Impact of spray-drying on the pili of Lactobacillus rhamnosus GG.

The preservation of the viability of microorganisms in probiotic formulations is the most important parameter ensuring the adequate concentration of live microorganisms at the time of administration. The formulation and processing techniques used to produce these probiotic formulations can influence the preservation of the microbial viability. However, it is also required that the bacteria maintain their key probiotic capacities during processing, formulation and shelf life. In this study, we investigated the impact of spray-drying on different cell wall properties of the model probiotic strain Lactobacillus rhamnosus GG, including its adherence to intestinal epithelial cells. The dltD gene knock-out mutant, L. rhamnosus GG CMPG5540, displaying modified cell wall lipoteichoic acids, showed significantly increased colony-forming units after spray-drying and subsequent storage under standard conditions compared to wild-type L. rhamnosus GG. In contrast, disruption of the biosynthesis of exopolysaccharides or pili expression did not impact survival. However, spray-drying did significantly affect the adherence capacity of L. rhamnosus GG. Scanning electron microscopy confirmed that the pili, key surface factors for adherence to intestinal cells and mucus, were sheared off during the spray-drying process. These data thus highlight that both the functionality and viability of probiotics should be assessed during the spray-drying process and subsequent storage.

Piliation of Lactobacillus rhamnosus GG promotes adhesion, phagocytosis, and cytokine modulation in macrophages.

Recently, spaCBA-encoded pili on the cell surface of Lactobacillus rhamnosus GG were identified to be key molecules for binding to human intestinal mucus and Caco-2 intestinal epithelial cells. Here, we investigated the role of the SpaCBA pilus of L. rhamnosus GG in the interaction with macrophages in vitro by comparing the wild type with surface mutants. Our results show that SpaCBA pili play a significant role in the capacity for adhesion to macrophages and also promote bacterial uptake by these phagocytic cells. Interestingly, our data suggest that SpaCBA pili also mediate anti-inflammatory effects by induction of interleukin-10 (IL-10) mRNA and reduction of interleukin-6 (IL-6) mRNA in a murine RAW 264.7 macrophage cell line. These pili appear to mediate these effects indirectly by promoting close contact with the macrophages, facilitating the exertion of anti-inflammatory effects by other surface molecules via yet unknown mechanisms. Blockage of complement receptor 3 (CR3), previously identified to be a receptor for streptococcal pili, significantly decreased the uptake of pilus-expressing strains in RAW 264.7 cells, while the expression of IL-10 and IL-6 mRNA by these macrophages was not affected by this blocking. On the other hand, blockage of Toll-like receptor 2 (TLR2) significantly reduced the expression of IL-6 mRNA irrespective of the presence of pili.

The highly autoaggregative and adhesive phenotype of the vaginal Lactobacillus plantarum strain CMPG5300 is sortase dependent.

Lactobacilli are important for the maintenance of a healthy ecosystem in the human vagina. Various mechanisms are postulated but so far are poorly substantiated by molecular studies, such as mutant analysis. Bacterial autoaggregation is an interesting phenomenon that can promote adhesion to host cells and displacement of pathogens. In this study, we report on the identification of a human vaginal isolate, Lactobacillus plantarum strain CMPG5300, which shows high autoaggregative and adhesive capacity. To investigate the importance of sortase-dependent proteins (SDPs) in these phenotypes, a gene deletion mutant was constructed for srtA, the gene encoding the housekeeping sortase that covalently anchors these SDPs to the cell surface. This mutant lost the capacity to autoaggregate, showed a decrease in adhesion to vaginal epithelial cells, and lost biofilm-forming capacity under the conditions tested. These results indicate that the housekeeping sortase SrtA of CMPG5300 is a key determinant of the peculiar surface properties of this vaginal Lactobacillus strain.

Analysis of the peptidoglycan hydrolase complement of Lactobacillus casei and characterization of the major γ-D-glutamyl-L-lysyl-endopeptidase.

Peptidoglycan (PG) is the major component of Gram positive bacteria cell wall and is essential for bacterial integrity and shape. Bacteria synthesize PG hydrolases (PGHs) which are able to cleave bonds in their own PG and play major roles in PG remodelling required for bacterial growth and division. Our aim was to identify the main PGHs in Lactobacillus casei BL23, a lactic acid bacterium with probiotic properties.The PGH complement was first identified in silico by amino acid sequence similarity searches of the BL23 genome sequence. Thirteen PGHs were detected with different predicted hydrolytic specificities. Transcription of the genes was confirmed by RT-PCR. A proteomic analysis combining the use of SDS-PAGE and LC-MS/MS revealed the main seven PGHs synthesized during growth of L. casei BL23. Among these PGHs, LCABL_02770 (renamed Lc-p75) was identified as the major one. This protein is the homolog of p75 (Msp1) major secreted protein of Lactobacillus rhamnosus GG, which was shown to promote survival and growth of intestinal epithelial cells. We identified its hydrolytic specificity on PG and showed that it is a γ-D-glutamyl-L-lysyl-endopeptidase. It has a marked specificity towards PG tetrapeptide chains versus tripeptide chains and for oligomers rather than monomers. Immunofluorescence experiments demonstrated that Lc-p75 localizes at cell septa in agreement with its role in daughter cell separation. It is also secreted under an active form as detected in zymogram. Comparison of the muropeptide profiles of wild type and Lc-p75-negative mutant revealed a decrease of the amount of disaccharide-dipeptide in the mutant PG in agreement with Lc-p75 activity. As a conclusion, Lc-p75 is the major L. casei BL23 PGH with endopeptidase specificity and a key role in daughter cell separation. Further studies will aim at investigating the role of Lc-p75 in the anti-inflammatory potential of L. casei BL23.

