Isolates Bifidobacterium thermoacidophilum RBL81, RBL82 and RBL64 from newborn infant faeces were identified by fructose-6-phosphate phosphoketolase assay and by the PCR method of Touré et al. (2003). These isolates and Bifidobacterium lactis Bb12 (Chr. Hansen, Moersolm, Denmark) were individually subcultured anaerobically for 18 hours at 37 °C in 300 mL of De Man-Rogosa-Sharpe broth (MRS) (Rosell Institute Inc., Montreal, Canada) supplemented with 0.05 % cysteine-HCl (v/v) to lower the oxidation-reduction potential of the medium and thereby improve anaerobic growth (Sreekumar & Hosono, 1998). Tween 80 was added at a concentration of 1 % (v/v). Three hundred mL of each pre-culture was transferred to 270 mL of broth in separate 1 L flasks (3 flasks for each culture), which were incubated for 16 h at 37 °C in an anaerobic jar containing a carbon dioxide generating sachet (AnaeroGen, OXOID Ltd., Basingstoke, England).

After incubation (16 h), bifidobacteria cultures were used to extract cytoplasmic content according to method of Fernandez-Espla et al. (2000) with some modifications. Briefly, cells were collected at the end of exponential growth phase (16 h) by centrifugation at 5,000 x g for 10 min at 4 °C and washed three times with 0.01 M potassium phosphate buffer (PBS) at pH 7.0. The contents of bacterial cells were released by disruption of pelleted cells manually for 30 min at 4 °C with three or four volumes of alumina powder (Sigma Co., Saint Louis, Mo, USA) in a mortar laid in ice. PBS (1 mL) was added to the mixture to facilitate extraction. The alumina was separated from the suspension of disrupted cells by centrifugation at 1,000 x g for 15 min at 4 °C. Finally, the preparations were re-centrifuged at 30,000 x g for 30 min at 4 °C in order to pellet cell walls and obtain cytoplasmic extract in the supernatant fraction. The protein content of the cytoplasmic extract was determined by the Lowry method. All samples were stored at – 20 °C until they were used.

SDS-PAGE analysis was carried out to compare the peptide and protein profile of cytoplasm from bifidobacterial strains used. The analysis was carried out using 10 % polyacrylamide gel electrophoresis (PAGE) performed in the presence of sodium dodecyl sulfate (SDS) according to Laemmli (1970). Molecular markers Bio-Rad (Broad Range) with molecular mass ranging from 21.5 to 97.5 kDa were used.

Cytoplasmic content was separated by ampholyte-free isoelectric focusing using the method described by Groleau et al. (2002). Briefly, cytoplasmic extracts (0.1 g ) were resuspended in 40 mL of deionized water and fractionated for 2 h at 4 ^oC by liquid-phase isoelectric focusing in a preparative Rotofor cell (Bio-Rad Laboratories, Hercules, CA) at constant power (15 W). Twenty fractions were collected and the pH of each fraction was measured to determine the isoelectric pH (pI). The fractions were then pooled to obtain the acidic fraction (pI from 2 to 5) and basic fraction (pI from 5 to 12). Acidic and basic fractions were dried by Speed Vac concentrator (Savant Instrument Inc., Farmingdale, NY) and stored at 4 ⁰C until use.

