Borja Sánchez, Université de Bordeaux, UMR 5248 CNRS, UBX1-ENITAB, ENITAB, 1 cours du Général de Gaulle, 33175 Gradignan Cedex, France. E-mail: firstname.lastname@example.org
Aims: To identify novel proteins secreted by the probiotic bacterium Lactobacillus rhamnosus GG after growth in de Mann-Rogosa-Sharpe broth (MRS), a complex medium often used for the culture of Lactobacillus.
Methods and Results: The proteins secreted by L. rhamnosus GG strain were precipitated using a trichloroacetic acid-based protocol, resolved by SDS-PAGE, and identified by tandem mass spectrometry (MS/MS). Among the proteins secreted by this bacterium, a leukocyte elastase inhibitor, already present in the MRS broth, was identified. Other proteins such as cell wall hydrolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase, and an extracellular transcriptional regulator have been also identified.
Conclusions: Lactobacillus rhamnosus GG secretes several proteins during its growth in MRS, some of them with assigned functions in the prevention of the molecular mechanisms that lead to damage in the epithelial barrier (cell wall hydrolase) and in adhesion (GAPDH). The rest of the proteins require further genetic analysis in order to establish their precise roles. None of the proteins bound to mucin or fibronectin.
Significance and Impact of the Study: Some of these secreted proteins could be involved in the probiotic effects exerted by L. rhamnosus GG strain, their identification being the first step towards in depth functional studies.
Lactobacillus rhamnosus GG is a probiotic strain often included in the elaboration of fermented milks that was isolated from the intestinal tract of a healthy individual (Gorbach et al. 1987). This strain, which is acid and bile resistant, shows a high affinity to intestinal cells and is claimed to be able to colonize the human gastrointestinal tract (Conway et al. 1987). In the latter, L. rhamnosus GG is able to balance the intestinal microbiota, it produces host immunomodulation and reduces the symptoms of a wide range of gastrointestinal disorders, including rotavirus diarrhoea and aiding the treatment of gastrointestinal carriage of vancomycin-resistant enterococci in renal patients (Manley et al. 2007; Pant et al. 2007; Szajewska et al. 2007).
Some of the probiotic effects of this strain may be due to its secreted proteins. These proteins are accumulated in the culture media during bacterial growth. Secreted proteins have been related to the molecular intercommunication between bacteria and also to the monitoring of the bacterial environment (van Pijkeren et al. 2006). It has been proposed that secreted proteins could play crucial roles in the cross-talking of probiotic bacteria with the human host, as well as being responsible for certain probiotic traits such as pathogen inhibition and immunomodulation (Buck et al. 2005; Sánchez et al. 2008a). For example, it is known that secreted low-molecular-weight peptides induce heat shock protein expression in intestinal epithelial cells, being induction time- and concentration-dependent (Tao et al. 2006).
To date, L. rhamnosus GG has been shown to secrete two proteins: cell wall hydrolase and a homolog of a protein of unknown function from L. casei ATCC 334 (GenBank accession no. YP_805328), named p75 and p40, respectively (Yan et al. 2007). The mechanisms of action of these two proteins include the promotion of epithelial cell grown and inhibition of the apoptosis mediated by tumour necrosis factor. Moreover, p75 and p40 also reduced damage in the epithelial barrier induced by hydrogen peroxide by a MAP kinase-dependent molecular mechanism (Seth et al. 2008).
Identification of the proteins secreted by L. rhamnosus GG during its growth is important for understanding L. rhamnosus GG ecology and physiology in the human gastrointestinal tract. In this short communication, novel proteins secreted by L. rhamnosus GG grown in MRS have been precipitated, resolved by SDS-PAGE and identified. The most relevant results are discussed below.
Materials and Methods
Strain L. rhamnosus GG (ATCC 53103, FlorVis GG; Novartis, Origgio, Italy) was grown aerobically at 37°C in MRS broth (Becton Dickinson France SAS, Le Pont-De-Claix, France). About 50 ml of fresh MRS broth were inoculated (0·1% v/v) from an overnight culture, and cells were grown aerobically at 37°C until the early stationary phase (around 11 h of culture), as monitored by the by absorption at 600 nm.
