fatty acid moieties (42) and since fatty acid binding proteins are expressed only on differentiated IECs in the upper portion of the intestinal villus (12), we also examined the effects of LTA and milk on differentiated HT29 cells. Purified LTA from both L. johnsonii strain La1 and L. acidophilus strain La10 failed to trigger the release of IL-8 from either undifferentiated or differentiated HT29 cells. This is in accordance with the observation that HT29 cells do not express the Toll-like receptor 2 (TLR2) protein (9), the mediator required to respond to LTAs (1, 32, 39, 47). The potential stimulatory activity of LTAs from these two strains was confirmed by their capacity to induce the release of IL-8 in human peripheral blood mononuclear cells which express TLR2.

It has been shown that only certain LTAs were inducers of cytokine release (2, 6, 28, 30, 41). However, no structural motif explaining the divergent stimulatory potentials of these molecules has been identified. In nearly all cases, very large amounts of LTA (1 to 10 µg/ml) were required to stimulate responses of monocytes or macrophages in vitro. This is in contrast to LPS, which elicited responses in the picogram to nanogram per milliliter range. This raises the possibility that a trace contaminant in the LTA preparations could be responsible for the stimulatory activity observed.

LTAs from some sources have previously been shown to antagonize LPS-induced adhesion molecule expression and IL-8 release in human lung endothelial cells (7). Other work suggests that both antagonistic and agonistic effects are possible, depending on the level of CD14 and the source of LTA (41). Here we show that purified LTAs from both L. johnsonii strain La1 and L. acidophilus strain La10 inhibited LPS and bacterial stimulation of TNF-, IL-8, and ENA-78 expression by IECs. Moreover, LTA from strain La1 could also inhibit the response of IECs to LPS from pathogenic gram-negative bacterial species, as well as LPS-induced TNF- secretion by blood monocytes. As shown in previous studies on cultured monocytes (6, 30), LTA from Staphylococcus aureus did not induce cytokine release from IECs but rather displayed an inhibitory activity. Taken together, the present results indicate that rather than inducing inflammatory responses in IECs, LTAs from these gram-positive bacterial strains temper the LPS-mediated activation of these cells.

The mode of binding of LTAs to IECs and the events underlying the inhibitory effect on LPS activity need further study. The latter required coincubation with LPS and HM as a source of sCD14. The inability of LTA to directly trigger signaling mechanisms in IECs which block subsequent responsiveness to LPS may be due to differential expression of TLRs on the IECs and their interaction with CD14/LPS/LTA complexes. Certainly, HT29 cells do not express the TLR2 protein that is required to respond to LTA (9) but do display TLR4, identified as a major LPS signal transducer (for a review, see reference 5). However, since LTAs share with LPS the potential of binding CD14, the inhibitory effects of LTAs may simply be due to competitive binding to the CD14 molecule. Since the antagonistic activity of LTA was lost upon deacylation, the fatty acid moiety of the molecule must be implicated in the binding to the CD14 molecule.

In conclusion, our results demonstrate that LTAs from two gram-positive bacteria inhibit the responsiveness of IECs and blood monocytes to CD14-dependent effects of gram-negative bacteria. Our observations lend further support to the use of L. johnsonii strain La1 as a probiotic organism. Furthermore, they suggest a role for the LTAs from both L. johnsonii strain La1 and L. acidophilus strain La10 in maintaining gut homeostasis and as a therapy in diseases mediated by gram-negative bacteria or their components.

 
We are grateful to Nicole Duc, Patrick Serrant, and Janine Horlbeck for skillful technical assistance and to Irene Corthesy for helpful discussions and careful reading and improvement of the manuscript.

 
* Corresponding author. Mailing address: Food Immunology Group, Nestec Limited, Nestlé Research Center, Vers-chez-les-Blanc, P.O. Box 44, CH-1000 Lausanne 26, Switzerland. Phone: 41 21 785 82 65. Fax: 41 21 785 89 25. E-mail: karine.vidal{at}rdls.nestle.com.