A proliferation assay was carried out to evaluate the mononuclear cell response to cytoplasm fractions from B. lactis Bb12. Spleens were removed aseptically from euthanized Balb/C mice and single cell suspensions were prepared by mechanically disrupting the tissues through a cell strainer into RPMI 1640 medium (Gibco BRL Inc., Paisley, Scotland). Erythrocytes were removed from cell suspension by osmotic shock with 2 mL of 0.87 % NH₄Cl solution for 2 min at 37^oC. Spleen cells were then washed three times at 4°C, suspended in RPMI 1640 complete medium (with 10% fetal calf serum (FSC), 0.1 mL mL^-1 50 mM mercaptoethanol , 100 U mL^-1 penicillin, and 100 µg mL^-1 streptomycin, all from Gibco). The crude cytoplasmic extract and acidic and basic fractions were diluted in supplemented RPMI to 0.8, 8, 80 and 200 μg/mL; whereas the fractionated components (peaks) were diluted in supplemented RPMI at 0.8, 8 and 80 μg/mL. All the samples were filtered (0.22 μm). Cells were plated at a final concentration of 5x10⁵ cells mL^-1. A mitogenic agent, phytohemagglutinin (PHA) (10 µg mL^-1) was used alone as control for cell proliferation. Medium alone, PHA or above-mentioned concentrations of bifidobacterial components were added to wells in duplicate. Finally, AlamarBlue indicator (BioSource International, Inc., USA) was added to wells (20 µL/well) to measure cell proliferation. AlamarBlue system incorporates an oxidation-reduction indicator that both fluoresces and changes color in response to chemical reduction of culture media resulting from cell growth (Lancaster, 1996). Cell cultures were incubated for 48 h in a humidified 5 % CO₂ atmosphere at 37 °C. At the end of incubation, the microplates were centrifuged and supernatants were transferred to a black opaque 96-well microplate (Fisher Scientific, Pittsburgh, USA) to determine fluorescence values. Fluorescence measurements were made by FLUOstar spectrofluorometric microtiter well plate reader (BMG Labtechnologies, Offenburg, Germany) (excitation: 544 nm; emission: 610 nm; gain 026). Proliferation responses were expressed as a stimulation index calculated as the ratio of mean fluorescence value obtained for stimulated culture to the mean fluorescence value obtained for nonstimulated control cell cultures.

In order to evaluate cytokine production, a separate microplate was prepared without adding AlamarBlue. Cytokine production was measured in splenocytes cultured in conditions described above and added with bifidobacterial components and 10 μg/mL PHA, at which concentration the stimulation index was not affected. Interferon gamma (IFN-γ) and interleukin 4 (IL-4), in supernatants of splenocyte cultures, were evaluated by cytokine-specific sandwich ELISAs. Antibodies R 4-6A2 and biotinylated XMG1.2 were used for IFN-γ while MM-011 and biotinylated MM-16E3 were used for IL-4 (all from Endogen, Woburn, MA, USA). Coating antibodies were diluted to 2 µg mL^-1 and conjugate antibodies were diluted to 0.5 µg mL^-1. Cytokine concentrations were derived from linear dose-response standard curves obtained using dilutions of recombinant mouse IFN-γ (BD Pharmigen, San Diego, CA, USA) or recombinant mouse IL- (Endogen). Optical densities were measured at 450 nm after 15 min with TMB (tetramethylbenzidine) peroxidase substrate (Kirkegaard & Perry Laboratories) and H₂O₂ at room temperature. The reaction was stopped by addition of 1 M H₂SO₄. Fresh cell-free complete RPMI 1640 was used as negative control.

Splenocyte proliferation assay and cytokines evaluation were carried out in triplicate for each bacterial extract, and all analysis was done in duplicate. Statistical analyses were performed with STATGRAPHICS plus 4.1 (Manugistics Inc., Rockville, MD, USA). Significant differences between treatments were tested by analysis of variance (ANOVA). The treatments were compared using Fisher’s least significant difference (LSD) method, with a level of significance of P < 0.05.

To compare the cytoplasmic peptide and protein profile of three isolates of bifidobacteria and B. lactis Bb12 cytoplasm extracts were analysed by SDS-PAGE. The results reported in Figure 3.1 showed difference and similarities in protein profile between the strains tested.