The major secreted protein Msp1/p75 is O-glycosylated in Lactobacillus rhamnosus GG.

BACKGROUND: Although the occurrence, biosynthesis and possible functions of glycoproteins are increasingly documented for pathogens, glycoproteins are not yet widely described in probiotic bacteria. Nevertheless, knowledge of protein glycosylation holds important potential for better understanding specific glycan-mediated interactions of probiotics and for glycoengineering in food-grade microbes. RESULTS: Here, we provide evidence that the major secreted protein Msp1/p75 of the probiotic Lactobacillus rhamnosus GG is glycosylated. Msp1 was shown to stain positive with periodic-acid Schiff staining, to be susceptible to chemical deglycosylation, and to bind with the mannose-specific Concanavalin A (ConA) lectin. Recombinant expression in Escherichia coli resulted in a significant reduction in molecular mass, loss of ConA reactivity and increased sensitivity towards pronase E and proteinase K. Mass spectrometry showed that Msp1 is O-glycosylated and identified a glycopeptide TVETPSSA (amino acids 101-108) bearing hexoses presumably linked to the serine residues. Interestingly, these serine residues are not present in the homologous protein of several Lactobacillus casei strains tested, which also did not bind to ConA. The role of the glycan substitutions in known functions of Msp1 was also investigated. Glycosylation did not seem to impact significantly on the peptidoglycan hydrolase activity of Msp1. In addition, the glycan chain appeared not to be required for the activation of Akt signaling in intestinal epithelial cells by Msp1. On the other hand, examination of different cell extracts showed that Msp1 is a glycosylated protein in the supernatant, but not in the cell wall and cytosol fraction, suggesting a link between glycosylation and secretion of this protein. CONCLUSIONS: In this study we have provided the first evidence of protein O-glycosylation in the probiotic L rhamnosus GG. The major secreted protein Msp1 is glycosylated with ConA reactive sugars at the serine residues at 106 and 107. Glycosylation is not required for the peptidoglycan hydrolase activity of Msp1 nor for Akt activation capacity in epithelial cells, but appears to be important for its stability and protection against proteases.

Characterization of MabA, a modulator of Lactobacillus rhamnosus GG adhesion and biofilm formation.

Abstract The probiotic Lactobacillus rhamnosus GG, first isolated from healthy human gut microbiota, has been reported to adhere very well to components of the intestinal mucosa, thereby enabling transient colonization of the gastrointestinal tract (GIT). In a search for the genes responsible for the good adherence capacity of this strain, a genomic region encoding a protein with homology to putative adhesion proteins (LGG_01865) and its putative regulator (LGG_01866) was identified. The sequence of the L. rhamnosus GG LGG_01865 encodes a polypeptide of 2419 amino acid residues containing 26 repetitive DUF1542 domains and a C-terminal LPxTG cell wall-anchoring motif. Phenotypic analyses of a dedicated LGG_01865 knockout mutant revealed a reduced biofilm formation capacity on abiotic surfaces and decreased adhesion to intestinal epithelial cells and tissues of the murine GIT. This suggests a modulating role for LGG_01865 in L. rhamnosus GG-host interactions. Therefore, we propose a new name for LGG_01865, i.e. MabA, modulator of adhesion and biofilm. Expression analysis indicated that LGG_01866 plays a conditional role in the regulation of LGG_01865 expression, i.e. when cells are grown under conditions of sugar starvation.

Impact of luxS and suppressor mutations on the gastrointestinal transit of Lactobacillus rhamnosus GG.

It is generally believed that probiotic bacteria need to survive gastrointestinal transit to exert a health-promoting effect. In this study, a genuine luxS mutant and a luxS mutant containing unknown suppressor mutations of the probiotic strain Lactobacillus rhamnosus GG were compared to the wild type for survival and persistence in the murine gastrointestinal tract. The LuxS enzyme, catalyzing the production of the autoinducer-2 signaling molecule, also forms an integral part of the activated methyl cycle and the metabolism of methionine and cysteine. The genuine luxS mutant CMPG5412 showed drastically reduced persistence in mice, which was related to less survival in simulated gastric juice, indicating that LuxS metabolism is crucial for the gastric stress resistance of L. rhamnosus GG. The suppressor mutations in the other luxS mutant, CMPG5413, appear to compensate for the metabolic defects of the luxS mutation and to restore the resistance to gastric juice but cause a defect in adherence, biofilm formation, and exopolysaccharide production. The shorter residence time of this suppressor mutant in the murine gastrointestinal tract indicates a role for biofilm formation and exopolysaccharides in the persistence capacity of L. rhamnosus GG.