Editor: E. I. Tuomanen

Top Abstract Introduction Materials and Methods Results Discussion References

 

1
1. Aderem, A., and R. J. Ulevitch. 2000. Toll-like receptors in the induction of the innate immune response. Nature 406:782-787.[CrossRef][Medline]
2
2. Arakaki, R., S. Sugawara, H. Nakashima, S. Kotani, and H. Takada. 1998. A lipoteichoic acid fraction of Enterococcus hirae activates cultured human monocytic cells via a CD14-independent pathway to promote cytokine production, and the activity is inhibited by serum components. FEMS Immunol. Med. Microbiol. 22:283-291.[CrossRef][Medline]
3
3. Arditi, M., J. Zhou, R. Dorio, G. W. Rong, S. M. Goyert, and K. S. Kim. 1993. Endotoxin-mediated endothelial cell injury and activation: role of soluble CD14. Infect. Immun. 61:3149-3156.[Abstract/Free Full Text]
4
4. Bazil, V., V. Horejsi, M. Baudys, H. Kristofova, J. L. Strominger, W. Kostka, and I. Hilgert. 1986. Biochemical characterization of a soluble form of the 53-kDa monocyte surface antigen. Eur. J. Immunol. 16:1583-1589.[Medline]
5
5. Beutler, B. 2000. Tlr4: central component of the sole mammalian LPS sensor. Curr. Opin. Immunol. 12:20-26.[CrossRef][Medline]
6
6. Bhakdi, S., T. Klonisch, P. Nuber, and W. Fischer. 1991. Stimulation of monokine production by lipoteichoic acids. Infect. Immun. 59:4614-4620.[Abstract/Free Full Text]
7
7. Blease, K., Y. Chen, P. G. Hellewell, and A. Burke-Gaffney. 1999. Lipoteichoic acid inhibits lipopolysaccharide-induced adhesion molecule expression and IL-8 release in human lung microvascular endothelial cells. J. Immunol. 163:6139-6147.[Abstract/Free Full Text]
8
8. Brassart, D., and E. J. Schiffrin. 1997. The use of probiotics to reinforce mucosal defence mechanisms. Trends Food Sci. Technol. 9:321-326.[CrossRef]
9
9. Cario, E., I. M. Rosenberg, S. L. Brandwein, P. L. Beck, H. C. Reinecker, and D. K. Podolsky. 2000. Lipopolysaccharide activates distinct signaling pathways in intestinal epithelial cell lines expressing Toll-like receptors. J. Immunol. 164:966-972.[Abstract/Free Full Text]
10
10. Chen, P. S., T. Y. Toribara, and H. Warner. 1956. Microdetermination of phosphorus. Anal. Chem. 28:1756-1758.[CrossRef]
11
11. Cleveland, M. G., J. D. Gorham, T. L. Murphy, E. Tuomanen, and K. M. Murphy. 1996. Lipoteichoic acid preparations of gram-positive bacteria induce interleukin-12 through a CD14-dependent pathway. Infect. Immun. 64:1906-1912.[Abstract]
12
12. Cohn, S. M., T. C. Simon, K. A. Roth, E. H. Birkenmeier, and J. I. Gordon. 1992. Use of transgenic mice to map cis-acting elements in the intestinal fatty acid binding protein gene (Fabpi) that control its cell lineage-specific and regional patterns of expression along the duodenal-colonic and crypt-villus axes of the gut epithelium. J. Cell Biol. 119:27-44.[Abstract/Free Full Text]
13
13. Danforth, J. M., R. M. Strieter, S. L. Kunkel, D. A. Arenberg, G. M. VanOtteren, and T. J. Standiford. 1995. Macrophage inflammatory protein-1 alpha expression in vivo and in vitro: the role of lipoteichoic acid. Clin. Immunol. Immunopathol. 74:77-83.[CrossRef][Medline]
14
14. Diamond, G., J. P. Russell, and C. L. Bevins. 1996. Inducible expression of an antibiotic peptide gene in lipopolysaccharide-challenged tracheal epithelial cells. Proc. Natl. Acad. Sci. USA 93:5156-5160.[Abstract/Free Full Text]
15
15. Dubois, M. A., K. A. Gilles, J. K. Hamilton, P. A. Rebers, and F. Smith. 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28:350-356.[CrossRef]
16
16. Dziarski, R., A. J. Ulmer, and D. Gupta. 2000. Interactions of CD14 with components of gram-positive bacteria. Chem. Immunol. 74:83-107.[Medline]
17
17. Filipp, D., K. Alizadeh-Khiavi, C. Richardson, A. Palma, N. Paredes, O. Takeuchi, S. Akira, and M. Julius. 2001. Soluble CD14 enriched in colostrum and milk induces B cell growth and differentiation. Proc. Natl. Acad. Sci. USA 98:603-608.[Abstract/Free Full Text]