The band corresponding to the molecular weight (MW) of 75 kDa was shown only by B. lactis Bb12. While, a common band with MW of 34 kDa was found to be characteristic to the isolates RBL81, RBL82, and RBL64. Bands with high MW (>45 kDa) and weak MW (<31 kDa) allowing discrimination between B. lactis Bb12 and isolates tested were also observed. More proteins and peptides appear to be biosynthesized by B. lactis Bb12 compared to other bifidobacterial strains. However, as can be seen in the Figure 3.1, peptides seem to be produced in quantities non detectable by blue coomassie staining compared to proteins. Marasco et al. (1984) reported that bacteria elaborate in very low concentrations numerous peptides of similar composition, which may differ markedly in their specific biological activities. The differences observed in protein and peptides profile of bifidobacteria should be attributed to the variability and diversity of strain proteome. Champomier-Verges et al. (2002) mentioned that lactic acid bacteria strains grown in the same rich synthetic MRS medium show some common (a dozen) proteins which represent almost 20% of the total protein detected on the acidic proteomic map. However, lactic acid bacteria indicating metabolic adaptation to media conditions show some differences in protein content. Indeed, different stress treatments (e.g., heat, low pH, osmotic shock, etc.) have been reported to induce transiently general and specific proteins (e.g., heat shock proteins) by physiological changes enhancing bacterial ability to survive in adverse environmental conditions (Ang et al., 1991; Prasad et al., 2003, De Angelis et al., 2004).

Figure 3.1

Peptide and protein profile of cytoplasmic extracts from strains B. thermoacidophilum RBL64, RBL82 and RBL84, and B. lactis Bb12 obtained by SDS-PAGE using 10 % acrylamide gel.

The band corresponding to the molecular weight (MW) of 75 kDa was shown only by B. lactis Bb12. While, a common band with MW of 34 kDa was found to be characteristic to the isolates RBL81, RBL82, and RBL64. Bands with high MW (>45 kDa) and weak MW (<31 kDa) allowing discrimination between B. lactis Bb12 and isolates tested were also observed. More proteins and peptides appear to be biosynthesized by B. lactis Bb12 compared to other bifidobacterial strains. However, as can be seen in the Figure 3.1, peptides seem to be produced in quantities non detectable by blue coomassie staining compared to proteins. Marasco et al. (1984) reported that bacteria elaborate in very low concentrations numerous peptides of similar composition, which may differ markedly in their specific biological activities. The differences observed in protein and peptides profile of bifidobacteria should be attributed to the variability and diversity of strain proteome. Champomier-Verges et al. (2002) mentioned that lactic acid bacteria strains grown in the same rich synthetic MRS medium show some common (a dozen) proteins which represent almost 20% of the total protein detected on the acidic proteomic map. However, lactic acid bacteria indicating metabolic adaptation to media conditions show some differences in protein content. Indeed, different stress treatments (e.g., heat, low pH, osmotic shock, etc.) have been reported to induce transiently general and specific proteins (e.g., heat shock proteins) by physiological changes enhancing bacterial ability to survive in adverse environmental conditions (Ang et al., 1991; Prasad et al., 2003, De Angelis et al., 2004).

Peptides and proteins from B. lactis Bb12 were separated into different fractions by liquid-phase isoelectric focusing. This preparative technique allowed separation of amphoteric molecules such as proteins and peptides according to their isoelectric point (pI). Charged molecules migrate through a pH gradient in an electric field and reach their zwitterionic state at pH corresponding to their pI (Laas, 1998).

In order to separate several peptides and proteins with subtle differences in their amino acid composition, the acidic and basic fractions were analysed individually by RP-HPLC. The chromatogram of each fraction is shown in Figure 3.2 and Figure 3.3. Chromatography system resulted in marked differences in retention time for several peptides and proteins when applied to the column as a mixture. In both fractions, we observed numerous peaks throughout the elution gradient indicating the presence of heterogeneous group of peptides and proteins. However, most of materials that are eluted earlier (5 to 10 mn) gave high absorbance (A210 > 1.0).