Secreted protein extraction and protein manipulations
Aliquots of 5 ml of cultures at stationary phase were harvested by centrifugation (10 min, 3500 g, 4°C), and the supernatant was filtered (0·45 μm). About 10 ml of sodium deoxycholate were added and the mix was incubated at 4°C for 30 min. Chilled trichloroacetic acid was added to a final concentration of 60 g l−1, and proteins were allowed to precipitate for 2 h at 4°C. Proteins were recovered by centrifugation (10 min, 9300 g, 4°C), and the resulting pellets were washed twice with 2 ml of chilled acetone. The pellets were allowed to dry at room temperature and proteins were re-solubilized in an ultrasonic bath over 10 min (Deltasonic, Meaux, France) in 40 μl of Laemmli buffer (Laemmli 1970). These 40 μl were resolved by SDS-PAGE in 12·5% (w/v) polyacrylamide gels. Cytoplasmic extracts were obtained by sonication (Vibracell 75021 Ultrasonic Processor; Fisher Scientific Bioblock, Illkirch, France) for 3–7 cycles of 3 min (amplitude 12, duty 33%). Extracts were centrifuged (10 min, 10 000 g, 4°C) to eliminate cells and cellular debris, and protein concentration was calculated using the BCA Protein Assay kit (Pierce, Rockford, IL, USA). The most intense bands were excised from gels and digested with trypsin, and the resulting peptide mixture was analysed by tandem mass spectrometry (MS/MS) using MALDI Q-Tof Premier (Waters, Manchester, UK). Proteins were identified using the MS/MS search module from mascot software (http://www.matrixscience.com) against the non-redundant protein NCBI database. Gels were repeated three times from independent cultures.
About 50 μg per well of fibronectine (BD Biosciences, Bedford, MA, USA) or 3 mg per well of mucin, dissolved in phosphate buffered saline (PBS), were bound to 96-well sterile polystyrene plates (F96 Maxisorp Immunoplate, Nunc, Roskilde, Denmark) for 1 h at 37°C followed by an overnight incubation at 4°C. Non-bound protein was then removed, and the wells were washed twice with PBS. The wells were then blocked with 10 g l−1 BSA in PBS at 37°C for 2 h, and then washed twice with PBS. About 100 μg of L. rhamnosus GG secreted proteins, extracted as described before, were added to the wells and incubated at 37°C for 2 h. The wells were washed twice with PBS in order to eliminate unbound proteins. About 60 μl of 10 g l−1 SDS were added to each well and incubated at 37°C for 2 h under gentle agitation. The wells were then dried and bound proteins were solubilized in sample buffer containing 60 mmol l−1 Tris–HCl pH 6·8, 10% (v/v) glycerol, 10 g l−1β-mercaptoethanol and 5 mg l−1 bromophenol blue. Proteins were analysed by SDS-PAGE and then visualized by standard silver staining. Experiments were repeated three times from independent secreted protein extractions.
Figure 1a shows a representative polyacrylamide gel with the proteins secreted by L. rhamnosus GG strain after growth until stationary phase in MRS at 37°C. The corresponding protein identification parameters are listed in Table 1. Secreted protein profiles were different enough from cytoplasmic extracts to exclude cell lysis. It is important to note that these are crude extracts that also include proteins derived from the growth medium. In this way, MRS contained abundant amounts of a porcine serine protease inhibitor (serpin B1) (Fig. 1a, lane 2), which is, more precisely, a neutrophil elastase inhibitor (Remold-O’Donnell et al. 1992). The same serpin was also present among the proteins secreted by L. rhamnosus GG, bands G2 and G4 being thus identified. Band G4, with a lower molecular mass, is probably a proteolytic product of the heaviest band cleaved by a L. rhamnosus GG protease.
Table 1. Proteins secreted by the bacterium Lactobacillus rhamnosus GG
††Final subcellular localization was predicted using the psortb package (Gardy et al. 2005).
Cell wall-associated hydrolase
L. casei ATCC 334
L. casei ATCC 334
L. casei ATCC 334
L. casei ATCC 334
The rest of the bands were identified against the genome of the bacterium L. casei ATCC 334. Bands G3 and G5 were identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoglycerate kinase, respectively, which catalyse consecutive and reversible reactions in the glycolytic pathway. Another two proteins, G1 and G6, were predicted as extracellular using the PSORTb software (Gardy et al. 2005). G1 was identified as cell wall hydrolase, and G6 showed homology with a L. casei ATCC 334 transcriptional regulator.
Finally, and as is seen in Fig. 1b, none of the proteins secreted by the L. rhamnosus GG strain was bound to fibronectin or mucin after the binding assays.
In this work, novel proteins secreted by the probiotic bacterium L. rhamnosus GG after growing in MRS broth have been identified. These proteins may play important roles in the physiology of this probiotic bacterium, their identification being a necessary first step for the development of further works. Therefore, the aim of this short communication was to present such proteins.