18
18. Fischer, W. 1990. Bacterial phosphoglycolipids and lipoteichoic acids, p. 123-234. In M. Kates and D. J. Hanahan (ed.), Glycolipids, phosphoglycolipids and sulfoglycolipids. Plenum Press, New York, N.Y.
19
19. Fischer, W., H. U. Koch, and R. Haas. 1983. Improved preparation of lipoteichoic acids. Eur. J. Biochem. 133:523-530.[Medline]
20
20. Fischer, W., H. U. Koch, P. Rosel, and F. Fiedler. 1980. Alanine ester-containing native lipoteichoic acids do not act as lipoteichoic acid carrier. Isolation, structural and functional characterization. J. Biol. Chem. 255:4557-4562.[Free Full Text]
21
21. Folch, J., M. Less, and G. H. Sloane Stanley. 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497-509.[Free Full Text]
22
22. Frey, E. A., D. S. Miller, T. G. Jahr, A. Sundan, V. Bazil, T. Espevik, B. B. Finlay, and S. D. Wright. 1992. Soluble CD14 participates in the response of cells to lipopolysaccharide. J. Exp. Med. 176:1665-1671.[Abstract/Free Full Text]
23
23. Gibson, G. R., and X. Wang. 1994. Regulatory effects of bifidobacteria on the growth of other colonic bacteria. J. Appl. Bacteriol. 77:412-420.[Medline]
24
24. Granato, D., F. Perotti, I. Masserey, M. Rouvet, M. Golliard, A. Servin, and D. Brassart. 1999. Cell surface-associated lipoteichoic acid acts as an adhesion factor for attachment of Lactobacillus johnsonii La1 to human enterocyte-like Caco-2 cells. Appl. Environ. Microbiol. 65:1071-1077.[Abstract/Free Full Text]
25
25. Hayashi, J., T. Masaka, I. Saito, and I. Ishikawa. 1996. Soluble CD14 mediates lipopolysaccharide-induced intercellular adhesion molecule 1 expression in cultured human gingival fibroblasts. Infect. Immun. 64:4946-4951.[Abstract]
26
26. Haziot, A., G. W. Rong, J. Silver, and S. M. Goyert. 1993. Recombinant soluble CD14 mediates the activation of endothelial cells by lipopolysaccharide. J. Immunol. 151:1500-1507.[Abstract]
27
27. Keates, S., A. C. Keates, E. Mizoguchi, A. Bhan, and C. P. Kelly. 1997. Enterocytes are the primary source of the chemokine ENA-78 in normal colon and ulcerative colitis. Am. J. Physiol. 273:G75-G82.[Abstract/Free Full Text]
28
28. Keller, R., W. Fischer, R. Keist, and S. Bassetti. 1992. Macrophage response to bacteria: induction of marked secretory and cellular activities by lipoteichoic acids. Infect. Immun. 60:3664-3672.[Abstract/Free Full Text]
29
29. Krutzik, S. R., P. A. Sieling, and R. L. Modlin. 2001. The role of Toll-like receptors in host defense against microbial infection. Curr. Opin. Immunol. 13:104-108.[CrossRef][Medline]
30
30. Kusunoki, T., E. Hailman, T. S. Juan, H. S. Lichenstein, and S. D. Wright. 1995. Molecules from Staphylococcus aureus that bind CD14 and stimulate innate immune responses. J. Exp. Med. 182:1673-1682.[Abstract/Free Full Text]
31
31. Labeta, M. O., K. Vidal, J. E. Nores, M. Arias, N. Vita, B. P. Morgan, J. C. Guillemot, D. Loyaux, P. Ferrara, D. Schmid, M. Affolter, L. K. Borysiewicz, A. Donnet-Hughes, and E. J. Schiffrin. 2000. Innate recognition of bacteria in human milk is mediated by a milk-derived highly expressed pattern recognition receptor, soluble CD14. J. Exp. Med. 191:1807-1812.[Abstract/Free Full Text]
32
32. Lien, E., T. J. Sellati, A. Yoshimura, T. H. Flo, G. Rawadi, R. W. Finberg, J. D. Carroll, T. Espevik, R. R. Ingalls, J. D. Radolf, and D. T. Golenbock. 1999. Toll-like receptor 2 functions as a pattern recognition receptor for diverse bacterial products. J. Biol. Chem. 274:33419-33425.[Abstract/Free Full Text]
33
33. Loppnow, H., F. Stelter, U. Schonbeck, C. Schluter, M. Ernst, C. Schutt, and H. D. Flad. 1995. Endotoxin activates human vascular smooth muscle cells despite lack of expression of CD14 mRNA or endogenous membrane CD14. Infect. Immun. 63:1020-1026.[Abstract]