Figure 3.2

Peptide and protein profile of acidic fraction ( B. lactis Bb12) analysed by RP-HPLC (C18 column). Labelled peaks are selected and collected using an appropriated colum.

Figure 3.3

Peptide and protein profile of basic fraction ( B. lactis Bb12) analysed by RP-HPLC (C18 column). Labelled peaks are selected and collected using an appropriated colum.

Table 3.1. Average molecular weight of most abundant peptides or proteins detected in each labelled peak from acidic and basic fractions.

To determine the effect of cytoplasmic peptides and proteins from B. lactis B12 on cytokines secreted by activated lymphocytes, we analyzed the production of IFN-γ and interleukin IL-4. It is well established now that Helper T cells (Th) are heterogeneous with regard to cytokine secretion and their functions. Th₁ cells produce mainly IL-₂ and IFN-γ, while Th₂ cells predominantly produce IL-4 and IL-6. These two cytokines are shown to have antagonistic functions ( Yazdanbakhsh et al.,1999; Valentini et al., 2001).

The production of IFN-γ and IL-4 by splenocytes cultured with various concentration of cytoplasmic extract, and the acidic and basic fractions is illustrated by Figure 3.5 and Figure 3.6.

Figure 3.5

Effect of acidic fraction, basic fraction, and crude cytoplasmic extract from B. lactis Bb12 on splenocyte IFN-γ production. The cytoplasmic preparation was normalized on the basis of its protein content. Columns with different letters are significantly different.

Figure 3.6

Effect of acidic and basic fraction, and crude cytoplasmic extract from the strains B. lactis Bb12 on splenocyte IL-4 production. The cytoplasmic preparation was normalized on the basis of its protein content. Columns with different letters are significantly different.

According to these results, both crude cytoplasmic extract and basic fraction induce a bell-shaped dose-response curve. As shown in Figure 3.5, splenocytes stimulated by cyplasmic extract from B. lactis Bb12 secreted approximately twofold IFN-γ (1199,57 ±127,60) higher than when stimulated by the basic fraction (578,55±195,16) and fourfold when cultured with the acidic fraction (191,08±7,50 pg/ml). While, IFN-γ production obtained with PHA was estimated at 127,10±101,50 pg/ml. Th₁ cells appear to be more stimulated by basic fraction compared to acidic fraction.

The results reported in Figure 3.6 demonstrate low IL-4 production compared to those of IFN-γ obtained by cytoplasmic content, and both acidic and basic fractions. Hence, Th₂ cells seem to be weakly stimulated. The most significant production of IL-4 was obtained by crude cytoplasmic extract (9,14±4,04) and basic fraction (4,14±2,02). While, the production of IL-4 increased slightly upon treatment with acidic fraction (< 3,86 pg/ml). Th₁ lymphocytes stimulate cell-mediated immunity, which is characterized by intense phagocytic activity. Conversely, Th₂ cells stimulate humoral immunity, which is characterized by high antibody production. Evidence suggested that coinduction of T2 immunity maintains immune homeostasis during T1-mediated defence reactions. Th₁ cells also stimulate moderate levels of antibody production, whereas Th₂ cells actively suppress phagocytosis (Spellberg and Edwards, 2001; Bot et al., 2004).

It has been demonstrated that synthetic peptides stimulate T cell chemotaxis showing unique signalling and provide a helpful tool to understand the T cell activation mechanism (Zachariae et al., 1992; Kim et al., 2001). The synthetic peptides were shown to stimulate the formation of inositol phosphates in lymphocyte cells (Baek et al., 1996). Wang et al. (2004) reported that a fungal immunomodulatory protein from Flammulina velutipes (FIP-fve) exhibited potent mitogenic effects on human peripheral blood lymphocytes, inducing G1/G0 to S phase proliferation. The activation of T cells resulted in significant production and secretion of IFN-γ associated with intercellular adhesion molecule 1 expression but low detectable levels of interleukin-4 in vitro or in vivo .