Moonlighting proteins are present among the proteins secreted by L. rhamnosus GG
Two bands, G3 and G5, were identified as GAPDH and phosphoglycerate kinase. As previously stated, they catalyse consecutive reactions in the glycolysis pathway, and their conjunct metabolic activity converts dihydroxyacetone phosphate into 3-phosphoglycerate. Since GAPDH preserves its metabolic activity when is secreted (Sánchez et al., unpublished results), it is worthwhile determining if such reactions could take place within the human gastrointestinal tract. On the other hand, and in spite of its cytoplasmic prediction, it is known that GAPDH is present on the surface of a wide variety of micro-organisms, including commensal and pathogenic bacteria (Hurmalainen et al. 2007). It has been suggested that, besides their metabolic functions, proteins such as GAPDH could undergo ‘moonlighting’ when they are exposed on the bacterial surface, developing additional functions (Jeffery 2003). For example, it has been shown that GAPDH adheres to host components such blood antigens (Kinoshita et al. 2008b).
Preserving its metabolic activity or not, this is, to our knowledge, the first time that GAPDH is described as being secreted by a probiotic bacterium.
Extracellular proteins secreted by L. rhamnosus GG
Some proteins predicted as extracellular were also detected as secreted by L. rhamnosus GG. As mentioned above, band G6 was identified as a transcriptional regulator that carried within its sequence a ‘cell envelope-related transcriptional attenuator’ domain. This domain of unknown function is found in the extracellular domain of some membrane-bound proteins. One of these proteins is the protein LytR, which belongs to a family of proteins involved in cell-wall structural maintenance through autolysin regulation (Chatfield et al. 2005). Another protein having this domain is CpsA, a putative regulatory protein involved in exopolysaccharide biosynthesis (Hathaway et al. 2007). In consequence, G6 could develop physiological functions on the surface of L. rhamnosus GG strain.
In contrast, band G1 was identified as a cell wall-associated hydrolase. The amino acidic sequence of the L. casei ATCC 334 homolog was shown to contain a NPLC/P60 domain which, presumably, would allow this protein to attach itself to the cell wall by means of non-covalent interactions. In addition, the listerial homolog of this protein is involved in the process of invasion and replication within intestinal cells (Faith et al. 2007) and, recently, a homolog protein of Bifidobacterium longum NCIMB 8809 has been found to be secreted (Sánchez et al. 2008b). Taken together, these facts suggest that this protein might play important roles in the physiology of several Gram-positive bacteria.
Proteins already detected in media conditioned by L. rhamnosus GG
To date, two proteins, called p75 and p40, have been shown to be secreted by L. rhamnosus GG (Yan et al. 2007). These proteins seem to reduce the molecular reorganization that leads to the damage of intestinal epithelial tight junctions after hydrogen peroxide exposure, following a MAP kinase-dependent mechanism (Seth et al. 2008).
In the aforementioned works, proteins were identified after growing L. rhamnosus GG in MRS, cell washing with PBS, cell resuspension in cell culture medium and further incubation for 2 h at 37°C (Yan et al. 2007; Seth et al. 2008). Thus, information about the proteins accumulated in the supernatant during L. rhamnosus GG growth in MRS is lacking. Moreover, it is not clear whether p75 and p40 were secreted by the bacterium or simply washed from its surface. There were clear differences in the proteins identified by both methods, and though our approach identified protein p75, no traces of p40 were found among LGG secreted proteins. However, p40 could be detected by following the procedure of Yan et al. (2007) (data not shown).
Nevertheless, the results presented in both scientific papers strongly suggest that epithelial cells are an important target for the proteins secreted by L. rhamnosus GG strain and, in general, for the proteins secreted by probiotic bacteria. Protein binding assays were therefore conducted to establish whether these proteins bound to mucin or fibronectin, two common attachment sites on the gastrointestinal tract. As is seen in Fig. 1b, none of the proteins were bound to the matrices, suggesting that their intestinal targets are different from mucin or fibronectin. In this way, it has been shown that surface-associated GAPDH was able to bind intestinal receptors such as plasminogen, extracellular matrix proteins and human colonic mucin, though our approach failed to reproduce the latter in the L. rhamnosus GG strain (Hurmalainen et al. 2007; Kinoshita et al. 2008a,b).
In conclusion, some novel proteins secreted by the probiotic bacterium L. rhamnosus GG have been identified. Some of these secreted proteins have already been shown to promote intestinal epithelial integrity, and they may be responsible for other beneficial effects of L. rhamnosus GG strain, through their interaction with host cells. Further research will be needed to identify their mode of action and the molecular mechanisms by which these proteins might interact with the human host.
Borja Sánchez was the recipient of a Clarín postdoctoral contract from the Gobierno del Principado de Asturias funded by the Plan de Ciencia, Tecnología e Innovación (PCTI) de Asturias 2006–2009.