34
34. Mackie, R., A. Sghir, and H. R. Gaskins. 1999. Developmental microbial ecology of the neonatal gastrointestinal tract. Am. J. Clin. Nutr. 69:1035S-1045S.[Abstract/Free Full Text]
35
35. Mata, L. J., and J. J. Urrutia. 1971. Intestinal colonisation of breast-fed children in the rural area of low socioeconomic level. Ann. N. Y. Acad. Sci. 176:93-108.[CrossRef]
36
36. Mattsson, E., L. Verhage, J. Rollof, A. Fleer, J. Verhoef, and H. van Dijk. 1993. Peptidoglycan and teichoic acid from Staphylococcus epidermidis stimulate human monocytes to release tumour necrosis factor-alpha, interleukin-1 beta and interleukin-6. FEMS Immunol. Med. Microbiol. 7:281-287.[Medline]
37
37. Pugin, J., I. D. Heumann, A. Tomasz, V. V. Kravchenko, Y. Akamatsu, M. Nishijima, M. P. Glauser, P. S. Tobias, and R. J. Ulevitch. 1994. CD14 is a pattern recognition receptor. Immunity 1:509-516.[CrossRef][Medline]
38
38. Pugin, J., C. C. Schurer-Maly, D. Leturcq, A. Moriarty, R. J. Ulevitch, and P. S. Tobias. 1993. Lipopolysaccharide activation of human endothelial and epithelial cells is mediated by lipopolysaccharide-binding protein and soluble CD14. Proc. Natl. Acad. Sci. USA 90:2744-2748.[Abstract/Free Full Text]
39
39. Schwandner, R., R. Dziarski, H. Wesche, M. Rothe, and C. J. Kirschning. 1999. Peptidoglycan- and lipoteichoic acid-induced cell activation is mediated by toll-like receptor 2. J. Biol. Chem. 274:17406-17409.[Abstract/Free Full Text]
40
40. Standiford, T. J., D. A. Arenberg, J. M. Danforth, S. L. Kunkel, G. M. VanOtteren, and R. M. Strieter. 1994. Lipoteichoic acid induces secretion of interleukin-8 from human blood monocytes: a cellular and molecular analysis. Infect. Immun. 62:119-125.[Abstract/Free Full Text]
41
41. Sugawara, S., R. Arakaki, H. Rikiishi, and H. Takada. 1999. Lipoteichoic acid acts as an antagonist and an agonist of lipopolysaccharide on human gingival fibroblasts and monocytes in a CD14-dependent manner. Infect. Immun. 67:1623-1632.[Abstract/Free Full Text]
42
42. Teti, G., F. Tomasello, M. S. Chiofalo, G. Orefici, and P. Mastroeni. 1987. Adherence of group B streptococci to adult and neonatal epithelial cells mediated by lipoteichoic acid. Infect. Immun. 55:3057-3064.[Abstract/Free Full Text]
43
43. Ulevitch, R. J., and P. S. Tobias. 1995. Receptor-dependent mechanisms of cell stimulation by bacterial endotoxin. Annu. Rev. Immunol. 13:437-457.[CrossRef][Medline]
44
44. Vidal, K., M. O. Labeta, E. J. Schiffrin, and A. Donnet-Hughes. 2001. Soluble CD14 in human breast milk and its role in innate immune responses. Acta Odontol. Scand. 59:330-334.[CrossRef][Medline]
45
45. Wang, J. E., P. F. J/orgensen, M. Almlöf, C. Thiemermann, S. J. Foster, A. O. Aasen, and R. Solberg. 2000. Peptidoglycan and lipoteichoic acid from Staphylococcus aureus induce tumor necrosis factor alpha, interleukin 6 (IL-6), and IL-10 production in both T cells and monocytes in a human whole blood model. Infect. Immun. 68:3965-3970.[Abstract/Free Full Text]
46
46. Wright, S. D., R. A. Ramos, P. S. Tobias, R. J. Ulevitch, and J. C. Mathison. 1990. CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science 249:1431-1433.[Abstract/Free Full Text]
47
47. Yoshimura, A., E. Lien, R. R. Ingalls, E. Tuomanen, R. Dziarski, and D. Golenbock. 1999. Cutting edge: recognition of Gram-positive bacterial cell wall components by the innate immune system occurs via Toll-like receptor 2. J. Immunol. 163:1-5.[Abstract/Free Full Text]

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Infection and Immunity, April 2002, p. 2057-2064, Vol. 70, No. 4
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.4.2057-2064.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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